# Patent

## Title
Modulation of factor 11 expression

## Abstract

Disclosed herein are antisense compounds and methods for decreasing Factor 11 and treating or preventing thromboembolic complications in an individual in need thereof. Examples of disease conditions that can be ameliorated with the administration of antisense compounds targeted to Factor 11 include thrombosis, embolism, and thromboembolism, such as, deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke. Antisense compounds targeting Factor 11 can also be used as a prophylactic treatment to prevent individuals at risk for thrombosis and embolism.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 61/105,772, filed Oct. 15, 2008 and U.S. Provisional Application No. 61/174,461, filed Apr. 30, 2009. Each of the above applications is herein incorporated by reference in its entirety.
SEQUENCE LISTING
The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 20091015_BIOL0107USSEQ.txt created Oct. 15, 2009, which is 92 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.


FIELD OF THE INVENTION
Embodiments of the present invention provide methods, compounds, and compositions for reducing expression of Factor 11 mRNA and protein in an animal. Such methods, compounds, and compositions are useful to treat, prevent, or ameliorate thromboembolic complications.
BACKGROUND OF THE INVENTION
The circulatory system requires mechanisms that prevent blood loss, as well as those that counteract inappropriate intravascular obstructions. Generally, coagulation comprises a cascade of reactions culminating in the conversion of soluble fibrinogen to an insoluble fibrin gel. The steps of the cascade involve the conversion of an inactive zymogen to an activated enzyme. The active enzyme then catalyzes the next step in the cascade.
Coagulation Cascade
The coagulation cascade may be initiated through two branches, the tissue factor pathway (also “extrinsic pathway”), which is the primary pathway, and the contact activation pathway (also “intrinsic pathway”).
The tissue factor pathway is initiated by the cell surface receptor tissue factor (TF, also referred to as factor III), which is expressed constitutively by extravascular cells (pericytes, cardiomyocytes, smooth muscle cells, and keratinocytes) and expressed by vascular monocytes and endothelial cells upon induction by inflammatory cytokines or endotoxin. (Drake et al., Am J Pathol 1989, 134:1087-1097). TF is the high affinity cellular receptor for coagulation factor VIIa, a serine protease. In the absence of TF, VIIa has very low catalytic activity, and binding to TF is necessary to render VIIa functional through an allosteric mechanism. (Drake et al., Am J Pathol 1989, 134:1087-1097). The TF-VIIa complex activates factor X to Xa. Xa in turn associates with its co-factor factor Va into a prothrombinase complex which in turn activates prothrombin, (also known as factor II or factor 2) to thrombin (also known as factor IIa, or factor 2a). Thrombin activates platelets, converts fibrinogen to fibrin and promotes fibrin cross-linking by activating factor XIII, thus forming a stable plug at sites where TF is exposed on extravascular cells. In addition, thrombin reinforces the coagulation cascade response by activating factors V and VIII.
The contact activation pathway is triggered by activation of factor XII to XIIa. Factor XIIa converts XI to XIa, and XIa converts IX to IXa. IXa associates with its cofactor VIIIa to convert X to Xa. The two pathways converge at this point as factor Xa associates factor Va to activate prothrombin (factor II) to thrombin (factor IIa).
Inhibition of Coagulation.
At least three mechanisms keep the coagulation cascade in check, namely the action of activated protein C, antithrombin, and tissue factor pathway inhibitor. Activated protein C is a serine protease that degrades cofactors Va and VIIIa. Protein C is activated by thrombin with thrombomodulin, and requires coenzyme Protein S to function. Antithrombin is a serine protease inhibitor (serpin) that inhibits serine proteases: thrombin, Xa, XIIa, XIa and IXa. Tissue factor pathway inhibitor inhibits the action of Xa and the TF-VIIa complex. (Schwartz A L et al., Trends Cardiovasc Med. 1997; 7:234-239.)
Disease
Thrombosis is the pathological development of blood clots, and an embolism occurs when a blood clot migrates to another part of the body and interferes with organ function. Thromboembolism may cause conditions such as deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke. Significantly, thromboembolism is a major cause of morbidity affecting over 2 million Americans every year. (Adcock et al. American Journal of Clinical Pathology. 1997; 108:434-49). While most cases of thrombosis are due to acquired extrinsic problems, for example, surgery, cancer, immobility, some cases are due to a genetic predisposition, for example, antiphospholipid syndrome and the autosomal dominant condition, Factor V Leiden. (Bertina R M et al. Nature 1994; 369:64-67.)
Treatment.
The most commonly used anticoagulants, warfarin, heparin, and low molecular weight heparin (LMWH) all possess significant drawbacks.
Warfarin is typically used to treat patients suffering from atrial fibrillation. The drug interacts with vitamin K-dependent coagulation factors which include factors II, VII, IX and X. Anticoagulant proteins C and S are also inhibited by warfarin. Drug therapy using warfarin is further complicated by the fact that warfarin interacts with other medications, including drugs used to treat atrial fibrillation, such as amiodarone. Because therapy with warfarin is difficult to predict, patients must be carefully monitored in order to detect any signs of anomalous bleeding.
Heparin functions by activating antithrombin which inhibits both thrombin and factor X. (Bjork I, Lindahl U. Mol Cell Biochem. 1982 48: 161-182.) Treatment with heparin may cause an immunological reaction that makes platelets aggregate within blood vessels that can lead to thrombosis. This side effect is known as heparin-induced thrombocytopenia (HIT) and requires patient monitoring. Prolonged treatment with heparin may also lead to osteoporosis. LMWH can also inhibit Factor 2, but to a lesser degree than unfractioned heparin (UFH). LMWH has been implicated in the development of HIT.
Thus, current anticoagulant agents lack predictability and specificity and, therefore, require careful patient monitoring to prevent adverse side effects, such as bleeding complications. There are currently no anticoagulants which target only the intrinsic or extrinsic pathway.
SUMMARY OF THE INVENTION
Provided herein are methods, compounds, and compositions for modulating expression of Factor 11 mRNA and protein. In certain embodiments, Factor 11 specific inhibitors modulate expression of Factor 11 mRNA and protein. In certain embodiments, Factor 11 specific inhibitors are nucleic acids, proteins, or small molecules.
In certain embodiments, modulation can occur in a cell or tissue. In certain embodiments, the cell or tissue is in an animal. In certain embodiments, the animal is a human. In certain embodiments, Factor 11 mRNA levels are reduced. In certain embodiments, Factor 11 protein levels are reduced. Such reduction can occur in a time-dependent manner or in a dose-dependent manner.
Also provided are methods, compounds, and compositions useful for preventing, treating, and ameliorating diseases, disorders, and conditions. In certain embodiments, such diseases, disorders, and conditions are thromboembolic complications. Such thromboembolic complications include the categories of thrombosis, embolism, and thromboembolism. In certain embodiments such thromboembolic complications include deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke.
Such diseases, disorders, and conditions can have one or more risk factors, causes, or outcomes in common. Certain risk factors and causes for development of a thromboembolic complication include immobility, surgery (particularly orthopedic surgery), malignancy, pregnancy, older age, use of oral contraceptives, atrial fibrillation, previous thromboembolic complication, chronic inflammatory disease, and inherited or acquired prothrombotic clotting disorders. Certain outcomes associated with development of a thromboembolic complication include decreased blood flow through an affected vessel, death of tissue, and death.
In certain embodiments, methods of treatment include administering a Factor 11 specific inhibitor to an individual in need thereof.


DETAILED DESCRIPTION OF THE INVENTION
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “including” as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit, unless specifically stated otherwise.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference for the portions of the document discussed herein, as well as in their entirety.
DEFINITIONS
Unless specific definitions are provided, the nomenclature utilized in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical synthesis, and chemical analysis. Where permitted, all patents, applications, published applications and other publications, GENBANK Accession Numbers and associated sequence information obtainable through databases such as National Center for Biotechnology Information (NCBI) and other data referred to throughout in the disclosure herein are incorporated by reference for the portions of the document discussed herein, as well as in their entirety.
Unless otherwise indicated, the following terms have the following meanings:
“2′-O-methoxyethyl” (also 2′-MOE and 2′-O(CH2)2—OCH3) refers to an O-methoxy-ethyl modification of the 2′ position of a furosyl ring. A 2′-O-methoxyethyl modified sugar is a modified sugar.
“2′-O-methoxyethyl nucleotide” means a nucleotide comprising a 2′-O-methoxyethyl modified sugar moiety.
“5-methylcytosine” means a cytosine modified with a methyl group attached to the 5′ position. A 5-methylcytosine is a modified nucleobase.
“Active pharmaceutical agent” means the substance or substances in a pharmaceutical composition that provide a therapeutic benefit when administered to an individual. For example, in certain embodiments an antisense oligonucleotide targeted to Factor 11 is an active pharmaceutical agent.
“Active target region” or “target region” means a region to which one or more active antisense compounds is targeted. “Active antisense compounds” means antisense compounds that reduce target nucleic acid levels or protein levels.
“Administered concomitantly” refers to the co-administration of two agents in any manner in which the pharmacological effects of both are manifest in the patient at the same time. Concomitant administration does not require that both agents be administered in a single pharmaceutical composition, in the same dosage form, or by the same route of administration. The effects of both agents need not manifest themselves at the same time. The effects need only be overlapping for a period of time and need not be coextensive.
“Administering” means providing a pharmaceutical agent to an individual, and includes, but is not limited to administering by a medical professional and self-administering.
“Amelioration” refers to a lessening of at least one indicator, sign, or symptom of an associated disease, disorder, or condition. The severity of indicators may be determined by subjective or objective measures, which are known to those skilled in the art.
“Animal” refers to a human or non-human animal, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including, but not limited to, monkeys and chimpanzees.
“Antidote compound” refers to a compound capable of decreasing the intensity or duration of any antisense-mediated activity.
“Antidote oligonucleotide” means an antidote compound comprising an oligonucleotide that is complementary to and capable of hybridizing with an antisense compound.
“Antidote protein” means an antidote compound comprising a peptide.
“Antibody” refers to a molecule characterized by reacting specifically with an antigen in some way, where the antibody and the antigen are each defined in terms of the other. Antibody may refer to a complete antibody molecule or any fragment or region thereof, such as the heavy chain, the light chain, Fab region, and Fc region.
“Antisense activity” means any detectable or measurable activity attributable to the hybridization of an antisense compound to its target nucleic acid. In certain embodiments, antisense activity is a decrease in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid.
“Antisense compound” means an oligomeric compound that is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.
“Antisense inhibition” means reduction of target nucleic acid levels or target protein levels in the presence of an antisense compound complementary to a target nucleic acid compared to target nucleic acid levels or target protein levels in the absence of the antisense compound.
“Antisense oligonucleotide” means a single-stranded oligonucleotide having a nucleobase sequence that permits hybridization to a corresponding region or segment of a target nucleic acid.
“Bicyclic sugar” means a furosyl ring modified by the bridging of two non-geminal ring atoms. A bicyclic sugar is a modified sugar.
“Bicyclic nucleic acid” or “BNA” refers to a nucleoside or nucleotide wherein the furanose portion of the nucleoside or nucleotide includes a bridge connecting two carbon atoms on the furanose ring, thereby forming a bicyclic ring system.
“Cap structure” or “terminal cap moiety” means chemical modifications, which have been incorporated at either terminus of an antisense compound.
“Chemically distinct region” refers to a region of an antisense compound that is in some way chemically different than another region of the same antisense compound. For example, a region having 2′-O-methoxyethyl nucleotides is chemically distinct from a region having nucleotides without 2′-O-methoxyethyl modifications.
“Chimeric antisense compound” means an antisense compound that has at least two chemically distinct regions.
“Co-administration” means administration of two or more pharmaceutical agents to an individual. The two or more pharmaceutical agents may be in a single pharmaceutical composition, or may be in separate pharmaceutical compositions. Each of the two or more pharmaceutical agents may be administered through the same or different routes of administration. Co-administration encompasses parallel or sequential administration.
“Coagulation factor” means any of factors I, II, III, IV, V, VII, VIII, IX, X, XI, XII, XIII, or TAFI in the blood coagulation cascade. “Coagulation factor nucleic acid” means any nucleic acid encoding a coagulation factor. For example, in certain embodiments, a coagulation factor nucleic acid includes, without limitation, a DNA sequence encoding a coagulation factor (including genomic DNA comprising introns and exons), an RNA sequence transcribed from DNA encoding a coagulation factor, and an mRNA sequence encoding a coagulation factor. “Coagulation factor mRNA” means an mRNA encoding a coagulation factor protein.
“Complementarity” means the capacity for pairing between nucleobases of a first nucleic acid and a second nucleic acid.
“Contiguous nucleobases” means nucleobases immediately adjacent to each other.
“Diluent” means an ingredient in a composition that lacks pharmacological activity, but is pharmaceutically necessary or desirable. For example, the diluent in an injected composition may be a liquid, e.g. saline solution.
“Dose” means a specified quantity of a pharmaceutical agent provided in a single administration, or in a specified time period. In certain embodiments, a dose may be administered in one, two, or more boluses, tablets, or injections. For example, in certain embodiments where subcutaneous administration is desired, the desired dose requires a volume not easily accommodated by a single injection, therefore, two or more injections may be used to achieve the desired dose. In certain embodiments, the pharmaceutical agent is administered by infusion over an extended period of time or continuously. Doses may be stated as the amount of pharmaceutical agent per hour, day, week, or month.
“Effective amount” means the amount of active pharmaceutical agent sufficient to effectuate a desired physiological outcome in an individual in need of the agent. The effective amount may vary among individuals depending on the health and physical condition of the individual to be treated, the taxonomic group of the individuals to be treated, the formulation of the composition, assessment of the individual's medical condition, and other relevant factors.
“Factor 11 nucleic acid” or “Factor XI nucleic acid” or “F 11 nucleic acid” or “F XI nucleic acid” means any nucleic acid encoding Factor 11. For example, in certain embodiments, a Factor 11 nucleic acid includes a DNA sequence encoding Factor 11, an RNA sequence transcribed from DNA encoding Factor 11 (including genomic DNA comprising introns and exons), and an mRNA sequence encoding Factor 11. “Factor 11 mRNA” means an mRNA encoding a Factor 11 protein.
“Factor 11 specific inhibitor” refers to any agent capable of specifically inhibiting the expression of Factor 11 mRNA and/or Factor 11 protein at the molecular level. For example, Factor 11 specific inhibitors include nucleic acids (including antisense compounds), peptides, antibodies, small molecules, and other agents capable of inhibiting the expression of Factor 11 mRNA and/or Factor 11 protein. In certain embodiments, by specifically modulating Factor 11 mRNA expression and/or Factor 11 protein expression, Factor 11 specific inhibitors may affect other components of the coagulation cascade including downstream components. Similarly, in certain embodiments, Factor 11 specific inhibitors may affect other molecular processes in an animal.
“Factor 11 specific inhibitor antidote” means a compound capable of decreasing the effect of a Factor 11 specific inhibitor. In certain embodiments, a Factor 11 specific inhibitor antidote is selected from a Factor 11 peptide; a Factor 11 antidote oligonucleotide, including a Factor 11 antidote compound complementary to a Factor 11 antisense compound; and any compound or protein that affects the intrinsic or extrinsic coagulation pathway.
“Fully complementary” or “100% complementary” means each nucleobase of a first nucleic acid has a complementary nucleobase in a second nucleic acid. In certain embodiments, a first nucleic acid is an antisense compound and a target nucleic acid is a second nucleic acid.
“Gapmer” means a chimeric antisense compound in which an internal region having a plurality of nucleosides that support RNase H cleavage is positioned between external regions having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from the nucleoside or nucleosides comprising the external regions. The internal region may be referred to as a “gap segment” and the external regions may be referred to as “wing segments.”
“Gap-widened” means a chimeric antisense compound having a gap segment of 12 or more contiguous 2′-deoxyribonucleosides positioned between and immediately adjacent to 5′ and 3′ wing segments having from one to six nucleosides.
“Hybridization” means the annealing of complementary nucleic acid molecules. In certain embodiments, complementary nucleic acid molecules include an antisense compound and a target nucleic acid.
“Identifying an animal at risk for thromboembolic complications” means identifying an animal having been diagnosed with a thromboembolic complication or identifying an animal predisposed to develop a thromboembolic complication. Individuals predisposed to develop a thromboembolic complication include those having one or more risk factors for thromboembolic complications including immobility, surgery (particularly orthopedic surgery), malignancy, pregnancy, older age, use of oral contraceptives, and inherited or acquired prothrombotic clotting disorders. Such identification may be accomplished by any method including evaluating an individual's medical history and standard clinical tests or assessments.
“Immediately adjacent” means there are no intervening elements between the immediately adjacent elements.
“Individual” means a human or non-human animal selected for treatment or therapy.
“Internucleoside linkage” refers to the chemical bond between nucleosides.
“Linked nucleosides” means adjacent nucleosides which are bonded together.
“Mismatch” or “non-complementary nucleobase” refers to the case when a nucleobase of a first nucleic acid is not capable of pairing with the corresponding nucleobase of a second or target nucleic acid.
“Modified internucleoside linkage” refers to a substitution or any change from a naturally occurring internucleoside bond (i.e. a phosphodiester internucleoside bond).
“Modified nucleobase” refers to any nucleobase other than adenine, cytosine, guanine, thymidine, or uracil. An “unmodified nucleobase” means the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).
“Modified nucleotide” means a nucleotide having, independently, a modified sugar moiety, modified internucleoside linkage, or modified nucleobase. A “modified nucleoside” means a nucleoside having, independently, a modified sugar moiety or modified nucleobase.
“Modified oligonucleotide” means an oligonucleotide comprising a modified internucleoside linkage, a modified sugar, or a modified nucleobase.
“Modified sugar” refers to a substitution or change from a natural sugar.
“Motif” means the pattern of chemically distinct regions in an antisense compound.
“Naturally occurring internucleoside linkage” means a 3′ to 5′ phosphodiester linkage.
“Natural sugar moiety” means a sugar found in DNA (2′-H) or RNA (2′-OH).
“Nucleic acid” refers to molecules composed of monomeric nucleotides. A nucleic acid includes ribonucleic acids (RNA), deoxyribonucleic acids (DNA), single-stranded nucleic acids, double-stranded nucleic acids, small interfering ribonucleic acids (siRNA), and microRNAs (miRNA).
“Nucleobase” means a heterocyclic moiety capable of pairing with a base of another nucleic acid.
“Nucleobase sequence” means the order of contiguous nucleobases independent of any sugar, linkage, or nucleobase modification.
“Nucleoside” means a nucleobase linked to a sugar.
“Nucleoside mimetic” includes those structures used to replace the sugar or the sugar and the base and not necessarily the linkage at one or more positions of an oligomeric compound such as for example nucleoside mimetics having morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclo or tricyclo sugar mimetics e.g. non furanose sugar units. Nucleotide mimetic includes those structures used to replace the nucleoside and the linkage at one or more positions of an oligomeric compound such as for example peptide nucleic acids or morpholinos (morpholinos linked by —N(H)—C(═O)—O— or other non-phosphodiester linkage). Sugar surrogate overlaps with the slightly broader term nucleoside mimetic but is intended to indicate replacement of the sugar unit (furanose ring) only. The tetrahydropyranyl rings provided herein are illustrative of an example of a sugar surrogate wherein the furanose sugar group has been replaced with a tetrahydropyranyl ring system.
“Nucleotide” means a nucleoside having a phosphate group covalently linked to the sugar portion of the nucleoside.
“Oligomeric compound” or “oligomer” means a polymer of linked monomeric subunits which is capable of hybridizing to at least a region of a nucleic acid molecule.
“Oligonucleotide” means a polymer of linked nucleosides each of which can be modified or unmodified, independent one from another.
“Parenteral administration” means administration through injection or infusion. Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, or intracranial administration, e.g. intrathecal or intracerebroventricular administration.
“Peptide” means a molecule formed by linking at least two amino acids by amide bonds. Peptide refers to polypeptides and proteins.
“Pharmaceutical composition” means a mixture of substances suitable for administering to an individual. For example, a pharmaceutical composition may comprise one or more active pharmaceutical agents and a sterile aqueous solution.
“Pharmaceutically acceptable salts” means physiologically and pharmaceutically acceptable salts of antisense compounds, i.e., salts that retain the desired biological activity of the parent oligonucleotide and do not impart undesired toxicological effects thereto.
“Phosphorothioate linkage” means a linkage between nucleosides where the phosphodiester bond is modified by replacing one of the non-bridging oxygen atoms with a sulfur atom. A phosphorothioate linkage (P═S) is a modified internucleoside linkage.
“Portion” means a defined number of contiguous (i.e. linked) nucleobases of a nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of a target nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of an antisense compound.
“Prevent” refers to delaying or forestalling the onset or development of a disease, disorder, or condition for a period of time from minutes to indefinitely. Prevent also means reducing risk of developing a disease, disorder, or condition.
“Prodrug” means a therapeutic agent that is prepared in an inactive form that is converted to an active form within the body or cells thereof by the action of endogenous enzymes or other chemicals or conditions.
“Side effects” means physiological responses attributable to a treatment other than the desired effects. In certain embodiments, side effects include injection site reactions, liver function test abnormalities, renal function abnormalities, liver toxicity, renal toxicity, central nervous system abnormalities, myopathies, and malaise. For example, increased aminotransferase levels in serum may indicate liver toxicity or liver function abnormality. For example, increased bilirubin may indicate liver toxicity or liver function abnormality.
“Single-stranded oligonucleotide” means an oligonucleotide which is not hybridized to a complementary strand.
“Specifically hybridizable” refers to an antisense compound having a sufficient degree of complementarity between an antisense oligonucleotide and a target nucleic acid to induce a desired effect, while exhibiting minimal or no effects on non-target nucleic acids under conditions in which specific binding is desired, i.e. under physiological conditions in the case of in vivo assays and therapeutic treatments.
“Targeting” or “targeted” means the process of design and selection of an antisense compound that will specifically hybridize to a target nucleic acid and induce a desired effect.
“Target nucleic acid,” “target RNA,” and “target RNA transcript” all refer to a nucleic acid capable of being targeted by antisense compounds.
“Target segment” means the sequence of nucleotides of a target nucleic acid to which an antisense compound is targeted. “5′ target site” refers to the 5′-most nucleotide of a target segment. “3′ target site” refers to the 3′-most nucleotide of a target segment.
“Therapeutically effective amount” means an amount of a pharmaceutical agent that provides a therapeutic benefit to an individual.
“Thromboembolic complication” means any disease, disorder, or condition involving an embolism caused by a thrombus. Examples of such diseases, disorders, and conditions include the categories of thrombosis, embolism, and thromboembolism. In certain embodiments, such disease disorders, and conditions include deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke.
“Treat” refers to administering a pharmaceutical composition to effect an alteration or improvement of a disease, disorder, or condition.
“Unmodified nucleotide” means a nucleotide composed of naturally occurring nucleobases, sugar moieties, and internucleoside linkages. In certain embodiments, an unmodified nucleotide is an RNA nucleotide (i.e. β-D-ribonucleosides) or a DNA nucleotide (i.e. β-D-deoxyribonucleoside).
Certain Embodiments
Embodiments of the present invention provide methods, compounds, and compositions for decreasing Factor 11 mRNA and protein expression.
Embodiments of the present invention provide methods, compounds, and compositions for the treatment, prevention, or amelioration of diseases, disorders, and conditions associated with Factor 11 in an individual in need thereof. Also contemplated are methods and compounds for the preparation of a medicament for the treatment, prevention, or amelioration of a disease, disorder, or condition associated with Factor 11. Factor 11 associated diseases, disorders, and conditions include thromboembolic complications such as thrombosis, embolism, thromboembolism, deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke.
Embodiments of the present invention provide a Factor 11 specific inhibitor for use in treating, preventing, or ameliorating a Factor 11 associated disease. In certain embodiments, Factor 11 specific inhibitors are nucleic acids (including antisense compounds), peptides, antibodies, small molecules, and other agents capable of inhibiting the expression of Factor 11 mRNA and/or Factor 11 protein.
In certain embodiments of the present invention, Factor 11 specific inhibitors are peptides or proteins, such as, but not limited to, alpha 1 protease inhibitors, antithrombin III, C1 inhibitors, and alpha 2 plasmin inhibitors as described in J Clin Invest 1982, 69:844-852; alpha 1 antitrypsin (alpha 1AT) as described in Thromb Res 1987, 48:145-151; Factor 11 peptide inhibitors as described in USPPN 2008/021998 and Blood 1998, 92:4198-206; MAP4-RGKWC as described in Thromb Res 2001, 104:451-465; beta 2 GPI as described in Proc Natl Acad Sci 2004, 101:3939-44; Lentinus proteinase inhibitor as described in Eur J Biochem 1999, 262:915-923; protease nexin-2/amyloid beta protein precursor Kunitz domain inhibitor (APPI) and antithrombin (AT) as described in J Biol Chem 2004, 279:29485-29492; and aprotinin as described in J Biol Chem 2005, 280:23523-30.
In certain embodiments of the present invention, Factor 11 specific inhibitors are antibodies, such as, but not limited to, Winston-Salem (IgG3 kappa) and Baltimore (IgG1 kappa) as described in Blood 1988, 72:1748-54; 5F4, 3C1, and 1F1 as described in J Biol Chem 1985, 260:10714-719; monoclonal antibodies as described in Throm Haemost 1990, 63:417-23; XI-5108 as described in J Thromb Haem 2006, 4:1496-1501; monoclonal antibodies 4-1 as described in Thromb Res 1986, 42:225-34; and abcixmab antibody as described in Example 19 of U.S. Pat. No. 6,566,140.
In certain embodiments of the present invention, Factor 11 specific inhibitors are small molecules, such as, but not limited to, diisopropyl fluorophosphates (DFP); the small molecule inhibitors as described in Examples 1-7 of USPPN 2004/0180855; and p-aminobenzamidine (pAB) as described in J Biol Chem 2005, 280:23523-30.
Embodiments of the present invention provide a Factor 11 specific inhibitor, as described herein, for use in treating, preventing, or ameliorating thromboembolic complications such as thrombosis, embolism, thromboembolism, deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke.
Embodiments of the present invention provide the use of Factor 11 specific inhibitors as described herein in the manufacture of a medicament for treating, ameliorating, or preventing a thromboembolic complication such as thrombosis, embolism, thromboembolism, deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke.
Embodiments of the present invention provide a Factor 11 specific inhibitor as described herein for use in treating, preventing, or ameliorating a thromboembolic complication as described herein by combination therapy with an additional agent or therapy as described herein. Agents or therapies can be co-administered or administered concomitantly.
Embodiments of the present invention provide the use of a Factor 11 specific inhibitor as described herein in the manufacture of a medicament for treating, preventing, or ameliorating a thromboembolic complication as described herein by combination therapy with an additional agent or therapy as described herein. Agents or therapies can be co-administered or administered concomitantly.
Embodiments of the present invention provide the use of a Factor 11 specific inhibitor as described herein in the manufacture of a medicament for treating, preventing, or ameliorating a thromboembolic complication as described herein in a patient who is subsequently administered an additional agent or therapy as described herein.
Embodiments of the present invention provide a kit for treating, preventing, or ameliorating a thromboembolic complication as described herein wherein the kit comprises:
(i) a Factor 11 specific inhibitor as described herein; and alternatively
(ii) an additional agent or therapy as described herein.
A kit of the present invention may further include instructions for using the kit to treat, prevent, or ameliorate a thromboembolic complication as described herein by combination therapy as described herein.
Embodiments of the present invention provide antisense compounds targeted to a Factor 11 nucleic acid. In certain embodiments, the Factor 11 nucleic acid is any of the sequences set forth in GENBANK Accession No. NM—000128.3 (incorporated herein as SEQ ID NO: 1), GENBANK Accession No. NT—022792.17, truncated from 19598000 to 19624000, (incorporated herein as SEQ ID NO: 2), GENBANK Accession No. NM—028066.1 (incorporated herein as SEQ ID NO: 6), exons 1-15 GENBANK Accession No. NW—001118167.1 (incorporated herein as SEQ ID NO: 274).
In certain embodiments, the invention provides a compound comprising a modified oligonucleotide. In certain embodiments, the compound of the invention comprises a modified oligonucleotide consisting of 12 to 30 linked nucleosides.
In certain embodiments, the compound of the invention may comprise a modified oligonucleotide comprising a nucleobase sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to an equal length portion of SEQ ID NO: 1. In certain embodiments, the compound of the invention may comprise a modified oligonucleotide comprising a nucleobase sequence 100% complementary to an equal length portion of SEQ ID NO: 1.
In certain embodiments, the invention provides a compound comprising a modified oligonucleotide comprising a nucleobase sequence complementary to at least a portion of nucleobases 656 to 676 of SEQ ID NO: 1. Said modified oligonucleotide may comprise at least 8, at least 10, at least 12, at least 14, at least 16, at least 18 or 20 contiguous nucleobases complementary to an equal length portion of nucleobases 656 to 676 of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence 100% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may achieve at least 80% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 3).
In certain embodiments, the invention provides a compound comprising a modified oligonucleotide comprising a nucleobase sequence complementary to at least a portion of nucleobases 665 to 687 of SEQ ID NO: 1. Said modified oligonucleotide may comprise at least 8, at least 10, at least 12, at least 14, at least 16, at least 18 or 20 contiguous nucleobases complementary to an equal length portion of nucleobases 665 to 687 of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence 100% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may achieve at least 50% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 3).
In certain embodiments, the invention provides a compound comprising a modified oligonucleotide comprising a nucleobase sequence complementary to at least a portion of nucleobases 675 to 704 of SEQ ID NO: 1. Said modified oligonucleotide may comprise at least 8, at least 10, at least 12, at least 14, at least 16, at least 18 or 20 contiguous nucleobases complementary to an equal length portion of nucleobases 675 to 704 of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence 100% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may achieve at least 50% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 3).
In certain embodiments, the invention provides a compound comprising a modified oligonucleotide comprising a nucleobase sequence complementary to at least a portion of nucleobases 677 to 704 of SEQ ID NO: 1. Said modified oligonucleotide may comprise at least 8, at least 10, at least 12, at least 14, at least 16, at least 18 or 20 contiguous nucleobases complementary to an equal length portion of nucleobases 677 to 704 of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence 100% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may achieve at least 60% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 3).
In certain embodiments, the invention provides a compound comprising a modified oligonucleotide comprising a nucleobase sequence complementary to at least a portion of nucleobases 678 to 697 of SEQ ID NO: 1. Said modified oligonucleotide may comprise at least 8, at least 10, at least 12, at least 14, at least 16, at least 18 or 20 contiguous nucleobases complementary to an equal length portion of nucleobases 678 to 697 of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence 100% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may achieve at least 70% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 3).
In certain embodiments, the invention provides a compound comprising a modified oligonucleotide comprising a nucleobase sequence complementary to at least a portion of nucleobases 680 to 703 of SEQ ID NO: 1. Said modified oligonucleotide may comprise at least 8, at least 10, at least 12, at least 14, at least 16, at least 18 or 20 contiguous nucleobases complementary to an equal length portion of nucleobases 680 to 703 of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence 100% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may achieve at least 80% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 3 and Example 30).
In certain embodiments, the invention provides a compound comprising a modified oligonucleotide comprising a nucleobase sequence complementary to at least a portion of nucleobases 683 to 702 of SEQ ID NO: 1. Said modified oligonucleotide may comprise at least 8, at least 10, at least 12, at least 14, at least 16, at least 18 or 20 contiguous nucleobases complementary to an equal length portion of nucleobases 683 to 702 of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence 100% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may achieve at least 90% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 3).
In certain embodiments, the invention provides a compound comprising a modified oligonucleotide comprising a nucleobase sequence complementary to at least a portion of nucleobases 738 to 759 of SEQ ID NO: 1. Said modified oligonucleotide may comprise at least 8, at least 10, at least 12, at least 14, at least 16, at least 18 or 20 contiguous nucleobases complementary to an equal length portion of nucleobases 738 to 759 of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence 100% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may achieve at least 80% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 3 and Example 30).
In certain embodiments, the invention provides a compound comprising a modified oligonucleotide comprising a nucleobase sequence complementary to at least a portion of nucleobases 738 to 760 of SEQ ID NO: 1. Said modified oligonucleotide may comprise at least 8, at least 10, at least 12, at least 14, at least 16, at least 18 or 20 contiguous nucleobases complementary to an equal length portion of nucleobases 738 to 760 of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence 100% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may achieve at least 60% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 3).
In certain embodiments, the invention provides a compound comprising a modified oligonucleotide comprising a nucleobase sequence complementary to at least a portion of nucleobases 738 to 762 of SEQ ID NO: 1. Said modified oligonucleotide may comprise at least 8, at least 10, at least 12, at least 14, at least 16, at least 18 or 20 contiguous nucleobases complementary to an equal length portion of nucleobases 738 to 762 of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence 100% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may achieve at least 45% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 3).
In certain embodiments, the invention provides a compound comprising a modified oligonucleotide comprising a nucleobase sequence complementary to at least a portion of nucleobases 1018 to 1042 of SEQ ID NO: 1. Said modified oligonucleotide may comprise at least 8, at least 10, at least 12, at least 14, at least 16, at least 18 or 20 contiguous nucleobases complementary to an equal length portion of nucleobases 1018 to 1042 of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence 100% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may achieve at least 80% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 3).
In certain embodiments, the invention provides a compound comprising a modified oligonucleotide comprising a nucleobase sequence complementary to at least a portion of nucleobases 1062 to 1089 of SEQ ID NO: 1. Said modified oligonucleotide may comprise at least 8, at least 10, at least 12, at least 14, at least 16, at least 18 or 20 contiguous nucleobases complementary to an equal length portion of nucleobases 1062 to 1089 of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence 100% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may achieve at least 70% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 3).
In certain embodiments, the invention provides a compound comprising a modified oligonucleotide comprising a nucleobase sequence complementary to at least a portion of nucleobases 1062 to 1090 of SEQ ID NO: 1. Said modified oligonucleotide may comprise at least 8, at least 10, at least 12, at least 14, at least 16, at least 18 or 20 contiguous nucleobases complementary to an equal length portion of nucleobases 1062 to 1090 of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence 100% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may achieve at least 60% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 3).
In certain embodiments, the invention provides a compound comprising a modified oligonucleotide comprising a nucleobase sequence complementary to at least a portion of nucleobases 1062 to 1091 of SEQ ID NO: 1. Said modified oligonucleotide may comprise at least 8, at least 10, at least 12, at least 14, at least 16, at least 18 or 20 contiguous nucleobases complementary to an equal length portion of nucleobases 1062 to 1091 of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence 100% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may achieve at least 20% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 3).
In certain embodiments, the invention provides a compound comprising a modified oligonucleotide comprising a nucleobase sequence complementary to at least a portion of nucleobases 1275 to 1301 of SEQ ID NO: 1. Said modified oligonucleotide may comprise at least 8, at least 10, at least 12, at least 14, at least 16, at least 18 or 20 contiguous nucleobases complementary to an equal length portion of nucleobases 1062 to 1091 of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence 100% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may achieve at least 80% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 3).
In certain embodiments, the invention provides a compound comprising a modified oligonucleotide comprising a nucleobase sequence complementary to at least a portion of nucleobases 1276 to 1301 of SEQ ID NO: 1. Said modified oligonucleotide may comprise at least 8, at least 10, at least 12, at least 14, at least 16, at least 18 or 20 contiguous nucleobases complementary to an equal length portion of nucleobases 1062 to 1091 of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence 100% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may achieve at least 80% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 30).
In certain embodiments, the invention provides a compound comprising a modified oligonucleotide comprising a nucleobase sequence complementary to at least a portion of nucleobases 1284 to 1308 of SEQ ID NO: 1. Said modified oligonucleotide may comprise at least 8, at least 10, at least 12, at least 14, at least 16, at least 18 or 20 contiguous nucleobases complementary to an equal length portion of nucleobases 1062 to 1091 of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence 100% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may achieve at least 80% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 3).
In certain embodiments, the invention provides a compound comprising a modified oligonucleotide comprising a nucleobase sequence complementary to at least a portion of nucleobases 1291 to 1317 of SEQ ID NO: 1. Said modified oligonucleotide may comprise at least 8, at least 10, at least 12, at least 14, at least 16, at least 18 or 20 contiguous nucleobases complementary to an equal length portion of nucleobases 1062 to 1091 of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence 100% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may achieve at least 80% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 3).
In certain embodiments, the invention provides a compound comprising a modified oligonucleotide comprising a nucleobase sequence complementary to at least a portion of nucleobases 1275 to 1318 of SEQ ID NO: 1. Said modified oligonucleotide may comprise at least 8, at least 10, at least 12, at least 14, at least 16, at least 18 or 20 contiguous nucleobases complementary to an equal length portion of nucleobases 1275 to 1318 of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence 100% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may achieve at least 70% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 3).
Embodiments of the present invention provide compounds comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, or 20 contiguous nucleobases of a nucleobase sequence selected from among the nucleobase sequences recited in SEQ ID NOs: 15 to 241.
Embodiments of the present invention provide compounds comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, or 20 contiguous nucleobases of a nucleobase sequence selected from among the nucleobase sequences recited in SEQ ID NOs: 15 to 269.
Embodiments of the present invention provide compounds comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, or 20 contiguous nucleobases of a nucleobase sequence selected from among the nucleobase sequences recited in SEQ ID NOs: 242 to 269.
In certain embodiments, the modified oligonucleotide comprises at least 8, at least 10, at least 12, at least 14, at least 16, or at least 18 nucleobases of a nucleobase sequence selected from SEQ ID NOs: 22, 31, 32, 34, 36 to 38, 40, 41, 43, 51 to 53, 55, 56, 59, 60, 64, 66, 71, 73, 75, 96, 98 to 103, 105 to 109, 113 to 117, 119, 124, 127, 129, 171, 172, 174, 176, 178, 179, 181 to 197, 199 to 211, and 213 to 232. In certain embodiments, the modified oligonucleotide comprises a nucleobase sequence selected from SEQ ID NOs: 22, 31, 32, 34, 36 to 38, 40, 41, 43, 51 to 53, 55, 56, 59, 60, 64, 66, 71, 73, 75, 96, 98 to 103, 105 to 109, 113 to 117, 119, 124, 127, 129, 171, 172, 174, 176, 178, 179, 181 to 197, 199 to 211, and 213 to 232. In certain embodiments, the modified oligonucleotide consists of a nucleobase sequence selected from SEQ ID NOs: 22, 31, 32, 34, 36 to 38, 40, 41, 43, 51 to 53, 55, 56, 59, 60, 64, 66, 71, 73, 75, 96, 98 to 103, 105 to 109, 113 to 117, 119, 124, 127, 129, 171, 172, 174, 176, 178, 179, 181 to 197, 199 to 211, and 213 to 232. Said modified oligonucleotide may achieve at least 70% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 3).
In certain embodiments, the modified oligonucleotide comprises at least 8, at least 10, at least 12, at least 14, at least 16, or at least 18 nucleobases of a nucleobase sequence selected from SEQ ID NOs: 22, 31, 34, 37, 40, 43, 51 to 53, 60, 98, 100 to 102, 105 to 109, 114, 115, 119, 171, 174, 176, 179, 181, 186, 188 to 193, 195, 196, 199 to 210, and 213 to 232. In certain embodiments, the modified oligonucleotide comprises a nucleobase sequence selected from SEQ ID NOs: 22, 31, 34, 37, 40, 43, 51 to 53, 60, 98, 100 to 102, 105 to 109, 114, 115, 119, 171, 174, 176, 179, 181, 186, 188 to 193, 195, 196, 199 to 210, and 213 to 232. In certain embodiments, the modified oligonucleotide consists of a nucleobase sequence selected from SEQ ID NOs: 22, 31, 34, 37, 40, 43, 51 to 53, 60, 98, 100 to 102, 105 to 109, 114, 115, 119, 171, 174, 176, 179, 181, 186, 188 to 193, 195, 196, 199 to 210, and 213 to 232. Said modified oligonucleotide may achieve at least 80% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 3).
In certain embodiments, the modified oligonucleotide comprises at least 8, at least 10, at least 12, at least 14, at least 16, or at least 18 nucleobases of a nucleobase sequence selected from SEQ ID NOs: 31, 37, 100, 105, 179, 190 to 193, 196, 202 to 207, 209, 210, 214 to 219, 221 to 224, 226, 227, 229, and 231. In certain embodiments, the modified oligonucleotide comprises a nucleobase sequence selected from SEQ ID NOs: 31, 37, 100, 105, 179, 190 to 193, 196, 202 to 207, 209, 210, 214 to 219, 221 to 224, 226, 227, 229, and 231. In certain embodiments, the modified oligonucleotide consists of a nucleobase sequence selected from SEQ ID NOs: 31, 37, 100, 105, 179, 190 to 193, 196, 202 to 207, 209, 210, 214 to 219, 221 to 224, 226, 227, 229, and 231. Said modified oligonucleotide may achieve at least 90% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 3).
In certain embodiments, the modified oligonucleotide comprises at least 8, at least 10, at least 12, at least 14, at least 16, or at least 18 nucleobases of a nucleobase sequence selected from SEQ ID NOs: 34, 52, 53, 114, 115, 190, 213 to 232, 242 to 260, and 262 to 266. In certain embodiments, the modified oligonucleotide comprises a nucleobase sequence selected from SEQ ID NOs: 34, 52, 53, 114, 115, 190, 213 to 232, 242 to 260, and 262 to 266. In certain embodiments, the modified oligonucleotide consists of a nucleobase sequence selected from SEQ ID NOs: 34, 52, 53, 114, 115, 190, 213 to 232, 242 to 260, and 262 to 266. Said modified oligonucleotides may achieve at least 70% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 30).
In certain embodiments, the modified oligonucleotide comprises at least 8, at least 10, at least 12, at least 14, at least 16, or at least 18 nucleobases of a nucleobase sequence selected from SEQ ID NOs: 34, 52, 53, 114, 115, 190, 213 to 216, 218 to 226, 243 to 246, 248, 249, 252 to 259, 264, and 265. In certain embodiments, the modified oligonucleotide comprises a nucleobase sequence selected from SEQ ID NOs: 34, 52, 53, 114, 115, 190, 213 to 216, 218 to 226, 243 to 246, 248, 249, 252 to 259, 264, and 265. In certain embodiments, the modified oligonucleotide consists of a nucleobase sequence selected from SEQ ID NOs: 34, 52, 53, 114, 115, 190, 213 to 216, 218 to 226, 243 to 246, 248, 249, 252 to 259, 264, and 265. Said modified oligonucleotides may achieve at least 80% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 30).
In certain embodiments, the modified oligonucleotide comprises at least 8, at least 10, at least 12, at least 14, at least 16, or at least 18 nucleobases of a nucleobase sequence selected from SEQ ID NOs: 34, 190, 215, 222, 223, 226, 246, and 254. In certain embodiments, the modified oligonucleotide comprises a nucleobase sequence selected from SEQ ID NOs: 34, 190, 215, 222, 223, 226, 246, and 254. In certain embodiments, the modified oligonucleotide consists of a nucleobase sequence selected from SEQ ID NOs: 34, 190, 215, 222, 223, 226, 246, and 254. Said modified oligonucleotides may achieve at least 90% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 30).
In certain embodiments, the compound consists of a single-stranded modified oligonucleotide.
In certain embodiments, the modified oligonucleotide consists of 20 linked nucleosides.
In certain embodiments, the nucleobase sequence of the modified oligonucleotide is 100% complementary to a nucleobase sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 6 or SEQ ID NO: 274.
In certain embodiments, the compound has at least one modified internucleoside linkage. In certain embodiments, the internucleoside linkage is a phosphorothioate internucleoside linkage.
In certain embodiments, the compound has at least one nucleoside comprising a modified sugar. In certain embodiments, the at least one modified sugar is a bicyclic sugar. In certain embodiments, the at least one modified sugar comprises a 2′-O-methoxyethyl.
Embodiments of the present invention provide compounds comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, or 20 contiguous nucleobases of a nucleobase sequence selected from among the nucleobase sequences recited in SEQ ID NOs: 15 to 241, SEQ ID NOs: 15 to 269, or SEQ ID NOs: 242 to 269, wherein at least one nucleoside comprises a modified sugar.
In certain embodiments, said at least one at least one modified sugar is a bicyclic sugar.
In certain embodiments, said at least one bicyclic sugar comprises a 4′-(CH2)n—O-2′ bridge, wherein n is 1 or 2.
In certain embodiments, said at least one bicyclic sugar comprises a 4′-CH(CH3)—O-2′ bridge.
In certain embodiments, said at least one modified sugar comprises a 2′-O-methoxyethyl group.
Embodiments of the present invention provide compounds comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, or 20 contiguous nucleobases of a nucleobase sequence selected from among the nucleobase sequences recited in SEQ ID NOs: 15 to 241, SEQ ID NOs: 15 to 269, or SEQ ID NOs: 242 to 269, comprising at least one tetrahydropyran modified nucleoside wherein a tetrahydropyran ring replaces the furanose ring.
In certain embodiments, said at least one tetrahydropyran modified nucleoside has the structure:




In certain embodiments, the compound has at least one nucleoside comprising a modified nucleobase. In certain embodiments, the modified nucleobase is a 5-methylcytosine.
In certain embodiments, the modified oligonucleotide of the compound comprises:
(i) a gap segment consisting of linked deoxynucleosides;
(ii) a 5′ wing segment consisting of linked nucleosides;
(iii) a 3′ wing segment consisting of linked nucleosides, wherein the gap segment is positioned immediately adjacent to and between the 5′ wing segment and the 3′ wing segment and wherein each nucleoside of each wing segment comprises a modified sugar. In some such embodiments, each cytosine in the modified oligonucleotide is a 5-methylcytosine.
In certain embodiments, the modified oligonucleotide of the compound comprises:
(i) a gap segment consisting of ten linked deoxynucleosides;
(ii) a 5′ wing segment consisting of five linked nucleosides;
(iii) a 3′ wing segment consisting of five linked nucleosides, wherein the gap segment is positioned immediately adjacent to and between the 5′ wing segment and the 3′ wing segment, wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar; and wherein each internucleoside linkage is a phosphorothioate linkage. In some such embodiments, each cytosine in the modified oligonucleotide is a 5-methylcytosine.
In certain embodiments, the modified oligonucleotide of the compound comprises:
(i) a gap segment consisting of fourteen linked deoxynucleosides;
(ii) a 5′ wing segment consisting of three linked nucleosides;
(iii) a 3′ wing segment consisting of three linked nucleosides, wherein the gap segment is positioned immediately adjacent to and between the 5′ wing segment and the 3′ wing segment, wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar; and wherein each internucleoside linkage is a phosphorothioate linkage. In some such embodiments, each cytosine in the modified oligonucleotide is a 5-methylcytosine.
In certain embodiments, the modified oligonucleotide of the compound comprises:
(i) a gap segment consisting of thirteen linked deoxynucleosides;
(ii) a 5′ wing segment consisting of two linked nucleosides;
(iii) a 3′ wing segment consisting of five linked nucleosides, wherein the gap segment is positioned immediately adjacent to and between the 5′ wing segment and the 3′ wing segment, wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar; and wherein each internucleoside linkage is a phosphorothioate linkage. In some such embodiments, each cytosine in the modified oligonucleotide is a 5-methylcytosine.
Embodiments of the present invention provide a composition comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 12 contiguous nucleobases of a nucleobase sequence selected from among the nucleobase sequences recited in SEQ ID NOs: 15 to 241 or a salt thereof and a pharmaceutically acceptable carrier or diluent.
Embodiments of the present invention provide a composition comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 12 contiguous nucleobases of a nucleobase sequence selected from among the nucleobase sequences recited in SEQ ID NOs: 15 to 269 or a salt thereof and a pharmaceutically acceptable carrier or diluent.
Embodiments of the present invention provide a composition comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 12 contiguous nucleobases of a nucleobase sequence selected from among the nucleobase sequences recited in SEQ ID NOs: 241 to 269 or a salt thereof and a pharmaceutically acceptable carrier or diluent.
Embodiments of the present invention provide methods comprising administering to an animal a compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8 contiguous nucleobases of a nucleobase sequence selected from among the nucleobase sequences recited in SEQ ID NOs: 15 to 241.
Embodiments of the present invention provide methods comprising administering to an animal a compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8 contiguous nucleobases of a nucleobase sequence selected from among the nucleobase sequences recited in SEQ ID NOs: 15 to 269.
Embodiments of the present invention provide methods comprising administering to an animal a compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8 contiguous nucleobases of a nucleobase sequence selected from among the nucleobase sequences recited in SEQ ID NOs: 241 to 269.
In certain embodiments, the animal is a human.
In certain embodiments, the administering prevents deep vein thrombosis or pulmonary embolism.
In certain embodiments, the compound is co-administered with any of the group selected from aspirin, clopidogrel, dipyridamole, heparin, lepirudin, ticlopidine, warfarin, apixaban, rivaroxaban, and LOVENOX.
In certain embodiments, the compound is co-administered with any Factor Xa inhibitor.
In certain embodiment, the Factor Xa inhibitor is any of Rivaroxaban, LY517717, YM150, apixaban, PRT054021, and DU-176b.
In certain embodiments, the compound is administered concomitantly with any of the group selected from aspirin, clopidogrel, dipyridamole, heparin, lepirudin, ticlopidine, warfarin, apixaban, rivaroxaban, and LOVENOX are administered concomitantly.
In certain embodiments, the administering is parenteral administration. In certain embodiments, the parenteral administration is any of subcutaneous or intravenous administration.
Embodiments of the present invention provide methods comprising identifying an animal at risk for developing thromboembolic complications and administering to the at risk animal a therapeutically effective amount of a compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides, wherein the modified oligonucleotide is complementary to a Factor 11 nucleic acid.
In certain embodiments, the thromboembolic complication is deep vein thrombosis, pulmonary embolism, or a combination thereof.
Embodiments of the present invention provide methods comprising identifying an animal having a clotting disorder by administering to the animal a therapeutically effective amount of a compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides, wherein the modified oligonucleotide is complementary to a Factor 11 nucleic acid.
In certain embodiments, the compound is co-administered with any of the group selected from aspirin, clopidogrel, dipyridamole, heparin, lepirudin, ticlopidine, warfarin, apixaban, rivaroxaban, and LOVENOX.
In certain embodiments, the compound is administered concomitantly with any of the group selected from aspirin, clopidogrel, dipyridamole, heparin, lepirudin, ticlopidine, warfarin, apixaban, rivaroxaban, and LOVENOX are administered concomitantly.
Embodiments of the present invention provide methods comprising reducing the risk for thromboembolic complications in an animal by administering to the animal a therapeutically effective amount of a compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides, wherein the modified oligonucleotide is complementary to a Factor 11 nucleic acid.
Embodiments of the present invention provide methods comprising treating a clotting disorder in an animal by administering to the animal a therapeutically effective amount of a compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides, wherein the modified oligonucleotide is complementary to a Factor 11 nucleic acid.
Embodiments of the present invention provide methods comprising inhibiting Factor 11 expression in an animal by administering to the animal a therapeutically effective amount of a compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides, wherein the modified oligonucleotide is complementary to a Factor 11 nucleic acid.
In certain embodiments, the Factor 11 inhibition in the animal is reversed by administering an antidote to the modified oligonucleotide.
In certain embodiments, the antidote is an oligonucleotide complementary to the modified oligonucleotide.
Antisense Compounds
Oligomeric compounds include, but are not limited to, oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics, antisense compounds, antisense oligonucleotides, and siRNAs. An oligomeric compound may be “antisense” to a target nucleic acid, meaning that is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.
In certain embodiments, an antisense compound has a nucleobase sequence that, when written in the 5′ to 3′ direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted. In certain such embodiments, an antisense oligonucleotide has a nucleobase sequence that, when written in the 5′ to 3′ direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted.
In certain embodiments, an antisense compound targeted to a Factor 11 nucleic acid is 12 to 30 subunits in length. In other words, such antisense compounds are from 12 to 30 linked subunits. In other embodiments, the antisense compound is 8 to 80, 12 to 50, 15 to 30, 18 to 24, 19 to 22, or linked subunits. In certain such embodiments, the antisense compounds are 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 linked subunits in length, or a range defined by any two of the above values. In some embodiments the antisense compound is an antisense oligonucleotide, and the linked subunits are nucleotides.
In certain embodiments anitsense oligonucleotides targeted to a Factor 11 nucleic acid may be shortened or truncated. For example, a single subunit may be deleted from the 5′ end (5′ truncation), or alternatively from the 3′ end (3′ truncation). A shortened or truncated antisense compound targeted to a Factor 11 nucleic acid may have two subunits deleted from the 5′ end, or alternatively may have two subunits deleted from the 3′ end, of the antisense compound. Alternatively, the deleted nucleosides may be dispersed throughout the antisense compound, for example, in an antisense compound having one nucleoside deleted from the 5′ end and one nucleoside deleted from the 3′ end.
When a single additional subunit is present in a lengthened antisense compound, the additional subunit may be located at the 5′ or 3′ end of the antisense compound. When two or more additional subunits are present, the added subunits may be adjacent to each other, for example, in an antisense compound having two subunits added to the 5′ end (5′ addition), or alternatively to the 3′ end (3′ addition), of the antisense compound. Alternatively, the added subunits may be dispersed throughout the antisense compound, for example, in an antisense compound having one subunit added to the 5′ end and one subunit added to the 3′ end.
It is possible to increase or decrease the length of an antisense compound, such as an antisense oligonucleotide, and/or introduce mismatch bases without eliminating activity. For example, in Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a series of antisense oligonucleotides 13-25 nucleobases in length were tested for their ability to induce cleavage of a target RNA in an oocyte injection model. Antisense oligonucleotides 25 nucleobases in length with 8 or 11 mismatch bases near the ends of the antisense oligonucleotides were able to direct specific cleavage of the target mRNA, albeit to a lesser extent than the antisense oligonucleotides that contained no mismatches. Similarly, target specific cleavage was achieved using 13 nucleobase antisense oligonucleotides, including those with 1 or 3 mismatches.
Gautschi et al (J. Natl. Cancer Inst. 93:463-471, March 2001) demonstrated the ability of an oligonucleotide having 100% complementarity to the bcl-2 mRNA and having 3 mismatches to the bcl-xL mRNA to reduce the expression of both bcl-2 and bcl-xL in vitro and in vivo. Furthermore, this oligonucleotide demonstrated potent anti-tumor activity in vivo.
Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988) tested a series of tandem 14 nucleobase antisense oligonucleotides, and a 28 and 42 nucleobase antisense oligonucleotides comprised of the sequence of two or three of the tandem antisense oligonucleotides, respectively, for their ability to arrest translation of human DHFR in a rabbit reticulocyte assay. Each of the three 14 nucleobase antisense oligonucleotides alone was able to inhibit translation, albeit at a more modest level than the 28 or 42 nucleobase antisense oligonucleotides.
Antisense Compound Motifs
In certain embodiments, antisense compounds targeted to a Factor 11 nucleic acid have chemically modified subunits arranged in patterns, or motifs, to confer to the antisense compounds properties such as enhanced the inhibitory activity, increased binding affinity for a target nucleic acid, or resistance to degradation by in vivo nucleases.
Chimeric antisense compounds typically contain at least one region modified so as to confer increased resistance to nuclease degradation, increased cellular uptake, increased binding affinity for the target nucleic acid, and/or increased inhibitory activity. A second region of a chimeric antisense compound may optionally serve as a substrate for the cellular endonuclease RNase H, which cleaves the RNA strand of an RNA:DNA duplex.
Antisense compounds having a gapmer motif are considered chimeric antisense compounds. In a gapmer an internal region having a plurality of nucleotides that supports RNaseH cleavage is positioned between external regions having a plurality of nucleotides that are chemically distinct from the nucleosides of the internal region. In the case of an antisense oligonucleotide having a gapmer motif, the gap segment generally serves as the substrate for endonuclease cleavage, while the wing segments comprise modified nucleosides. In certain embodiments, the regions of a gapmer are differentiated by the types of sugar moieties comprising each distinct region. The types of sugar moieties that are used to differentiate the regions of a gapmer may in some embodiments include β3-D-ribonucleosides, β-D-deoxyribonucleosides, 2′-modified nucleosides (such 2′-modified nucleosides may include 2′-MOE, and 2′-O—CH3, among others), and bicyclic sugar modified nucleosides (such bicyclic sugar modified nucleosides may include those having a 4′-(CH2)n—O-2′ bridge, where n=1 or n=2). Preferably, each distinct region comprises uniform sugar moieties. The wing-gap-wing motif is frequently described as “X-Y-Z”, where “X” represents the length of the 5′ wing region, “Y” represents the length of the gap region, and “Z” represents the length of the 3′ wing region. As used herein, a gapmer described as “X-Y-Z” has a configuration such that the gap segment is positioned immediately adjacent each of the 5′ wing segment and the 3′ wing segment. Thus, no intervening nucleotides exist between the 5′ wing segment and gap segment, or the gap segment and the 3′ wing segment. Any of the antisense compounds described herein can have a gapmer motif. In some embodiments, X and Z are the same, in other embodiments they are different. In a preferred embodiment, Y is between 8 and 15 nucleotides. X, Y or Z can be any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more nucleotides. Thus, gapmers of the present invention include, but are not limited to, for example 5-10-5, 4-8-4, 4-12-3, 4-12-4, 3-14-3, 2-13-5, 2-16-2, 1-18-1, 3-10-3, 2-10-2, 1-10-1, 2-8-2, 5-8-5, or 6-8-6.
In certain embodiments, the antisense compound has a “wingmer” motif, having a wing-gap or gap-wing configuration, i.e. an X-Y or Y-Z configuration as described above for the gapmer configuration. Thus, wingmer configurations of the present invention include, but are not limited to, for example 5-10, 8-4, 4-12, 12-4, 3-14, 16-2, 18-1, 10-3, 2-10, 1-10, 8-2, 2-13, 5-13, 5-8, or 6-8.
In certain embodiments, antisense compounds targeted to a Factor 11 nucleic acid possess a 5-10-5 gapmer motif.
In certain embodiments, antisense compounds targeted to a Factor 11 nucleic acid possess a 3-14-3 gapmer motif.
In certain embodiments, antisense compounds targeted to a Factor 11 nucleic acid possess a 2-13-5 gapmer motif.
In certain embodiments, antisense compounds targeted to a Factor 11 nucleic acid possess a 5-8-5 gapmer motif.
In certain embodiments, antisense compounds targeted to a Factor 11 nucleic acid possess a 6-8-6 gapmer motif.
In certain embodiments, an antisense compound targeted to a Factor 11 nucleic acid has a gap-widened motif.
In certain embodiments, a gap-widened antisense oligonucleotide targeted to a Factor 11 nucleic acid has a gap segment of fourteen 2′-deoxyribonucleotides positioned immediately adjacent to and between wing segments of three chemically modified nucleosides. In certain embodiments, the chemical modification comprises a 2′-sugar modification. In another embodiment, the chemical modification comprises a 2′-MOE sugar modification.
In certain embodiments, a gap-widened antisense oligonucleotide targeted to a Factor 11 nucleic acid has a gap segment of thirteen 2′-deoxyribonucleotides positioned immediately adjacent to and between a 5′ wing segment of two chemically modified nucleosides and a 3′ wing segment of five chemically modified nucleosides. In certain embodiments, the chemical modification comprises a 2′-sugar modification. In another embodiment, the chemical modification comprises a 2′-MOE sugar modification.
Target Nucleic Acids, Target Regions and Nucleotide Sequences
Nucleotide sequences that encode Factor 11 include, without limitation, the following: GENBANK Accession No. NM—000128.3, first deposited with GENBANK on Mar. 24, 1999 incorporated herein as SEQ ID NO: 1; NT—022792.17, truncated from 19598000 to 19624000, first deposited with GENBANK on Nov. 29, 2000, and incorporated herein as SEQ ID NO: 2; GENBANK Accession No. NM—028066.1, first deposited with GENBANK on Jun. 2, 2002, incorporated herein as SEQ ID NO: 6; and exons 1-15 GENBANK Accession No. NW—001118167.1, first deposited with GENBANK on Mar. 28, 2006, incorporated herein as SEQ ID NO: 274.
It is understood that the sequence set forth in each SEQ ID NO in the Examples contained herein is independent of any modification to a sugar moiety, an internucleoside linkage, or a nucleobase. As such, antisense compounds defined by a SEQ ID NO may comprise, independently, one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase. Antisense compounds described by Isis Number (Isis No) indicate a combination of nucleobase sequence and motif.
In certain embodiments, a target region is a structurally defined region of the target nucleic acid. For example, a target region may encompass a 3′ UTR, a 5′ UTR, an exon, an intron, an exon/intron junction, a coding region, a translation initiation region, translation termination region, or other defined nucleic acid region. The structurally defined regions for Factor 11 can be obtained by accession number from sequence databases such as NCBI and such information is incorporated herein by reference. In certain embodiments, a target region may encompass the sequence from a 5′ target site of one target segment within the target region to a 3′ target site of another target segment within the same target region.
Targeting includes determination of at least one target segment to which an antisense compound hybridizes, such that a desired effect occurs. In certain embodiments, the desired effect is a reduction in mRNA target nucleic acid levels. In certain embodiments, the desired effect is reduction of levels of protein encoded by the target nucleic acid or a phenotypic change associated with the target nucleic acid.
A target region may contain one or more target segments. Multiple target segments within a target region may be overlapping. Alternatively, they may be non-overlapping. In certain embodiments, target segments within a target region are separated by no more than about 300 nucleotides. In certain embodiments, target segments within a target region are separated by a number of nucleotides that is, is about, is no more than, is no more than about, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides on the target nucleic acid, or is a range defined by any two of the preceeding values. In certain embodiments, target segments within a target region are separated by no more than, or no more than about, 5 nucleotides on the target nucleic acid. In certain embodiments, target segments are contiguous. Contemplated are target regions defined by a range having a starting nucleic acid that is any of the 5′ target sites or 3′ target sites listed herein.
Suitable target segments may be found within a 5′ UTR, a coding region, a 3′ UTR, an intron, an exon, or an exon/intron junction. Target segments containing a start codon or a stop codon are also suitable target segments. A suitable target segment may specifically exclude a certain structurally defined region such as the start codon or stop codon.
The determination of suitable target segments may include a comparison of the sequence of a target nucleic acid to other sequences throughout the genome. For example, the BLAST algorithm may be used to identify regions of similarity amongst different nucleic acids. This comparison can prevent the selection of antisense compound sequences that may hybridize in a non-specific manner to sequences other than a selected target nucleic acid (i.e., non-target or off-target sequences).
There may be variation in activity (e.g., as defined by percent reduction of target nucleic acid levels) of the antisense compounds within an active target region. In certain embodiments, reductions in Factor 11 mRNA levels are indicative of inhibition of Factor 11 expression. Reductions in levels of a Factor 11 protein are also indicative of inhibition of target mRNA expression. Further, phenotypic changes are indicative of inhibition of Factor 11 expression. For example, a prolonged aPTT time can be indicative of inhibition of Factor 11 expression. In another example, prolonged aPTT time in conjunction with a normal PT time can be indicative of inhibition of Factor 11 expression. In another example, a decreased quantity of Platelet Factor 4 (PF-4) can be indicative of inhibition of Factor 11 expression. In another example, reduced formation of thrombus or increased time for thrombus formation can be indicative of inhibition of Factor 11 expression.
Hybridization
In some embodiments, hybridization occurs between an antisense compound disclosed herein and a Factor 11 nucleic acid. The most common mechanism of hybridization involves hydrogen bonding (e.g., Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementary nucleobases of the nucleic acid molecules.
Hybridization can occur under varying conditions. Stringent conditions are sequence-dependent and are determined by the nature and composition of the nucleic acid molecules to be hybridized.
Methods of determining whether a sequence is specifically hybridizable to a target nucleic acid are well known in the art. In certain embodiments, the antisense compounds provided herein are specifically hybridizable with a Factor 11 nucleic acid.
Complementarily
An antisense compound and a target nucleic acid are complementary to each other when a sufficient number of nucleobases of the antisense compound can hydrogen bond with the corresponding nucleobases of the target nucleic acid, such that a desired effect will occur (e.g., antisense inhibition of a target nucleic acid, such as a Factor 11 nucleic acid).
Non-complementary nucleobases between an antisense compound and a Factor 11 nucleic acid may be tolerated provided that the antisense compound remains able to specifically hybridize to a target nucleic acid. Moreover, an antisense compound may hybridize over one or more segments of a Factor 11 nucleic acid such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure, mismatch or hairpin structure).
In certain embodiments, the antisense compounds provided herein, or a specified portion thereof, are, or are at least, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to a Factor 11 nucleic acid, a target region, target segment, or specified portion thereof. Percent complementarity of an antisense compound with a target nucleic acid can be determined using routine methods.
For example, an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an antisense compound which is 18 nucleobases in length having 4 (four) noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403 410; Zhang and Madden, Genome Res., 1997, 7, 649 656). Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482 489).
In certain embodiments, the antisense compounds provided herein, or specified portions thereof, are fully complementary (i.e. 100% complementary) to a target nucleic acid, or specified portion thereof. For example, antisense compound may be fully complementary to a Factor 11 nucleic acid, or a target region, or a target segment or target sequence thereof. As used herein, “fully complementary” means each nucleobase of an antisense compound is capable of precise base pairing with the corresponding nucleobases of a target nucleic acid. For example, a 20 nucleobase antisense compound is fully complementary to a target sequence that is 400 nucleobases long, so long as there is a corresponding 20 nucleobase portion of the target nucleic acid that is fully complementary to the antisense compound. Fully complementary can also be used in reference to a specified portion of the first and/or the second nucleic acid. For example, a 20 nucleobase portion of a 30 nucleobase antisense compound can be “fully complementary” to a target sequence that is 400 nucleobases long. The 20 nucleobase portion of the 30 nucleobase oligonucleotide is fully complementary to the target sequence if the target sequence has a corresponding 20 nucleobase portion wherein each nucleobase is complementary to the 20 nucleobase portion of the antisense compound. At the same time, the entire 30 nucleobase antisense compound may or may not be fully complementary to the target sequence, depending on whether the remaining 10 nucleobases of the antisense compound are also complementary to the target sequence.
The location of a non-complementary nucleobase may be at the 5′ end or 3′ end of the antisense compound. Alternatively, the non-complementary nucleobase or nucleobases may be at an internal position of the antisense compound. When two or more non-complementary nucleobases are present, they may be contiguous (i.e. linked) or non-contiguous. In one embodiment, a non-complementary nucleobase is located in the wing segment of a gapmer antisense oligonucleotide.
In certain embodiments, antisense compounds that are, or are up to 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length comprise no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as a Factor 11 nucleic acid, or specified portion thereof.
In certain embodiments, antisense compounds that are, or are up to 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length comprise no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as a Factor 11 nucleic acid, or specified portion thereof.
The antisense compounds provided herein also include those which are complementary to a portion of a target nucleic acid. As used herein, “portion” refers to a defined number of contiguous (i.e. linked) nucleobases within a region or segment of a target nucleic acid. A “portion” can also refer to a defined number of contiguous nucleobases of an antisense compound. In certain embodiments, the antisense compounds, are complementary to at least an 8 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 12 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 15 nucleobase portion of a target segment. Also contemplated are antisense compounds that are complementary to at least a 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleobase portion of a target segment, or a range defined by any two of these values.
Identity
The antisense compounds provided herein may also have a defined percent identity to a particular nucleotide sequence, SEQ ID NO, or compound represented by a specific Isis number, or portion thereof. As used herein, an antisense compound is identical to the sequence disclosed herein if it has the same nucleobase pairing ability. For example, a RNA which contains uracil in place of thymidine in a disclosed DNA sequence would be considered identical to the DNA sequence since both uracil and thymidine pair with adenine. Shortened and lengthened versions of the antisense compounds described herein as well as compounds having non-identical bases relative to the antisense compounds provided herein also are contemplated. The non-identical bases may be adjacent to each other or dispersed throughout the antisense compound. Percent identity of an antisense compound is calculated according to the number of bases that have identical base pairing relative to the sequence to which it is being compared.
In certain embodiments, the antisense compounds, or portions thereof, are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to one or more of the antisense compounds or SEQ ID NOs, or a portion thereof, disclosed herein.
In certain embodiments, a portion of the antisense compound is compared to an equal length portion of the target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equal length portion of the target nucleic acid.
In certain embodiments, a portion of the antisense oligonucleotide is compared to an equal length portion of the target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equal length portion of the target nucleic acid.
Modifications
A nucleoside is a base-sugar combination. The nucleobase (also known as base) portion of the nucleoside is normally a heterocyclic base moiety. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2′, 3′ or 5′ hydroxyl moiety of the sugar. Oligonucleotides are formed through the covalent linkage of adjacent nucleosides to one another, to form a linear polymeric oligonucleotide. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside linkages of the oligonucleotide.
Modifications to antisense compounds encompass substitutions or changes to internucleoside linkages, sugar moieties, or nucleobases. Modified antisense compounds are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target, increased stability in the presence of nucleases, or increased inhibitory activity.
Chemically modified nucleosides may also be employed to increase the binding affinity of a shortened or truncated antisense oligonucleotide for its target nucleic acid. Consequently, comparable results can often be obtained with shorter antisense compounds that have such chemically modified nucleosides.
Modified Internucleoside Linkages
The naturally occurring internucleoside linkage of RNA and DNA is a 3′ to 5′ phosphodiester linkage. Antisense compounds having one or more modified, i.e. non-naturally occurring, internucleoside linkages are often selected over antisense compounds having naturally occurring internucleoside linkages because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases.
Oligonucleotides having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom as well as internucleoside linkages that do not have a phosphorus atom. Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing linkages are well known.
In certain embodiments, antisense compounds targeted to a Factor 11 nucleic acid comprise one or more modified internucleoside linkages. In certain embodiments, the modified internucleoside linkages are phosphorothioate linkages. In certain embodiments, each internucleoside linkage of an antisense compound is a phosphorothioate internucleoside linkage.
Modified Sugar Moieties
Antisense compounds of the invention can optionally contain one or more nucleosides wherein the sugar group has been modified. Such sugar modified nucleosides may impart enhanced nuclease stability, increased binding affinity or some other beneficial biological property to the antisense compounds. In certain embodiments, nucleosides comprise a chemically modified ribofuranose ring moieties. Examples of chemically modified ribofuranose rings include without limitation, addition of substitutent groups (including 5′ and 2′ substituent groups, bridging of non-geminal ring atoms to form bicyclic nucleic acids (BNA), replacement of the ribosyl ring oxygen atom with S, N(R), or C(R1)(R)2 (R═H, C1-C12 alkyl or a protecting group) and combinations thereof. Examples of chemically modified sugars include 2′-F-5′-methyl substituted nucleoside (see PCT International Application WO 2008/101157 Published on Aug. 21, 2008 for other disclosed 5′,2′-bis substituted nucleosides) or replacement of the ribosyl ring oxygen atom with S with further substitution at the 2′-position (see published U.S. Patent Application US2005-0130923, published on Jun. 16, 2005) or alternatively 5′-substitution of a BNA (see PCT International Application WO 2007/134181 Published on Nov. 22, 2007 wherein LNA is substituted with for example a 5′-methyl or a 5′-vinyl group).
Examples of nucleosides having modified sugar moieties include without limitation nucleosides comprising 5′-vinyl, 5′-methyl (R or S), 4′-S, 2′-F, 2′-OCH3 and 2′-O(CH2)2PCH3 substituent groups. The substituent at the 2′ position can also be selected from allyl, amino, azido, thio, O-allyl, O—C1-C10 alkyl, OCF3, O(CH2)2SCH3, O(CH2)2-O—N(Rm)(Rn), and O—CH2-C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted C1-C10 alkyl.
Examples of bicyclic nucleic acids (BNAs) include without limitation nucleosides comprising a bridge between the 4′ and the 2′ ribosyl ring atoms. In certain embodiments, antisense compounds provided herein include one or more BNA nucleosides wherein the bridge comprises one of the formulas: 4′-(CH2)-O-2′ (LNA); 4′-(CH2)-S-2; 4′-(CH2)-O-2′ (LNA); 4′-(CH2)2-O-2′ (ENA); 4′-C(CH3)2-O-2′ (see PCT/US2008/068922); 4′-CH(CH3)-O-2′ and 4′-CH(CH2OCH3) -O-2′ (see U.S. Pat. No. 7,399,845, issued on Jul. 15, 2008); 4′-CH2-N(OCH3)-2′ (see PCT/US2008/064591); 4′-CH2-O—N(CH3)-2′ (see published U.S. Patent Application US2004-0171570, published Sep. 2, 2004); 4′-CH2-N(R)—O-2′ (see U.S. Pat. No. 7,427,672, issued on Sep. 23, 2008); 4′-CH2-C(CH3)-2′ and 4′-CH2-C(═CH2)-2′ (see PCT/US2008/066154); and wherein R is, independently, H, C1-C12 alkyl, or a protecting group. Each of the foregoing BNAs include various stereochemical sugar configurations including for example α-L-ribofuranose and β-D-ribofuranose (see PCT international application PCT/DK98/00393, published on Mar. 25, 1999 as WO 99/14226).
In certain embodiments, nucleosides are modified by replacement of the ribosyl ring with a sugar surrogate. Such modification includes without limitation, replacement of the ribosyl ring with a surrogate ring system (sometimes referred to as DNA analogs) such as a morpholino ring, a cyclohexenyl ring, a cyclohexyl ring or a tetrahydropyranyl ring such as one having one of the formula:




Many other bicyclo and tricyclo sugar surrogate ring systems are also know in the art that can be used to modify nucleosides for incorporation into antisense compounds (see for example review article: Leumann, J. C, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854). Such ring systems can undergo various additional substitutions to enhance activity.
Methods for the preparations of modified sugars are well known to those skilled in the art.
In nucleotides having modified sugar moieties, the nucleobase moieties (natural, modified or a combination thereof) are maintained for hybridization with an appropriate nucleic acid target.
In certain embodiments, antisense compounds targeted to a Factor 11 nucleic acid comprise one or more nucleotides having modified sugar moieties. In certain embodiments, the modified sugar moiety is 2′-MOE. In certain embodiments, the 2′-MOE modified nucleotides are arranged in a gapmer motif.
Modified Nucleobases
Nucleobase (or base) modifications or substitutions are structurally distinguishable from, yet functionally interchangeable with, naturally occurring or synthetic unmodified nucleobases. Both natural and modified nucleobases are capable of participating in hydrogen bonding. Such nucleobase modifications may impart nuclease stability, binding affinity or some other beneficial biological property to antisense compounds. Modified nucleobases include synthetic and natural nucleobases such as, for example, 5-methylcytosine (5-me-C). Certain nucleobase substitutions, including 5-methylcytosine substitutions, are particularly useful for increasing the binding affinity of an antisense compound for a target nucleic acid. For example, 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278).
Additional modified nucleobases include 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡C—CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.
Heterocyclic base moieties may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Nucleobases that are particularly useful for increasing the binding affinity of antisense compounds include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2 aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.
In certain embodiments, antisense compounds targeted to a Factor 11 nucleic acid comprise one or more modified nucleobases. In certain embodiments, gap-widened antisense oligonucleotides targeted to a Factor 11 nucleic acid comprise one or more modified nucleobases. In certain embodiments, the modified nucleobase is 5-methylcytosine. In certain embodiments, each cytosine is a 5-methylcytosine.
Compositions and Methods for Formulating Pharmaceutical Compositions
Antisense oligonucleotides may be admixed with pharmaceutically acceptable active or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions are dependent upon a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.
An antisense compound targeted to a Factor 11 nucleic acid can be utilized in pharmaceutical compositions by combining the antisense compound with a suitable pharmaceutically acceptable diluent or carrier. A pharmaceutically acceptable diluent includes phosphate-buffered saline (PBS). PBS is a diluent suitable for use in compositions to be delivered parenterally. Accordingly, in one embodiment, employed in the methods described herein is a pharmaceutical composition comprising an antisense compound targeted to a Factor 11 nucleic acid and a pharmaceutically acceptable diluent. In certain embodiments, the pharmaceutically acceptable diluent is PBS. In certain embodiments, the antisense compound is an antisense oligonucleotide.
Pharmaceutical compositions comprising antisense compounds encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other oligonucleotide which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.
A prodrug can include the incorporation of additional nucleosides at one or both ends of an antisense compound which are cleaved by endogenous nucleases within the body, to form the active antisense compound.
Conjugated Antisense Compounds
Antisense compounds may be covalently linked to one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the resulting antisense oligonucleotides. Typical conjugate groups include cholesterol moieties and lipid moieties. Additional conjugate groups include carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.
Antisense compounds can also be modified to have one or more stabilizing groups that are generally attached to one or both termini of antisense compounds to enhance properties such as, for example, nuclease stability. Included in stabilizing groups are cap structures. These terminal modifications protect the antisense compound having terminal nucleic acid from exonuclease degradation, and can help in delivery and/or localization within a cell. The cap can be present at the 5′-terminus (5′-cap), or at the 3′-terminus (3′-cap), or can be present on both termini. Cap structures are well known in the art and include, for example, inverted deoxy abasic caps. Further 3′ and 5′-stabilizing groups that can be used to cap one or both ends of an antisense compound to impart nuclease stability include those disclosed in WO 03/004602 published on Jan. 16, 2003.
Cell Culture and Antisense Compounds Treatment
The effects of antisense compounds on the level, activity or expression of Factor 11 nucleic acids can be tested in vitro in a variety of cell types. Cell types used for such analyses are available from commerical vendors (e.g. American Type Culture Collection, Manassus, Va.; Zen-Bio, Inc., Research Triangle Park, N.C.; Clonetics Corporation, Walkersville, Md.) and are cultured according to the vendor's instructions using commercially available reagents (e.g. Invitrogen Life Technologies, Carlsbad, Calif.). Illustrative cell types include, but are not limited to, HepG2 cells, Hep3B cells, and primary hepatocytes.
In Vitro Testing of Antisense Oligonucleotides
Described herein are methods for treatment of cells with antisense oligonucleotides, which can be modified appropriately for treatment with other antisense compounds.
In general, cells are treated with antisense oligonucleotides when the cells reach approximately 60-80% confluency in culture.
One reagent commonly used to introduce antisense oligonucleotides into cultured cells includes the cationic lipid transfection reagent LIPOFECTIN (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotides are mixed with LIPOFECTIN in OPTI-MEM 1 (Invitrogen, Carlsbad, Calif.) to achieve the desired final concentration of antisense oligonucleotide and a LIPOFECTIN concentration that typically ranges 2 to 12 ug/mL per 100 nM antisense oligonucleotide.
Another reagent used to introduce antisense oligonucleotides into cultured cells includes LIPOFECTAMINE (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotide is mixed with LIPOFECTAMINE in OPTI-MEM 1 reduced serum medium (Invitrogen, Carlsbad, Calif.) to achieve the desired concentration of antisense oligonucleotide and a LIPOFECTAMINE concentration that typically ranges 2 to 12 ug/mL per 100 nM antisense oligonucleotide.
Another technique used to introduce antisense oligonucleotides into cultured cells includes electroporation.
Cells are treated with antisense oligonucleotides by routine methods. Cells are typically harvested 16-24 hours after antisense oligonucleotide treatment, at which time RNA or protein levels of target nucleic acids are measured by methods known in the art and described herein. In general, when treatments are performed in multiple replicates, the data are presented as the average of the replicate treatments.
The concentration of antisense oligonucleotide used varies from cell line to cell line. Methods to determine the optimal antisense oligonucleotide concentration for a particular cell line are well known in the art. Antisense oligonucleotides are typically used at concentrations ranging from 1 nM to 300 nM when transfected with LIPOFECTAMINE. Antisense oligonucleotides are used at higher concentrations ranging from 625 to 20,000 nM when transfected using electroporation.
RNA Isolation
RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are well known in the art. RNA is prepared using methods well known in the art, for example, using the TRIZOL Reagent (Invitrogen, Carlsbad, Calif.) according to the manufacturer's recommended protocols.
Analysis of Inhibition of Target Levels or Expression
Inhibition of levels or expression of a Factor 11 nucleic acid can be assayed in a variety of ways known in the art. For example, target nucleic acid levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or quantitaive real-time PCR. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are well known in the art. Northern blot analysis is also routine in the art. Quantitative real-time PCR can be conveniently accomplished using the commercially available ABI PRISM 7600, 7700, or 7900 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions.
Quantitative Real-Time PCR Analysis of Target RNA Levels
Quantitation of target RNA levels may be accomplished by quantitative real-time PCR using the ABI PRISM 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. Methods of quantitative real-time PCR are well known in the art.
Prior to real-time PCR, the isolated RNA is subjected to a reverse transcriptase (RT) reaction, which produces complementary DNA (cDNA) that is then used as the substrate for the real-time PCR amplification. The RT and real-time PCR reactions are performed sequentially in the same sample well. RT and real-time PCR reagents are obtained from Invitrogen (Carlsbad, Calif.). RT real-time-PCR reactions are carried out by methods well known to those skilled in the art.
Gene (or RNA) target quantities obtained by real time PCR are normalized using either the expression level of a gene whose expression is constant, such as cyclophilin A, or by quantifying total RNA using RIBOGREEN (Invitrogen, Inc. Carlsbad, Calif.). Cyclophilin A expression is quantified by real time PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RIBOGREEN RNA quantification reagent (Invetrogen, Inc. Eugene, Oreg.). Methods of RNA quantification by RIBOGREEN are taught in Jones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374). A CYTOFLUOR 4000 instrument (PE Applied Biosystems) is used to measure RIBOGREEN fluorescence.
Probes and primers are designed to hybridize to a Factor 11 nucleic acid. Methods for designing real-time PCR probes and primers are well known in the art, and may include the use of software such as PRIMER EXPRESS Software (Applied Biosystems, Foster City, Calif.).
Analysis of Protein Levels
Antisense inhibition of Factor 11 nucleic acids can be assessed by measuring Factor 11 protein levels. Protein levels of Factor 11 can be evaluated or quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA), quantitative protein assays, protein activity assays (for example, caspase activity assays), immunohistochemistry, immunocytochemistry or fluorescence-activated cell sorting (FACS). Antibodies directed to a target can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional monoclonal or polyclonal antibody generation methods well known in the art. Antibodies useful for the detection of mouse, rat, monkey, and human Factor 11 are commercially available.
In Vivo Testing of Antisense Compounds
Antisense compounds, for example, antisense oligonucleotides, are tested in animals to assess their ability to inhibit expression of Factor 11 and produce phenotypic changes, such as, prolonged aPTT, prolonged aPTT time in conjunction with a normal PT, decreased quantity of Platelet Factor 4 (PF-4), and reduced formation of thrombus or increased time for thrombus formation. Testing may be performed in normal animals, or in experimental disease models. For administration to animals, antisense oligonucleotides are formulated in a pharmaceutically acceptable diluent, such as phosphate-buffered saline. Administration includes parenteral routes of administration, such as intraperitoneal, intravenous, and subcutaneous. Calculation of antisense oligonucleotide dosage and dosing frequency is within the abilities of those skilled in the art, and depends upon factors such as route of administration and animal body weight. Following a period of treatment with antisense oligonucleotides, RNA is isolated from liver tissue and changes in Factor 11 nucleic acid expression are measured. Changes in Factor 11 protein levels are also measured using a thrombin generation assay. In addition, effects on clot times, e.g. PT and aPTT, are determined using plasma from treated animals.
Tolerability
In certain embodiments, the compounds provided herein display minimal side effects. Side effects include responses to the administration of the antisense compound that are typically unrelated to the targeting of factor 11, such as an inflammatory response in the animal. In certain embodiments compounds are well tolerated by the animal. Increased tolerability can depend on a number of factors, including, but not limited to, the nucleotide sequence of the antisense compound, chemical modifications to the nucleotides, the particular motif of unmodified and modified nucleosides in the antisense compound, or combinations thereof. Tolerability may be determined by a number of factors. Such factors include body weight, organ weight, liver function, kidney function, platelet count, white blood cell count.
In certain embodiments, the compounds provided herein demonstrate minimal effect on organ weight. In certain embodiments, the compounds demonstrate less than a 7-fold, 6-fold, 5-fold, 4-fold, 3-fold, 2-fold or no significant increase in spleen and/or liver weight.
In certain embodiments, the compounds provided herein demonstrate minimal effect on liver function. Factors for the evaluation of liver function include ALT levels, AST levels, plasma bilirubin levels and plasma albumin levels. In certain embodiments the compounds provided herein demonstrate less than a 7-fold, less than a 6-fold, less than a 5-fold, less than a 4-fold, less than a 3-fold or less than a 2-fold or no significant increase in ALT or AST. In certain embodiments the compounds provided herein demonstrate less than a 3-fold, less than a 2-fold or no significant increase in plasma bilirubin levels.
In certain embodiments, the compounds provided herein demonstrate minimal effect on kidney function. In certain embodiments, the compounds provided herein demonstrate less than a 3-fold, less than a 2-fold, or no significant increase in plasma concentrations of blood urea nitrogen (BUN). In certain embodiments, the compounds provided herein demonstrate less than a 6-fold, 5-fold, 4-fold, 3-fold, 2-fold, or no significant increase in the ratio of urine protein to creatinine.
In certain embodiments, the compounds provided herein demonstrate minimal effect on hematological factors. In certain embodiments, the compounds provided herein demonstrate less than a 60%, 50%, 40%, 30%, 20%, 10% or 5% decrease in platelet count. In certain embodiments, the compounds provided herein demonstrate less than a 4-fold, less than a 3-fold, less than a 2-fold or no significant increase in monocyte count.
In certain embodiments compounds further display favorable pharmacokinetics. In certain embodiments, antisense compounds exhibit relatively high half-lives in relevant biological fluids or tissues.
In certain embodiments, compounds or compositions further display favorable viscosity. In certain embodiments, the viscosity of the compound or composition is no more than 40 cP at a concentration of 165-185 mg/mL.
In other embodiments, the compounds display combinations of the characteristics above and reduce factor 11 mRNA expression in an animal model with high efficiency.
Certain Indications
In certain embodiments, the invention provides methods of treating an individual comprising administering one or more pharmaceutical compositions of the present invention. In certain embodiments, the individual has a thromboembolic complication. In certain embodiments, the individual is at risk for a blood clotting disorder, including, but not limited to, infarct, thrombosis, embolism, thromboembolism such as deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke. This includes individuals with an acquired problem, disease, or disorder that leads to a risk of thrombosis, for example, surgery, cancer, immobility, sepsis, atherosclerosis atrial fibrillation, as well as genetic predisposition, for example, antiphospholipid syndrome and the autosomal dominant condition, Factor V Leiden. In certain embodiments, the individual has been identified as in need of anticoagulation therapy. Examples of such individuals include, but are not limited to, those undergoing major orthopedic surgery (e.g., hip/knee replacement or hip fracture surgery) and patients in need of chronic treatment, such as those suffering from arterial fibrillation to prevent stroke. In certain embodiments the invention provides methods for prophylactically reducing Factor 11 expression in an individual. Certain embodiments include treating an individual in need thereof by administering to an individual a therapeutically effective amount of an antisense compound targeted to a Factor 11 nucleic acid.
In one embodiment, administration of a therapeutically effective amount of an antisense compound targeted to a Factor 11 nucleic acid is accompanied by monitoring of Factor 11 levels in the serum of an individual, to determine an individual's response to administration of the antisense compound. An individual's response to administration of the antisense compound is used by a physician to determine the amount and duration of therapeutic intervention.
In certain embodiments, administration of an antisense compound targeted to a Factor 11 nucleic acid results in reduction of Factor 11 expression by at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of these values. In certain embodiments, administration of an antisense compound targeted to a Factor 11 nucleic acid results in a change in a measure of blood clotting as measured by a standard test, for example, but not limited to, activated partial thromboplastin time (aPTT) test, prothrombin time (PT) test, thrombin time (TCT), bleeding time, or D-dimer. In certain embodiments, administration of a Factor 11 antisense compound increases the measure by at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of these values. In some embodiments, administration of a Factor 11 antisense compound decreases the measure by at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of these values.
In certain embodiments, pharmaceutical compositions comprising an antisense compound targeted to Factor 11 are used for the preparation of a medicament for treating a patient suffering or susceptible to a thromboembolic complication.
Certain Combination Therapies
In certain embodiments, one or more pharmaceutical compositions of the present invention are co-administered with one or more other pharmaceutical agents. In certain embodiments, such one or more other pharmaceutical agents are designed to treat the same disease, disorder, or condition as the one or more pharmaceutical compositions of the present invention. In certain embodiments, such one or more other pharmaceutical agents are designed to treat a different disease, disorder, or condition as the one or more pharmaceutical compositions of the present invention. In certain embodiments, such one or more other pharmaceutical agents are designed to treat an undesired side effect of one or more pharmaceutical compositions of the present invention. In certain embodiments, one or more pharmaceutical compositions of the present invention are co-administered with another pharmaceutical agent to treat an undesired effect of that other pharmaceutical agent. In certain embodiments, one or more pharmaceutical compositions of the present invention are co-administered with another pharmaceutical agent to produce a combinational effect. In certain embodiments, one or more pharmaceutical compositions of the present invention are co-administered with another pharmaceutical agent to produce a synergistic effect.
In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are administered at the same time. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are administered at different times. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are prepared together in a single formulation. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are prepared separately.
In certain embodiments, pharmaceutical agents that may be co-administered with a pharmaceutical composition of the present invention include anticoagulant or antiplatelet agents. In certain embodiments, pharmaceutical agents that may be co-administered with a pharmaceutical composition of the present invention include NSAID/Cyclooxygenase inhibitors, such as, aspirin. In certain embodiments, pharmaceutical agents that may be co-administered with a pharmaceutical composition of the present invention include adenosine diphosphate (ADP) receptor inhibitors, such as, clopidogrel (PLAVIX) and ticlopidine (TICLID). In certain embodiments, pharmaceutical agents that may be co-administered with a pharmaceutical composition of the present invention include phosphodiesterase inhibitors, such as, cilostazol (PLETAL). In certain embodiments, pharmaceutical agents that may be co-administered with a pharmaceutical composition of the present invention include, glycoprotein IIB/IIIA inhibitors, such as, abciximab (REOPRO), eptifibatide (INTEGRILIN), tirofiban (AGGRASTAT), and defibrotide. In certain embodiments, pharmaceutical agents that may be co-administered with a pharmaceutical composition of the present invention include, adenosine reuptake inhibitors, such as, to dipyridamole (PERSANTINE). In certain embodiments, pharmaceutical agents that may be co-administered with a pharmaceutical composition of the present invention include, but are not limited to warfarin (and related coumarins), heparin, direct thrombin inhibitors (such as lepirudin, bivalirudin), apixaban, LOVENOX, and small molecular compounds that interfere directly with the enzymatic action of particular coagulation factors (e.g. rivaroxaban, which interferes with Factor Xa). In certain embodiments, pharmaceutical agents that may be co-administered with a Factor 11 specific inhibitor of the present invention include, but are not limited to, an additional Factor 11 inhibitor. In certain embodiments, the anticoagulant or antiplatelet agent is administered prior to administration of a pharmaceutical composition of the present invention. In certain embodiments, the anticoagulant or antiplatelet agent is administered following administration of a pharmaceutical composition of the present invention. In certain embodiments the anticoagulant or antiplatelet agent is administered at the same time as a pharmaceutical composition of the present invention. In certain embodiments the dose of a co-administered anticoagulant or antiplatelet agent is the same as the dose that would be administered if the anticoagulant or antiplatelet agent was administered alone. In certain embodiments the dose of a co-administered anticoagulant or antiplatelet agent is lower than the dose that would be administered if the anticoagulant or antiplatelet agent was administered alone. In certain embodiments the dose of a co-administered anticoagulant or antiplatelet agent is greater than the dose that would be administered if the anticoagulant or antiplatelet agent was administered alone.
In certain embodiments, the co-administration of a second compound enhances the anticoagulant effect of a first compound, such that co-administration of the compounds results in an anticoagulant effect that is greater than the effect of administering the first compound alone. In other embodiments, the co-administration results in anticoagulant effects that are additive of the effects of the compounds when administered alone. In certain embodiments, the co-administration results in anticoagulant effects that are supra-additive of the effects of the compounds when administered alone. In certain embodiments, the co-administration of a second compound increases antithrombotic activity without increased bleeding risk. In certain embodiments, the first compound is an antisense compound. In certain embodiments, the second compound is an antisense compound.
In certain embodiments, an antidote is administered anytime after the administration of a Factor 11 specific inhibitor. In certain embodiments, an antidote is administered anytime after the administration of an antisense oligonucleotide targeting Factor 11. In certain embodiments, the antidote is administered minutes, hours, days, weeks, or months after the administration of an antisense compound targeting Factor 11. In certain embodiments, the antidote is a complementary (e.g. the sense strand) to the antisense compound targeting Factor 11. In certain embodiments, the antidote is a Factor 7, Factor 7a, Factor 11, or Factor 11a protein. In certain embodiments, the Factor 7, Factor 7a, Factor 11, or Factor 11a protein is a human Factor 7, human Factor 7a, human Factor 11, or human Factor 11a protein. In certain embodiments, the Factor 7 protein is NOVOSEVEN.
Certain Co-Administered Antiplatelet Therapies
In certain embodiments, Factor 11 inhibitors are combined with antiplatelet therapies. In certain embodiments, administration of a Factor 11 inhibitor in combination with an antiplatelet therapy results in little to no appreciable or detectable increase in risk of bleeding as compared to antiplatelet therapy alone. In certain embodiments, the risk profile or risk indications are unchanged over antiplatelet therapy alone.
The combination of antiplatelet and anticoagulant therapy is used in clinical practice most frequently in patients diagnosed with, for example, thromboembolism, atrial fibrillation, a heart valve disorder, valvular heart disease, stroke, CAD, and in patients having a mechanical valve. The benefit of dual therapy relates to the probable additive effect of suppressing both platelet and coagulation factor activities. The risk of dual therapy is the potential for increased bleeding (Dowd, M. Plenary Sessions/Thrombosis Research 123 (2008)).
Prior combinations of antiplatelet and anticoagulant therapy have been shown to increase the risk of bleeding compared with anticoagulant or antiplatelet therapy alone. Such combinations include, FXa inhibitors (e.g., apixiban and rivaroxaban) with ADP receptor/P2Y12 inhibitors (Thienopyridines such as clopidogrel—also known as PLAVIX) and NSAIDs (e.g., aspirin and naproxen) (Kubitza, D. et al., Br. J. Clin. Pharmacol. 63:4 (2006); Wong, P. C. et al. Journal of Thrombosis and Haemostasis 6 (2008); FDA Advisory Committee Briefing Document for New Drug Application 22-406 (2009)). For example, Wong reports that addition of certain doses of apixaban to aspirin and to aspirin plus clopidogrel produced a significant increase in bleeding time compared with aspirin alone and asprin plus clopidogrel. Kubitza reports that the combination administration of rivaroxaban and naproxen significantly increased bleeding time over naproxen alone.
EXAMPLES
Non-Limiting Disclosure and Incorporation by Reference
While certain compounds, compositions and methods described herein have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds described herein and are not intended to limit the same. Each of the references recited in the present application is incorporated herein by reference in its entirety.
Example 1
Antisense Inhibition of Human Factor 11 in HepG2 Cells
Antisense oligonucleotides targeted to a Factor 11 nucleic acid were tested for their effects on Factor 11 mRNA in vitro. Cultured HepG2 cells at a density of 10,000 cells per well were transfected using lipofectin reagent with 75 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Factor 11 mRNA levels were measured by quantitative real time PCR. Factor 11 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of Factor 11, relative to untreated control cells.
The chimeric antisense oligonucleotides in Tables 1 and 2 were designed as 5-10-5 MOE gapmers. The gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 10 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising 5 nucleotides each. Each nucleotide in the 5′ wing segment and each nucleotide in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytidine residues throughout each gapmer are 5-methylcytidines. “Target start site” indicates the 5′-most nucleotide to which the gapmer is targeted. “Target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted. Each gapmer listed in Table 1 is targeted to SEQ ID NO: 1 (GENBANK Accession No. NM—000128.3) and each gapmer listed in Table 2 is targeted to SEQ ID NO: 2 (GENBANK Accession No. NT—022792.17, truncated from 19598000 to 19624000).







TABLE 1







Inhibition of human Factor 11 mRNA levels by chimeric


antisense oligonucleotides having 5-10-5


MOE wings and deoxy gap targeted to SEQ ID NO: 1













Target
Target





Oligo
Start
Stop


SEQ ID


ID
Site
Site
Sequence
% inhibition
NO















412187
38
57
TTCAAACAAGTGACATACAC
21
15


 


412188
96
115
TGAGAGAATTGCTTGCTTTC
21
16


 


412189
106
125
AAATATACCTTGAGAGAATT
8
17


 


412190
116
135
AGTATGTCAGAAATATACCT
24
18


 


412191
126
145
TTAAAATCTTAGTATGTCAG
14
19


 


412192
146
165
CAGCATATTTGTGAAAGTCG
44
20


 


412193
222
241
TGTGTAGGAAATGGTCACTT
38
21


 


412194
286
305
TGCAATTCTTAATAAGGGTG
80
22


 


412195
321
340
AAATCATCCTGAAAAGACCT
22
23


 


412196
331
350
TGATATAAGAAAATCATCCT
25
24


 


412197
376
395
ACACATTCACCAGAAACTGA
45
25


 


412198
550
569
TTCAGGACACAAGTAAACCA
21
26


 


412199
583
602
TTCACTCTTGGCAGTGTTTC
66
27


 


412200
612
631
AAGAATACCCAGAAATCGCT
59
28


 


412201
622
641
CATTGCTTGAAAGAATACCC
66
29


 


412202
632
651
TTGGTGTGAGCATTGCTTGA
65
30


 


412203
656
675
AATGTCTTTGTTGCAAGCGC
91
31


 


412204
676
695
TTCATGTCTAGGTCCACATA
74
32


 


412205
686
705
GTTTATGCCCTTCATGTCTA
69
33


 


412206
738
757
CCGTGCATCTTTCTTGGCAT
87
34


 


412207
764
783
CGTGAAAAAGTGGCAGTGGA
64
35


 


412208
811
830
AGACAAATGTTACGATGCTC
73
36


 


412209
821
840
GTGCTTCAGTAGACAAATGT
91
37


 


412210
896
915
TGCACAGGATTTCAGTGAAA
73
38


 


412211
906
925
GATTAGAAAGTGCACAGGAT
64
39


 


412212
1018
1037
CCGGGATGATGAGTGCAGAT
88
40


 


412213
1028
1047
AAACAAGCAACCGGGATGAT
71
41


 


412214
1048
1067
TCCTGGGAAAAGAAGGTAAA
58
42


 


412215
1062
1081
ATTCTTTGGGCCATTCCTGG
81
43


 


412216
1077
1096
AAAGATTTCTTTGAGATTCT
43
44


 


412217
1105
1124
AATCCACTCTCAGATGTTTT
47
45


 


412218
1146
1165
AACCAGAAAGAGCTTTGCTC
27
46


 


412219
1188
1207
GGCAGAACACTGGGATGCTG
56
47


 


412220
1204
1223
TGGTAAAATGAAGAATGGCA
58
48


 


412221
1214
1233
ATCAGTGTCATGGTAAAATG
48
49


 


412222
1241
1263
AACAATATCCAGTTCTTCTC
5
50


 


412223
1275
1294
ACAGTTTCTGGCAGGCCTCG
84
51


 


412224
1285
1304
GCATTGGTGCACAGTTTCTG
87
52


 


412225
1295
1314
GCAGCGGACGGCATTGGTGC
86
53


 


412226
1371
1390
TTGAAGAAAGCTTTAAGTAA
17
54


 


412227
1391
1410
AGTATTTTAGTTGGAGATCC
75
55


 


412228
1425
1444
ATGTGTATCCAGAGATGCCT
71
56


 


412229
1456
1475
GTACACTCATTATCCATTTT
64
57


 


412230
1466
1485
GATTTTGGTGGTACACTCAT
52
58


 


412231
1476
1495
TCCTGGGCTTGATTTTGGTG
74
59


 


412232
1513
1532
GGCCACTCACCACGAACAGA
80
60


 


412233
1555
1574
TGTCTCTGAGTGGGTGAGGT
64
61


 


412234
1583
1602
GTTTCCAATGATGGAGCCTC
60
62


 


412235
1593
1612
ATATCCACTGGTTTCCAATG
57
63


 


412236
1618
1637
CCATAGAAACAGTGAGCGGC
72
64


 


412237
1628
1647
TGACTCTACCCCATAGAAAC
48
65


 


412238
1642
1661
CGCAAAATCTTAGGTGACTC
71
66


 


412239
1673
1692
TTCAGATTGATTTAAAATGC
43
67


 


412240
1705
1724
TGAACCCCAAAGAAAGATGT
32
68


 


412241
1715
1734
TATTATTTCTTGAACCCCAA
41
69


 


412242
1765
1784
AACAAGGCAATATCATACCC
49
70


 


412243
1775
1794
TTCCAGTTTCAACAAGGCAA
70
71


 


412244
1822
1841
GAAGGCAGGCATATGGGTCG
53
72


 


412245
1936
1955
GTCACTAAGGGTATCTTGGC
75
73


 


412246
1992
2011
AGATCATCTTATGGGTTATT
68
74


 


412247
2002
2021
TAGCCGGCACAGATCATCTT
75
75


 


412248
2082
2101
CCAGATGCCAGACCTCATTG
53
76


 


412249
2195
2214
CATTCACACTGCTTGAGTTT
55
77


 


412250
2268
2287
TGGCACAGTGAACTCAACAC
63
78


 


412251
2326
2345
CTAGCATTTTCTTACAAACA
58
79


 


412252
2450
2469
TTATGGTAATTCTTGGACTC
39
80


 


412253
2460
2479
AAATATTGCCTTATGGTAAT
20
81


 


412254
2485
2504
TATCTGCCTATATAGTAATC
16
82


 


412255
2510
2529
GCCACTACTTGGTTATTTTC
38
83


 


412256
2564
2583
AACAAATCTATTTATGGTGG
39
84


 


412257
2622
2641
CTGCAAAATGGTGAAGACTG
57
85


 


412258
2632
2651
GTGTAGATTCCTGCAAAATG
44
86


 


412259
2882
2901
TTTTCAGGAAAGTGTATCTT
37
87


 


412260
2892
2911
CACAAATCATTTTTCAGGAA
27
88


 


412261
2925
2944
TCCCAAGATATTTTAAATAA
3
89


 


412262
3168
3187
AATGAGATAAATATTTGCAC
34
90


 


412263
3224
3243
TGAAAGCTATGTGGTGACAA
33
91


 


412264
3259
3278
CACACTTGATGAATTGTATA
27
92


 


413460
101
120
TACCTTGAGAGAATTGCTTG
40
93


 


413461
111
130
GTCAGAAATATACCTTGAGA
39
94


 


413462
121
140
ATCTTAGTATGTCAGAAATA
12
95


 


413463
381
400
GAGTCACACATTCACCAGAA
74
96


 


413464
627
646
GTGAGCATTGCTTGAAAGAA
42
97


 


413465
637
656
CTTATTTGGTGTGAGCATTG
80
98


 


413466
661
680
ACATAAATGTCTTTGTTGCA
79
99


 


413467
666
685
GGTCCACATAAATGTCTTTG
91
100


 


413468
671
690
GTCTAGGTCCACATAAATGT
84
101


 


413469
681
700
TGCCCTTCATGTCTAGGTCC
84
102


 


413470
692
711
GTTATAGTTTATGCCCTTCA
72
103


 


413471
816
835
TCAGTAGACAAATGTTACGA
67
104


 


413472
826
845
TGGGTGTGCTTCAGTAGACA
99
105


 


413473
911
930
AGCCAGATTAGAAAGTGCAC
80
106


 


413474
1023
1042
AGCAACCGGGATGATGAGTG
84
107


 


413475
1053
1072
GCCATTCCTGGGAAAAGAAG
80
108


 


413476
1067
1086
TTGAGATTCTTTGGGCCATT
88
109


 


413477
1151
1170
ACTGAAACCAGAAAGAGCTT
54
110


 


413478
1193
1212
AGAATGGCAGAACACTGGGA
53
111


 


413479
1209
1228
TGTCATGGTAAAATGAAGAA
40
112


 


413480
1219
1238
AAGAAATCAGTGTCATGGTA
71
113


 


413481
1280
1299
GGTGCACAGTTTCTGGCAGG
86
114


 


413482
1290
1309
GGACGGCATTGGTGCACAGT
85
115


 


413483
1300
1319
AACTGGCAGCGGACGGCATT
78
116


 


413484
1430
1449
CCTTAATGTGTATCCAGAGA
74
117


 


413485
1461
1480
TGGTGGTACACTCATTATCC
68
118


 


413486
1471
1490
GGCTTGATTTTGGTGGTACA
83
119


 


413487
1481
1500
AACGATCCTGGGCTTGATTT
57
120


 


413488
1560
1579
ACAGGTGTCTCTGAGTGGGT
49
121


 


413489
1588
1607
CACTGGTTTCCAATGATGGA
68
122


 


413490
1623
1642
CTACCCCATAGAAACAGTGA
57
123


 


413491
1633
1652
TTAGGTGACTCTACCCCATA
73
124


 


413492
1647
1666
AGAGACGCAAAATCTTAGGT
68
125


 


413493
1710
1729
TTTCTTGAACCCCAAAGAAA
65
126


 


413494
1780
1799
GTGGTTTCCAGTTTCAACAA
70
127


 


413495
1921
1940
TTGGCTTTCTGGAGAGTATT
58
128


 


413496
1997
2016
GGCACAGATCATCTTATGGG
72
129


 


413497
2627
2646
GATTCCTGCAAAATGGTGAA
39
130


 


413498
2637
2656
GCAGAGTGTAGATTCCTGCA
60
131


 


413499
2887
2906
ATCATTTTTCAGGAAAGTGT
52
132
















TABLE 2







Inhibition of human Factor 11 mRNA levels by chimeric


antisense oligonucleotides having 5-10-5


MOE wings and deoxy gap targeted to SEQ ID NO: 2













Target
Target





Oligo
Start
Stop


SEQ ID


ID
Site
Site
Sequence
% inhibition
NO















413500
1658
1677
GTGAGACAAATCAAGACTTC
15
133


 


413501
2159
2178
TTAGTTTACTGACACTAAGA
23
134


 


413502
2593
2612
CTGCTTTATGAAAAACCAAC
22
135


 


413503
3325
3344
ATACCTAGTACAATGTAAAT
29
136


 


413504
3548
3567
GGCTTGTGTGTGGTCAATAT
54
137


 


413505
5054
5073
TGGGAAAGCTTTCAATATTC
57
138


 


413506
6474
6493
ATGGAATTGTGCTTATGAGT
57
139


 


413507
7590
7609
TTTCAAGCTCAGGATGGGAA
55
140


 


413508
7905
7924
GTTGGTAAAATGCAACCAAA
64
141


 


413509
8163
8182
TCAGGACACAAGTAAACCTG
66
142


 


413510
9197
9216
TGCAAGCTGGAAATAAAAGC
17
143


 


413511
9621
9640
TGCCAATTTAAAAGTGTAGC
43
144


 


413512
9800
9819
ATATTTCAAAATCCAGTATG
39
145


 


413513
9919
9938
TTCTGAATATACAAATTAAT
27
146


 


413514
9951
9970
TTTACTATGAAAATCTAAAT
5
147


 


413515
11049
11068
GGTATCCTGAGTGAGATCTA
36
148


 


413516
11269
11288
CCAGCTATCAGGAAAATTCC
50
149


 


413517
12165
12184
AAAGCTATTGGAGACTCAGA
51
150


 


413518
12584
12603
ATGGAATCTCTTCATTTCAT
49
151


 


413519
12728
12747
ATGGAGACATTCATTTCCAC
59
152


 


413520
13284
13303
GCTCTGAGAGTTCCAATTCA
52
153


 


413521
14504
14523
CTGGGAAGGTGAATTTTTAG
62
154


 


413522
14771
14790
TCAAGAGTCTTCATGCTACC
42
155


 


413523
15206
15225
TCAGTTTACCTGGGATGCTG
61
156


 


413524
15670
15689
GACATTATACTCACCATTAT
7
157


 


413525
15905
15924
GTATAAATGTGTCAAATTAA
43
158


 


413526
16482
16501
GTAAAGTTTTACCTTAACCT
47
159


 


413527
17298
17317
CCATAATGAAGAAGGAAGGG
52
160


 


413528
17757
17776
TTAAGTTACATTGTAGACCA
48
161


 


413529
18204
18223
TGTGTGGGTCCTGAAATTCT
52
162


 


413530
18981
19000
ATCTTGTAATTACACACCCC
27
163


 


413531
19174
19193
GTACACTCTGCAACAGAAGC
47
164


 


413532
19604
19623
AGGGAATAACATGAAGGCCC
32
165


 


413533
20936
20955
ATCCAGTTCACCATTGGAGA
48
166


 


413534
21441
21460
TTTTCCAGAAGAGACTCTTC
31
167


 


413535
21785
21804
GTCACATTTAAAATTTCCAA
41
168


 


413536
23422
23441
TTAATATACTGCAGAGAACC
37
169


 


413537
25893
25912
AGAAATATCCCCAGACAGAG
16
170









Example 2
Dose-Dependent Antisense Inhibition of Human Factor 11 in HepG2 Cells
Twelve gapmers, exhibiting over 84 percent or greater in vitro inhibition of human Factor 11, were tested at various doses in HepG2 cells. Cells were plated at a density of 10,000 cells per well and transfected using lipofectin reagent with 9.375 nM, 18.75 nM, 37.5 nM, 75 nM, and 150 nM concentrations of antisense oligonucleotide, as specified in Table 3. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Factor 11 mRNA levels were measured by quantitative real-time PCR. Human Factor 11 primer probe set RTS 2966 (forward sequence: CAGCCTGGAGCATCGTAACA, incorporated herein as SEQ ID NO: 3; reverse sequence: TTTATCGAGCTTCGTTATTCTGGTT, incorporated herein as SEQ ID NO: 4; probe sequence: TTGTCTACTGAAGCACACCCAAACAGGGAX, incorporated herein as SEQ ID NO: 5) was used to measure mRNA levels. Factor 11 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of Factor 11, relative to untreated control cells. As illustrated in Table 3, Factor 11 mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells.







TABLE 3







Dose-dependent antisense inhibition


of human Factor 11 in HepG2 cells















9.375
18.75
37.5
75
150
IC50
SEQ ID



nM
nM
nM
nM
nM
(nM)
No.


















412203
29
15
61
77
82
33
31


412206
28
44
68
80
89
22
34


412212
28
45
59
73
88
25
40


412223
33
48
62
76
81
21
51


412224
24
45
57
70
81
28
52


412225
32
42
65
78
73
23
53


413467
2
35
49
61
47
43
100


413468
14
34
56
78
75
35
101


413469
24
33
53
70
84
33
102


413476
26
44
64
73
82
25
109


413481
22
38
56
67
83
32
114


413482
26
39
59
74
82
28
115









Example 3
Antisense Inhibition of Human Factor 11 in HepG2 Cells by Oligonucleotides Designed by Microwalk
Additional gapmers were designed based on the gapmers presented in Table 3. These gapmers were designed by creating gapmers shifted slightly upstream and downstream (i.e. “microwalk”) of the original gapmers from Table 3. Gapmers were also created with various motifs, e.g. 5-10-5 MOE, 3-14-3 MOE, and 2-13-5 MOE. These gapmers were tested in vitro. Cultured HepG2 cells at a density of 10,000 cells per well were transfected using lipofectin reagent with 75 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Factor 11 mRNA levels were measured by quantitative real-time PCR. Factor 11 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of Factor 11, relative to untreated control cells.
The in vitro inhibition data for the gapmers designed by microwalk were then compared with the in vitro inhibition data for the gapmers from Table 3, as indicated in Tables 4, 5, 6, 7, and 8. The oligonucleotides are displayed according to the region on the human mRNA (GENBANK Accession No. NM—000128.3) to which they map.
The chimeric antisense oligonucleotides in Table 4 were designed as 5-10-5 MOE, 3-14-3 MOE, and 2-13-5 MOE gapmers. The first listed gapmers in Table 4 are the original gapmers (see Table 3) from which the remaining gapmers were designed via microwalk and are designated by an asterisk. The 5-10-5 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 10 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising 5 nucleotides each. The 3-14-3 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 14 2′-deoxynucleotides and is flanked on both sides (in the 5′and 3′ directions) by wings comprising 3 nucleotides each. The 2-13-5 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 13 2′-deoxynucleotides. The central gap is flanked on the 5′ end with a wing comprising 2 nucleotides and on the 3′ end with a wing comprising 5 nucleotides. For each of the motifs (5-10-5, 3-14-3, and 2-13-5), each nucleotide in the 5′ wing segment and each nucleotide in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytidine residues throughout each gapmer are 5-methylcytidines. “Target start site” indicates the 5′-most nucleotide to which the gapmer is targeted. “Target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted. Each gapmer listed in Table 4 is targeted to SEQ ID NO: 1 (GENBANK Accession No. NM—000128.3).
As shown in Table 4, all of the 5-10-5 MOE gapmers, 3-14-3 MOE gapmers, and 2-13-5 MOE gapmers targeted to the target region beginning at target start site 656 and ending at the target stop site 704 (i.e. nucleobases 656-704) of SEQ ID NO: 1 exhibit at least 20% inhibition of Factor 11 mRNA. Many of the gapmers exhibit at least 60% inhibition. Several of the gapmers exhibit at least 80% inhibition, including ISIS numbers: 416806, 416809, 416811, 416814, 416821, 416825, 416826, 416827, 416828, 416868, 416869, 416878, 416879, 416881, 416883, 416890, 416891, 416892, 416893, 416894, 416895, 416896, 416945, 416946, 416969, 416970, 416971, 416972, 416973, 412203, 413467, 413468, and 413469. The following ISIS numbers exhibited at least 90% inhibition: 412203, 413467, 416825, 416826, 416827, 416868, 416878, 416879, 416892, 416893, 416895, 416896, 416945, 416972, and 416973. The following ISIS numbers exhibited at least 95% inhibition: 416878, 416892, 416895, and 416896.







TABLE 4







Inhibition of human Factor 11 mRNA levels by chimeric antisense


oligonucleotides targeted to nucleobases 656 to 704 of SEQ ID NO: 1


(GENBANK Accession No. NM_000128.3)














Target
Target

%

SEQ


ISIS No.
Start Site
Stop Site
Sequence (5′ to 3′)
inhibition
Motif
ID No.
















*412203
656
675
AATGTCTTTGTTGCAAGCGC
97
5-10-5
31


 


*413467
666
685
GGTCCACATAAATGTCTTTG
92
5-10-5
100


 


*413468
671
690
GTCTAGGTCCACATAAATGT
83
5-10-5
101


 


*413469
681
700
TGCCCTTCATGTCTAGGTCC
86
5-10-5
102


 


416868
656
675
AATGTCTTTGTTGCAAGCGC
93
3-14-3
31


 


416945
656
675
AATGTCTTTGTTGCAAGCGC
94
2-13-5
31


 


416806
657
676
AAATGTCTTTGTTGCAAGCG
86
5-10-5
171


 


416869
657
676
AAATGTCTTTGTTGCAAGCG
81
3-14-3
171


 


416946
657
676
AAATGTCTTTGTTGCAAGCG
86
2-13-5
171


 


416807
658
677
TAAATGTCTTTGTTGCAAGC
51
5-10-5
172


 


416870
658
677
TAAATGTCTTTGTTGCAAGC
76
3-14-3
172


 


416947
658
677
TAAATGTCTTTGTTGCAAGC
62
2-13-5
172


 


416808
659
678
ATAAATGTCTTTGTTGCAAG
55
5-10-5
173


 


416871
659
678
ATAAATGTCTTTGTTGCAAG
28
3-14-3
173


 


416948
659
678
ATAAATGTCTTTGTTGCAAG
62
2-13-5
173


 


416809
660
679
CATAAATGTCTTTGTTGCAA
86
5-10-5
174


 


416872
660
679
CATAAATGTCTTTGTTGCAA
20
3-14-3
174


 


416949
660
679
CATAAATGTCTTTGTTGCAA
64
2-13-5
174


 


416873
661
680
ACATAAATGTCTTTGTTGCA
51
3-14-3
99


 


416950
661
680
ACATAAATGTCTTTGTTGCA
71
2-13-5
99


 


416810
662
681
CACATAAATGTCTTTGTTGC
68
5-10-5
175


 


416874
662
681
CACATAAATGTCTTTGTTGC
49
3-14-3
175


 


416951
662
681
CACATAAATGTCTTTGTTGC
48
2-13-5
175


 


416811
663
682
CCACATAAATGTCTTTGTTG
84
5-10-5
176


 


416875
663
682
CCACATAAATGTCTTTGTTG
75
3-14-3
176


 


416952
663
682
CCACATAAATGTCTTTGTTG
51
2-13-5
176


 


416812
664
68
TCCACATAAATGTCTTTGTT
59
5-10-5
177


 


416876
664
683
TCCACATAAATGTCTTTGTT
37
3-14-3
177


 


416953
664
683
TCCACATAAATGTCTTTGTT
45
2-13-5
177


 


416813
665
684
GTCCACATAAATGTCTTTGT
70
5-10-5
178


 


416877
665
684
GTCCACATAAATGTCTTTGT
51
3-14-3
178


 


416954
665
684
GTCCACATAAATGTCTTTGT
61
2-13-5
178


 


416878
666
685
GGTCCACATAAATGTCTTTG
95
3-14-3
100


 


416955
666
685
GGTCCACATAAATGTCTTTG
75
2-13-5
100


 


416814
667
686
AGGTCCACATAAATGTCTTT
83
5-10-5
179


 


416879
667
686
AGGTCCACATAAATGTCTTT
92
3-14-3
179


 


416956
667
686
AGGTCCACATAAATGTCTTT
61
2-13-5
179


 


416815
668
687
TAGGTCCACATAAATGTCTT
63
5-10-5
180


 


416880
668
687
TAGGTCCACATAAATGTCTT
66
3-14-3
180


 


416957
668
687
TAGGTCCACATAAATGTCTT
59
2-13-5
180


 


416816
669
688
CTAGGTCCACATAAATGTCT
79
5-10-5
181


 


416881
669
688
CTAGGTCCACATAAATGTCT
81
3-14-3
181


 


416958
669
688
CTAGGTCCACATAAATGTCT
43
2-13-5
181


 


416817
670
689
TCTAGGTCCACATAAATGTC
74
5-10-5
182


 


416882
670
689
TCTAGGTCCACATAAATGTC
60
3-14-3
182


 


416959
670
689
TCTAGGTCCACATAAATGTC
25
2-13-5
182


 


416883
671
690
GTCTAGGTCCACATAAATGT
82
3-14-3
101


 


416960
671
690
GTCTAGGTCCACATAAATGT
60
2-13-5
101


 


416818
672
691
TGTCTAGGTCCACATAAATG
76
5-10-5
183


 


416884
672
691
TGTCTAGGTCCACATAAATG
69
3-14-3
183


 


416961
672
691
TGTCTAGGTCCACATAAATG
40
2-13-5
183


 


416819
673
692
ATGTCTAGGTCCACATAAAT
56
5-10-5
184


 


416885
673
692
ATGTCTAGGTCCACATAAAT
67
3-14-3
184


 


416962
673
692
ATGTCTAGGTCCACATAAAT
77
2-13-5
184


 


416820
674
693
CATGTCTAGGTCCACATAAA
77
5-10-5
185


 


416886
674
693
CATGTCTAGGTCCACATAAA
74
3-14-3
185


 


416963
674
693
CATGTCTAGGTCCACATAAA
48
2-13-5
185


 


416821
675
694
TCATGTCTAGGTCCACATAA
84
5-10-5
186


 


416964
675
694
TCATGTCTAGGTCCACATAA
69
2-13-5
186


 


412204
676
695
TTCATGTCTAGGTCCACATA
76
5-10-5
32


 


416888
676
695
TTCATGTCTAGGTCCACATA
76
3-14-3
32


 


416965
676
695
TTCATGTCTAGGTCCACATA
53
2-13-5
32


 


416822
677
696
CTTCATGTCTAGGTCCACAT
76
5-10-5
187


 


416889
677
696
CTTCATGTCTAGGTCCACAT
60
3-14-3
187


 


416966
677
696
CTTCATGTCTAGGTCCACAT
64
2-13-5
187


 


416823
678
697
CCTTCATGTCTAGGTCCACA
77
5-10-5
188


 


416890
678
697
CCTTCATGTCTAGGTCCACA
87
3-14-3
188


 


416967
678
697
CCTTCATGTCTAGGTCCACA
75
2-13-5
188


 


416824
679
698
CCCTTCATGTCTAGGTCCAC
64
5-10-5
189


 


416891
679
698
CCCTTCATGTCTAGGTCCAC
81
3-14-3
189


 


416968
679
698
CCCTTCATGTCTAGGTCCAC
73
2-13-5
189


 


416825
680
699
GCCCTTCATGTCTAGGTCCA
92
5-10-5
190


 


416892
680
699
GCCCTTCATGTCTAGGTCCA
100
3-14-3
190


 


416969
680
699
GCCCTTCATGTCTAGGTCCA
80
2-13-5
190


 


416893
681
700
TGCCCTTCATGTCTAGGTCC
90
3-14-3
102


 


416970
681
700
TGCCCTTCATGTCTAGGTCC
88
2-13-5
102


 


416826
682
701
ATGCCCTTCATGTCTAGGTC
94
5-10-5
191


 


416894
682
701
ATGCCCTTCATGTCTAGGTC
85
3-14-3
191


 


416971
682
701
ATGCCCTTCATGTCTAGGTC
83
2-13-5
191


 


416827
683
702
TATGCCCTTCATGTCTAGGT
93
5-10-5
192


 


416895
683
702
TATGCCCTTCATGTCTAGGT
95
3-14-3
192


 


416972
683
702
TATGCCCTTCATGTCTAGGT
90
2-13-5
192


 


416828
684
703
TTATGCCCTTCATGTCTAGG
87
5-10-5
193


 


416896
684
703
TTATGCCCTTCATGTCTAGG
95
3-14-3
193


 


416973
684
703
TTATGCCCTTCATGTCTAGG
92
2-13-5
193


 


416829
685
704
TTTATGCCCTTCATGTCTAG
72
5-10-5
194


 


416897
685
704
TTTATGCCCTTCATGTCTAG
66
3-14-3
194


 


416974
685
704
TTTATGCCCTTCATGTCTAG
73
2-13-5
194









The chimeric antisense oligonucleotides in Table 5 were designed as 5-10-5 MOE, 3-14-3 MOE, and 2-13-5 MOE gapmers. The first listed gapmer in Table 5 is the original gapmer (see Table 3) from which the remaining gapmers were designed via microwalk and is designated by an asterisk. The 5-10-5 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 10 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising 5 nucleotides each. The 3-14-3 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 14 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising 3 nucleotides each. The 2-13-5 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 13 2′-deoxynucleotides. The central gap is flanked on the 5′ end with a wing comprising 2 nucleotides and on the 3′ end with a wing comprising 5 nucleotides. For each of the motifs (5-10-5, 3-14-3, and 2-13-5), each nucleotide in the 5′ wing segment and each nucleotide in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytidine residues throughout each gapmer are 5-methylcytidines. “Target start site” indicates the 5′-most nucleotide to which the gapmer is targeted. “Target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted. Each gapmer listed in Table 5 is targeted to SEQ ID NO: 1 (GENBANK Accession No. NM—000128.3).
As shown in Table 5, all of the 5-10-5 MOE gapmers, 3-14-3 MOE gapmers, and 2-13-5 MOE gapmers targeted to the target region beginning at target start site 738 and ending at the target stop site 762 (i.e. nucleobases 738-762) of SEQ ID NO: 1 exhibit at least 45% inhibition of Factor 11 mRNA. Most of the gapmers exhibit at least 60% inhibition. Several of the gapmers exhibit at least 80% inhibition, including ISIS numbers: 412206, 416830, 416831, 416898, 416899, 416900, 416903, 416975, 416976, 416977, and 416980. The following ISIS numbers exhibited at least 90% inhibition: 412206, 416831, and 416900.







TABLE 5







Inhibition of human Factor 11 mRNA levels by chimeric antisense


oligonucleotides targeted to nucleobases 738 to 762 of


SEQ ID NO: 1 (GENBANK Accession No. NM_000128.3)














Target
Target

%

SEQ ID


ISIS No.
Start Site
Stop Site
Sequence (5′ to 3′)
inhibition
Motif
No.
















*412206
738
757
CCGTGCATCTTTCTTGGCAT
93
5-10-5
34


 


416898
738
757
CCGTGCATCTTTCTTGGCAT
88
3-14-3
34


 


416975
738
757
CCGTGCATCTTTCTTGGCAT
87
2-13-5
34


 


416830
739
758
TCCGTGCATCTTTCTTGGCA
81
5-10-5
195


 


416899
739
758
TCCGTGCATCTTTCTTGGCA
86
3-14-3
195


 


416976
739
758
TCCGTGCATCTTTCTTGGCA
83
2-13-5
195


 


416831
740
759
ATCCGTGCATCTTTCTTGGC
91
5-10-5
196


 


416900
740
759
ATCCGTGCATCTTTCTTGGC
90
3-14-3
196


 


416977
740
759
ATCCGTGCATCTTTCTTGGC
82
2-13-5
196


 


416832
741
760
CATCCGTGCATCTTTCTTGG
79
5-10-5
197


 


416901
741
760
CATCCGTGCATCTTTCTTGG
65
3-14-3
197


 


416978
741
760
CATCCGTGCATCTTTCTTGG
76
2-13-5
197


 


416833
742
761
TCATCCGTGCATCTTTCTTG
65
5-10-5
198


 


416902
742
761
TCATCCGTGCATCTTTCTTG
46
3-14-3
198


 


416979
742
761
TCATCCGTGCATCTTTCTTG
63
2-13-5
198


 


416834
743
762
GTCATCCGTGCATCTTTCTT
58
5-10-5
199


 


416903
743
762
GTCATCCGTGCATCTTTCTT
88
3-14-3
199


 


416980
743
762
GTCATCCGTGCATCTTTCTT
87
2-13-5
199









The chimeric antisense oligonucleotides in Table 6 were designed as 5-10-5 MOE, 3-14-3 MOE, and 2-13-5 MOE gapmers. The first listed gapmers in Table 6 are the original gapmers (see Table 3) from which the remaining gapmers were designed via microwalk and are designated by an asterisk. The 5-10-5 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 10 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising 5 nucleotides each. The 3-14-3 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 14 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising 3 nucleotides each. The 2-13-5 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 13 2′-deoxynucleotides. The central gap is flanked on the 5′ end with a wing comprising 2 nucleotides and on the 3′ end with a wing comprising 5 nucleotides. For each of the motifs (5-10-5, 3-14-3, and 2-13-5), each nucleotide in the 5′ wing segment and each nucleotide in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytidine residues throughout each gapmer are 5-methylcytidines. “Target start site” indicates the 5′-most nucleotide to which the gapmer is targeted. “Target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted. Each gapmer listed in Table 6 is targeted to SEQ ID NO: 1 (GENBANK Accession No. NM—000128.3).
As shown in Table 6, all of the 5-10-5 MOE gapmers, 3-14-3 MOE gapmers, and 2-13-5 MOE gapmers targeted to the target region beginning at target start site 1018 and ending at the target stop site 1042 (i.e. nucleobases 1018-1042) of SEQ ID NO: 1 exhibit at least 80% inhibition of Factor 11 mRNA. The following ISIS numbers exhibited at least 90% inhibition: 413474, 416837, 416838, 416904, 416907, and 416908.







TABLE 6







Inhibition of human Factor 11 mRNA levels by chimeric antisense


oligonucleotides targeted to nucleobases 1018 to 1042 of


SEQ ID NO: 1 (GENBANK Accession No. NM_000128.3)














Target Start
Target Stop

%

SEQ ID


ISIS No.
Site
Site
Sequence (5′ to 3′)
inhibition
Motif
No.
















*412212
1018
1037
CCGGGATGATGAGTGCAGAT
89
5-10-5
40


 


416904
1018
1037
CCGGGATGATGAGTGCAGAT
90
3-14-3
40


 


416981
1018
1037
CCGGGATGATGAGTGCAGAT
87
2-13-5
40


 


416835
1019
1038
ACCGGGATGATGAGTGCAGA
83
5-10-5
200


 


416905
1019
1038
ACCGGGATGATGAGTGCAGA
85
3-14-3
200


 


416982
1019
1038
ACCGGGATGATGAGTGCAGA
84
2-13-5
200


 


416836
1020
1039
AACCGGGATGATGAGTGCAG
89
5-10-5
201


 


416906
1020
1039
AACCGGGATGATGAGTGCAG
88
3-14-3
201


 


416983
1020
1039
AACCGGGATGATGAGTGCAG
86
2-13-5
201


 


416837
1021
1040
CAACCGGGATGATGAGTGCA
90
5-10-5
202


 


416907
1021
1040
CAACCGGGATGATGAGTGCA
90
3-14-3
202


 


416984
1021
1040
CAACCGGGATGATGAGTGCA
89
2-13-5
202


 


416838
1022
1041
GCAACCGGGATGATGAGTGC
94
5-10-5
203


 


416908
1022
1041
GCAACCGGGATGATGAGTGC
98
3-14-3
203


 


416985
1022
1041
GCAACCGGGATGATGAGTGC
88
2-13-5
203


 


413474
1023
1042
AGCAACCGGGATGATGAGTG
93
5-10-5
107









The chimeric antisense oligonucleotides in Table 7 were designed as 5-10-5 MOE, 3-14-3 MOE, and 2-13-5 MOE gapmers. The first listed gapmer in Table 7 is the original gapmer (see Table 3) from which the remaining gapmers were designed via microwalk and is designated by an asterisk. The 5-10-5 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 10 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising 5 nucleotides each. The 3-14-3 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 14 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising 3 nucleotides each. The 2-13-5 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 13 2′-deoxynucleotides. The central gap is flanked on the 5′ end with a wing comprising 2 nucleotides and on the 3′ end with a wing comprising 5 nucleotides. For each of the motifs (5-10-5, 3-14-3, and 2-13-5), each nucleotide in the 5′ wing segment and each nucleotide in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytidine residues throughout each gapmer are 5-methylcytidines. “Target start site” indicates the 5′-most nucleotide to which the gapmer is targeted. “Target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted. Each gapmer listed in Table 7 is targeted to SEQ ID NO: 1 (GENBANK Accession No. NM—000128.3).
As shown in Table 7, all of the 5-10-5 MOE gapmers, 3-14-3 MOE gapmers, and 2-13-5 MOE gapmers targeted to the target region beginning at target start site 1062 and ending at the target stop site 1091 (i.e. nucleobases 1062-1091) of SEQ ID NO: 1 exhibit at least 20% inhibition of Factor 11 mRNA. Many of the gapmers exhibit at least 50% inhibition, including: 412215, 413476, 413476, 416839, 416840, 416841, 416842, 416843, 416844, 416845, 416846, 416847, 416909, 416910, 416911, 416912, 416913, 416914, 416915, 416916, 416917, 416918, 416986, 416987, 416988, 416989, 416990, 416991, 416992, 416993, 416994, 416995. The following ISIS numbers exhibited at least 80% inhibition: 412215, 413476, 413476, 416839, 416840, 416841, 416842, 416843, 416844, 416845, 416910, 416911, 416912, 416913, 416914, 416916, 416917, 416986, 416987, 416989, 416991, 416992, 416993, and 416994. The following ISIS numbers exhibited at least 90% inhibition: 413476, 413476, 416842, 416844, 416910, 416911, 416912, 416913, 416916, 416917, and 416993.







TABLE 7







Inhibition of human Factor 11 mRNA levels by chimeric antisense


oligonucleotides targeted to nucleobases 1062 to 1091 of


SEQ ID NO: 1 (GENBANK Accession No. NM_000128.3)













ISIS
Target
Target

%

SEQ


No.
Start Site
Stop Site
Sequence (5′ to 3′)
inhibition
Motif
ID No.
















*413476
1067
1086
TTGAGATTCTTTGGGCCATT
93
5-10-5
109


 


412215
1062
1081
ATTCTTTGGGCCATTCCTGG
82
5-10-5
43


 


416909
1062
1081
ATTCTTTGGGCCATTCCTGG
78
3-14-3
43


 


416986
1062
1081
ATTCTTTGGGCCATTCCTGG
88
2-13-5
43


 


416839
1063
1082
GATTCTTTGGGCCATTCCTG
89
5-10-5
204


 


416910
1063
1082
GATTCTTTGGGCCATTCCTG
90
3-14-3
204


 


416987
1063
1082
GATTCTTTGGGCCATTCCTG
80
2-13-5
204


 


416840
1064
1083
AGATTCTTTGGGCCATTCCT
85
5-10-5
205


 


416911
1064
1083
AGATTCTTTGGGCCATTCCT
90
3-14-3
205


 


416988
1064
1083
AGATTCTTTGGGCCATTCCT
76
2-13-5
205


 


416841
1065
1084
GAGATTCTTTGGGCCATTCC
87
5-10-5
206


 


416912
1065
1084
GAGATTCTTTGGGCCATTCC
92
3-14-3
206


 


416989
1065
1084
GAGATTCTTTGGGCCATTCC
88
2-13-5
206


 


416842
1066
1085
TGAGATTCTTTGGGCCATTC
94
5-10-5
207


 


416913
1066
1085
TGAGATTCTTTGGGCCATTC
93
3-14-3
207


 


416990
1066
1085
TGAGATTCTTTGGGCCATTC
76
2-13-5
207


 


413476
1067
1086
TTGAGATTCTTTGGGCCATT
93
5-10-5
109


 


416914
1067
1086
TTGAGATTCTTTGGGCCATT
87
3-14-3
109


 


416991
1067
1086
TTGAGATTCTTTGGGCCATT
87
2-13-5
109


 


416843
1068
1087
TTTGAGATTCTTTGGGCCAT
89
5-10-5
208


 


416915
1068
1087
TTTGAGATTCTTTGGGCCAT
79
3-14-3
208


 


416992
1068
1087
TTTGAGATTCTTTGGGCCAT
84
2-13-5
208


 


416844
1069
1088
CTTTGAGATTCTTTGGGCCA
90
5-10-5
209


 


416916
1069
1088
CTTTGAGATTCTTTGGGCCA
91
3-14-3
209


 


416993
1069
1088
CTTTGAGATTCTTTGGGCCA
91
2-13-5
209


 


416845
1070
1089
TCTTTGAGATTCTTTGGGCC
86
5-10-5
210


 


416917
1070
1089
TCTTTGAGATTCTTTGGGCC
92
3-14-3
210


 


416994
1070
1089
TCTTTGAGATTCTTTGGGCC
83
2-13-5
210


 


416846
1071
1090
TTCTTTGAGATTCTTTGGGC
72
5-10-5
211


 


416918
1071
1090
TTCTTTGAGATTCTTTGGGC
63
3-14-3
211


 


416995
1071
1090
TTCTTTGAGATTCTTTGGGC
64
2-13-5
211


 


416847
1072
1091
TTTCTTTGAGATTCTTTGGG
50
5-10-5
212


 


416919
1072
1091
TTTCTTTGAGATTCTTTGGG
27
3-14-3
212


 


416996
1072
1091
TTTCTTTGAGATTCTTTGGG
22
2-13-5
212









The chimeric antisense oligonucleotides in Table 8 were designed as 5-10-5 MOE, 3-14-3 MOE, and 2-13-5 MOE gapmers. The first listed gapmers in Table 8 are the original gapmers (see Table 3) from which the remaining gapmers were designed via microwalk and are designated by an asterisk. The 5-10-5 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 10 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising 5 nucleotides each. The 3-14-3 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 14 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising 3 nucleotides each. The 2-13-5 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 13 2′-deoxynucleotides. The central gap is flanked on the 5′ end with a wing comprising 2 nucleotides and on the 3′ end with a wing comprising 5 nucleotides. For each of the motifs (5-10-5, 3-14-3, and 2-13-5), each nucleotide in the 5′ wing segment and each nucleotide in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytidine residues throughout each gapmer are 5-methylcytidines. “Target start site” indicates the 5′-most nucleotide to which the gapmer is targeted. “Target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted. Each gapmer listed in Table 8 is targeted to SEQ ID NO: 1 (GENBANK Accession No. NM—000128.3).
As shown in Table 8, all of the 5-10-5 MOE gapmers, 3-14-3 MOE gapmers, and 2-13-5 MOE gapmers targeted to the target region beginning at target start site 1275 and ending at the target stop site 1318 (i.e. nucleobases 1275-1318) of SEQ ID NO: 1 exhibit at least 70% inhibition of Factor 11 mRNA. Many of the gapmers exhibit at least 80% inhibition, including: 412223, 412224, 412225, 413482, 416848, 416849, 416850, 416851, 416852, 416853, 416854, 416855, 416856, 416857, 416858, 416859, 416860, 416861, 416862, 416863, 416864, 416865, 416866, 416867, 416920, 416921, 416922, 416923, 416924, 416925, 416926, 416927, 416928, 416929, 416930, 416931, 416932, 416933, 416934, 416935, 416936, 416937, 416938, 416939, 416940, 416941, 416942, 416943, 416944, 416997, 416998, 416999, 417000, 417001, 417002, 417003, 417004, 417006, 417007, 417008, 417009, 417010, 417011, 417013, 417014, 417015, 417016, 417017, 417018, 417019, and 417020. The following ISIS numbers exhibited at least 90% inhibition: 412224, 416850, 416853, 416856, 416857, 416858, 416861, 416862, 416864, 416922, 416923, 416924, 416925, 416926, 416928, 416931, 416932, 416933, 416934, 416935, 416937, 416938, 416940, 416941, 416943, 416999, 417002, 416854, and 416859.







TABLE 8







Inhibition of human Factor 11 mRNA levels by chimeric antisense


oligonucleotides targeted to nucleobases 1275 to 1318 of


SEQ ID NO: 1 (GENBANK Accession No. NM_000128.3)













ISIS
Target
Target

%

SEQ


No.
Start Site
Stop Site
Sequence (5′ to 3′)
inhibition
Motif
ID No.
















*412223
1275
1294
ACAGTTTCTGGCAGGCCTCG
85
5-10-5
51


 


*412224
1285
1304
GCATTGGTGCACAGTTTCTG
93
5-10-5
52


 


*413482
1290
1309
GGACGGCATTGGTGCACAGT
89
5-10-5
115


 


*412225
1295
1314
GCAGCGGACGGCATTGGTGC
86
5-10-5
53


 


416920
1275
1294
ACAGTTTCTGGCAGGCCTCG
88
3-14-3
51


 


416997
1275
1294
ACAGTTTCTGGCAGGCCTCG
84
2-13-5
51


 


416848
1276
1295
CACAGTTTCTGGCAGGCCTC
86
5-10-5
213


 


416921
1276
1295
CACAGTTTCTGGCAGGCCTC
88
3-14-3
213


 


416998
1276
1295
CACAGTTTCTGGCAGGCCTC
88
2-13-5
213


 


416849
1277
1296
GCACAGTTTCTGGCAGGCCT
88
5-10-5
214


 


416922
1277
1294
GCACAGTTTCTGGCAGGCCT
94
3-14-3
214


 


416999
1277
1296
GCACAGTTTCTGGCAGGCCT
92
2-13-5
214


 


416850
1278
1297
TGCACAGTTTCTGGCAGGCC
93
5-10-5
215


 


416923
1278
1297
TGCACAGTTTCTGGCAGGCC
96
3-14-3
215


 


417000
1278
1297
TGCACAGTTTCTGGCAGGCC
89
2-13-5
215


 


416851
1279
1298
GTGCACAGTTTCTGGCAGGC
88
5-10-5
216


 


416924
1279
1298
GTGCACAGTTTCTGGCAGGC
96
3-14-3
216


 


417001
1279
1298
GTGCACAGTTTCTGGCAGGC
83
2-13-5
216


 


416925
1280
1299
GGTGCACAGTTTCTGGCAGG
98
3-14-3
114


 


417002
1280
1299
GGTGCACAGTTTCTGGCAGG
92
2-13-5
114


 


416852
1281
1300
TGGTGCACAGTTTCTGGCAG
84
5-10-5
217


 


416926
1281
1300
TGGTGCACAGTTTCTGGCAG
93
3-14-3
217


 


417003
1281
1300
TGGTGCACAGTTTCTGGCAG
89
2-13-5
217


 


416853
1282
1301
TTGGTGCACAGTTTCTGGCA
91
5-10-5
218


 


416927
1282
1301
TTGGTGCACAGTTTCTGGCA
87
3-14-3
218


 


417004
1282
1301
TTGGTGCACAGTTTCTGGCA
86
2-13-5
218


 


416854
1283
1302
ATTGGTGCACAGTTTCTGGC
90
5-10-5
219


 


416928
1283
1302
ATTGGTGCACAGTTTCTGGC
91
3-14-3
219


 


417005
1283
1302
ATTGGTGCACAGTTTCTGGC
79
2-13-5
219


 


416855
1284
1303
CATTGGTGCACAGTTTCTGG
87
5-10-5
220


 


416929
1284
1303
CATTGGTGCACAGTTTCTGG
83
3-14-3
220


 


417006
1284
1303
CATTGGTGCACAGTTTCTGG
81
2-13-5
220


 


416930
1285
1304
GCATTGGTGCACAGTTTCTG
87
3-14-3
52


 


417007
1285
1304
GCATTGGTGCACAGTTTCTG
82
2-13-5
52


 


416856
1286
1305
GGCATTGGTGCACAGTTTCT
95
5-10-5
221


 


416931
1286
1305
GGCATTGGTGCACAGTTTCT
96
3-14-3
221


 


417008
1286
1305
GGCATTGGTGCACAGTTTCT
82
2-13-5
221


 


416857
1287
1306
CGGCATTGGTGCACAGTTTC
92
5-10-5
222


 


416932
1287
1306
CGGCATTGGTGCACAGTTTC
92
3-14-3
222


 


417009
1287
1306
CGGCATTGGTGCACAGTTTC
85
2-13-5
222


 


416858
1288
1307
ACGGCATTGGTGCACAGTTT
93
5-10-5
223


 


416933
1288
1307
ACGGCATTGGTGCACAGTTT
92
3-14-3
223


 


417010
1288
1307
ACGGCATTGGTGCACAGTTT
81
2-13-5
223


 


416859
1289
1308
GACGGCATTGGTGCACAGTT
90
5-10-5
224


 


416934
1289
1308
GACGGCATTGGTGCACAGTT
90
3-14-3
224


 


417011
1289
1308
GACGGCATTGGTGCACAGTT
86
2-13-5
224


 


416935
1290
1309
GGACGGCATTGGTGCACAGT
92
3-14-3
115


 


417012
1290
1309
GGACGGCATTGGTGCACAGT
72
2-13-5
115


 


416860
1291
1310
CGGACGGCATTGGTGCACAG
88
5-10-5
225


 


416936
1291
1310
CGGACGGCATTGGTGCACAG
89
3-14-3
225


 


417013
1291
1310
CGGACGGCATTGGTGCACAG
86
2-13-5
225


 


416861
1292
1311
GCGGACGGCATTGGTGCACA
92
5-10-5
226


 


416937
1292
1311
GCGGACGGCATTGGTGCACA
93
3-14-3
226


 


417014
1292
1311
GCGGACGGCATTGGTGCACA
87
2-13-5
226


 


416862
1293
1312
AGCGGACGGCATTGGTGCAC
90
5-10-5
227


 


416938
1293
1312
AGCGGACGGCATTGGTGCAC
90
3-14-3
227


 


417015
1293
1312
AGCGGACGGCATTGGTGCAC
87
2-13-5
227


 


416863
1294
1313
CAGCGGACGGCATTGGTGCA
83
5-10-5
228


 


416939
1294
1313
CAGCGGACGGCATTGGTGCA
88
3-14-3
228


 


417016
1294
1313
CAGCGGACGGCATTGGTGCA
85
2-13-5
228


 


416940
1295
1314
GCAGCGGACGGCATTGGTGC
92
3-14-3
53


 


417017
1295
1314
GCAGCGGACGGCATTGGTGC
82
2-13-5
53


 


416864
1296
1315
GGCAGCGGACGGCATTGGTG
93
5-10-5
229


 


416941
1296
1315
GGCAGCGGACGGCATTGGTG
95
3-14-3
229


 


417018
1296
1315
GGCAGCGGACGGCATTGGTG
82
2-13-5
229


 


416865
1297
1316
TGGCAGCGGACGGCATTGGT
88
5-10-5
230


 


416942
1297
1316
TGGCAGCGGACGGCATTGGT
85
3-14-3
230


 


417019
1297
1316
TGGCAGCGGACGGCATTGGT
84
2-13-5
230


 


416866
1298
1317
CTGGCAGCGGACGGCATTGG
88
5-10-5
231


 


416943
1298
1317
CTGGCAGCGGACGGCATTGG
92
3-14-3
231


 


417020
1298
1317
CTGGCAGCGGACGGCATTGG
84
2-13-5
231


 


416867
1299
1318
ACTGGCAGCGGACGGCATTG
83
5-10-5
232


 


416944
1299
1318
ACTGGCAGCGGACGGCATTG
83
3-14-3
232


 


417021
1299
1318
ACTGGCAGCGGACGGCATTG
74
2-13-5
232









Example 4
Dose-Dependent Antisense Inhibition of Human Factor 11 in HepG2 Cells
Gapmers from Example 3 (see Tables 4, 5, 6, 7, and 8), exhibiting in vitro inhibition of human Factor 11, were tested at various doses in HepG2 cells. Cells were plated at a density of 10,000 cells per well and transfected using lipofectin reagent with 9.375 nM, 18.75 nM, 37.5 nM and 75 nM concentrations of antisense oligonucleotide, as specified in Table 9. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Factor 11 mRNA levels were measured by quantitative real-time PCR. Human Factor 11 primer probe set RTS 2966 was used to measure mRNA levels. Factor 11 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of Factor 11, relative to untreated control cells. As illustrated in Table 9, Factor 11 mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells.







TABLE 9







Dose-dependent antisense inhibition of human Factor 11 in HepG2


cells via transfection of oligonucleotides with lipofectin















9.375
18.75
37.5
75

IC50
SEQ ID



nM
nM
nM
nM
Motif
(nM)
No.


















412203
33
40
62
74
5-10-5
24
31


412206
24
47
69
86
5-10-5
21
34


413467
35
51
62
69
5-10-5
20
100


413474
29
44
57
67
5-10-5
28
107


413476
24
58
62
77
5-10-5
21
109


416825
23
52
73
92
5-10-5
20
190


416826
8
36
58
84
5-10-5
29
191


416827
31
42
62
77
5-10-5
23
192


416838
31
51
64
86
5-10-5
19
203


416842
18
33
62
71
5-10-5
31
207


416850
4
30
67
84
5-10-5
29
215


416856
21
45
58
74
5-10-5
27
221


416858
0
28
54
82
5-10-5
33
223


416864
18
43
62
78
5-10-5
26
229


416878
22
34
60
82
5-10-5
27
100


416892
16
50
70
85
3-14-3
23
190


416895
39
57
66
71
3-14-3
15
192


416896
22
39
57
81
3-14-3
27
193


416908
36
57
67
76
3-14-3
16
203


416922
14
25
49
75
3-14-3
36
214


416923
36
47
60
67
3-14-3
23
215


416924
25
38
56
59
3-14-3
36
216


416925
13
38
59
75
3-14-3
30
114


416926
31
43
63
82
3-14-3
22
217


416931
44
39
57
71
3-14-3
22
221


416941
33
54
63
78
3-14-3
19
229


416945
34
45
62
65
2-13-5
24
31


416969
17
39
61
76
2-13-5
28
190


416972
32
40
60
69
2-13-5
26
192


416973
60
75
85
87
2-13-5
3
193


416984
26
50
62
81
2-13-5
22
202


416985
17
30
47
57
2-13-5
49
203


416989
18
41
62
83
2-13-5
26
206


416993
15
37
50
68
2-13-5
36
209


416999
24
37
55
73
2-13-5
30
214


417000
35
47
58
70
2-13-5
23
215


417002
35
52
67
70
2-13-5
19
114


417003
26
44
60
56
2-13-5
33
217









The gapmers were also transfected via electroporation and their dose dependent inhibition of human Factor 11 mRNA was measured. Cells were plated at a density of 20,000 cells per well and transfected via electroporation with 0.7 μM, 2.2 μM, 6.7 μM, and 20 μM concentrations of antisense oligonucleotide, as specified in Table 10. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Factor 11 mRNA levels were measured by quantitative real-time PCR. Human Factor 11 primer probe set RTS 2966 was used to measure mRNA levels. Factor 11 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of Factor 11, relative to untreated control cells. As illustrated in Table 10, Factor 11 mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells.







TABLE 10







Dose-dependent antisense inhibition of human Factor 11 in HepG2


cells via transfection of oligonucleotides with electroporation














0.7
2.2
6.7
20
IC50
SEQ ID



μM
μM
μM
μM
(μM)
No.

















412203
11
60
70
91
2.7
31


412206
22
39
81
94
2.7
34


413467
5
31
65
89
4.2
100


413474
0
5
52
81
6.9
107


413476
40
69
88
93
0.9
109


416825
27
74
92
98
1.3
190


416826
2
47
86
82
3.2
191


416827
37
68
87
92
1.1
192


416838
5
30
55
83
5.1
203


416842
0
10
66
92
5.0
207


416850
14
25
81
91
3.4
215


416856
0
29
47
93
5.1
221


416858
5
20
56
86
5.3
223


416864
32
65
78
90
1.4
229


416878
1
26
75
85
4.3
100


416892
14
52
82
92
2.5
190


416895
0
62
70
91
3.0
192


416896
12
35
81
89
3.2
193


416908
7
58
74
89
2.8
203


416922
35
51
77
91
1.7
214


416923
15
30
60
90
4.0
215


416924
22
40
63
70
4.1
216


416925
0
40
76
80
3.9
114


416926
47
71
91
94
0.6
217


416931
7
24
60
82
5.1
221


416941
16
38
79
89
3.0
229


416945
48
70
81
88
0.6
31


416969
25
34
86
92
2.5
190


416972
25
30
48
88
4.3
192


416973
20
48
86
93
2.3
193


416984
43
54
88
90
1.1
202


416985
12
48
45
69
5.8
203


416989
32
65
88
94
1.3
206


416993
22
48
87
92
2.2
209


416999
20
42
77
88
2.8
214


417000
46
73
76
89
0.6
215


417002
32
38
82
91
2.2
114


417003
0
34
75
89
3.9
217









Example 5
Selection and Confirmation of Effective Dose-Dependent Antisense Inhibition of Human Factor 11 in HepG2 Cells
Gapmers exhibiting significant dose-dependent inhibition of human Factor 11 in Example 4 were selected and tested at various doses in HepG2 cells. Cells were plated at a density of 10,000 cells per well and transfected using lipofectin reagent with 2.34 nM, 4.69 nM, 9.375 nM, 18.75 nM, 37.5 nM, and 75 nM concentrations of antisense oligonucleotide, as specified in Table 11. After a treatment period of approximately 16 hours, RNA was isolated from the cells and human Factor 11 mRNA levels were measured by quantitative real-time PCR. Human Factor 11 primer probe set RTS 2966 was used to measure mRNA levels. Factor 11 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of human Factor 11, relative to untreated control cells. As illustrated in Table 11, Factor 11 mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells compared to the control.







TABLE 11







Dose-dependent antisense inhibition of human Factor 11 in HepG2


cells via transfection of oligonucleotides with lipofectin

















2.34
4.69
9.375
18.75
37.5
75

IC50
SEQ ID



nM
nM
nM
nM
nM
nM
Motif
(nM)
No.




















416825
4
22
39
57
79
89
5-10-5
13
190


416826
15
22
32
54
76
90
5-10-5
15
191


416838
21
37
50
63
74
83
5-10-5
10
203


416850
24
31
49
55
70
77
5-10-5
13
215


416858
11
35
46
61
75
77
5-10-5
11
223


416864
13
34
42
65
68
80
5-10-5
15
229


416892
14
34
49
70
84
93
3-14-3
9
190


416925
24
34
45
56
67
72
3-14-3
13
114


416999
10
26
42
62
72
80
2-13-5
14
214


417002
17
26
49
61
81
84
2-13-5
12
114


417003
6
29
48
64
73
82
2-13-5
11
217









The gapmers were also transfected via electroporation and their dose dependent inhibition of human Factor 11 mRNA was measured. Cells were plated at a density of 20,000 cells per well and transfected via electroporation with 625 nM, 1250 nM, 2500 nM, 5,000 nM, 10,000 nM, and 20,000 nM concentrations of antisense oligonucleotide, as specified in Table 12. After a treatment period of approximately 16 hours, RNA was isolated from the cells and human Factor 11 mRNA levels were measured by quantitative real-time PCR. Human Factor 11 primer probe set RTS 2966 was used to measure mRNA levels. Factor 11 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of human Factor 11, relative to untreated control cells. As illustrated in Table 12, Factor 11 mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells compared to the control.







TABLE 12







Dose-dependent antisense inhibition of human Factor 11 in HepG2


cells via transfection of oligonucleotides with electroporation
















625
1250
2500
5000
10000
20000
IC50
SEQ ID



nM
nM
nM
nM
nM
nM
(μM)
No.



















416825
69
84
91
94
96
97
19
190


416826
67
82
89
92
95
97
33
191


416838
66
79
87
90
93
96
43
203


416850
69
80
87
90
93
96
25
215


416858
65
77
87
89
93
93
44
223


416864
45
74
84
87
92
94
338
229


416892
66
86
96
97
100
100
31
190


416925
64
80
88
91
95
96
51
114


416999
61
82
89
94
94
97
67
214


417002
59
72
86
90
94
96
156
114


417003
60
74
86
90
95
95
123
217









Example 6
Selection and Confirmation of Effective Dose-Dependent Antisense Inhibition of Human Factor 11 in Cyano Primary Hepatocytes
Gapmers from Example 4 exhibiting significant dose dependent in vitro inhibition of human Factor 11 were also tested at various doses in cyano primary hepatocytes. Cells were plated at a density of 35,000 cells per well and transfected via electroporation with 0.74 nM, 2.2 nM, 6.7 nM, 20 nM, 60 nM, and 180 nM concentrations of antisense oligonucleotide, as specified in Table 13. After a treatment period of approximately 16 hours, RNA was isolated from the cells and human Factor 11 mRNA levels were measured by quantitative real-time PCR. Human Factor 11 primer probe set RTS 2966 was used to measure mRNA levels. Factor 11 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of human Factor 11, relative to untreated control cells. As illustrated in Table 13, Factor 11 mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells compared to the control.







TABLE 13







Dose-dependent antisense inhibition of human


Factor 11 in cyano primary hepatocytes
















0.74
2.2
6.7
20
60
180
IC50
SEQ ID



nM
nM
nM
nM
nM
nM
(μM)
No.



















416825
5
22
51
61
77
84
1.0
190


416826
13
24
34
67
69
71
1.3
191


416838
0
0
21
34
48
62
6.9
203


416850
2
20
24
65
69
67
1.6
215


416858
2
13
22
44
63
68
3.7
223


416864
0
1
15
23
47
64
7.7
229


416892
20
20
43
62
88
92
1.0
190


416925
0
9
1
48
55
76
4.4
114


416999
3
40
36
62
67
82
1.3
214


417002
32
16
28
38
55
71
4.0
114


417003
12
18
19
39
58
74
4.1
217









Example 7
Selection and Confirmation of Effective Dose-Dependent Antisense Inhibition of Human Factor 11 in HepB3 Cells by Gapmers
Gapmers exhibiting in vitro inhibition of human Factor 11 in Example 4 were tested at various doses in human HepB3 cells. Cells were plated at a density of 4,000 cells per well and transfected using lipofectin reagent with 2.3 nM, 4.7 nM, 9.4 nM, 18.75 nM, 37.5 nM, and 75 nM concentrations of antisense oligonucleotide, as specified in Table 14. After a treatment period of approximately 16 hours, RNA was isolated from the cells and human Factor 11 mRNA levels were measured by quantitative real-time PCR. Human Factor 11 primer probe set RTS 2966 was used to measure mRNA levels. Factor 11 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of Factor 11, relative to untreated control cells. As illustrated in Table 14, Factor 11 mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells compared to the control.







TABLE 14







Dose-dependent antisense inhibition


of human Factor 11 in HepB3 cells
















2.3
4.7
9.4
18.75
37.5
75
IC50
SEQ ID


ISIS No.
nM
nM
nM
nM
nM
nM
(nM)
No.


















416825
0
15
34
36
53
59
35
190


416826
16
28
38
55
64
66
16
191


416838
23
34
43
59
71
56
11
203


416850
22
32
43
56
75
60
13
215


416858
17
34
43
57
74
62
12
223


416864
24
37
42
66
76
63
9
229


416892
28
34
50
68
82
72
9
190


416925
26
33
45
59
72
60
12
114


416999
19
33
42
60
71
59
12
214


417002
24
30
46
57
71
65
13
114


417003
11
28
40
40
63
58
17
217









The gapmers were also transfected via electroporation and their dose dependent inhibition of human Factor 11 mRNA was measured. Cells were plated at a density of 20,000 cells per well and transfected via electroporation with 41.15 nM, 123.457 nM, 370.37 nM, 1111.11 nM, 3333.33 nM, and 10,000 nM concentrations of antisense oligonucleotide, as specified in Table 15. After a treatment period of approximately 16 hours, RNA was isolated from the cells and human Factor 11 mRNA levels were measured by quantitative real-time PCR. Human Factor 11 primer probe set RTS 2966 was used to measure mRNA levels. Factor 11 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of human Factor 11, relative to untreated control cells. As illustrated in Table 15, Factor 11 mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells compared to the control.







TABLE 15







Dose-dependent antisense inhibition of human Factor 11 in HepB3 cells
















41.15
123.457
370.37
1111.11
3333.33
10000
IC50
SEQ ID



nM
nM
nM
nM
nM
nM
(μM)
No.



















416825
32
40
48
75
90
92
0.16
190


416826
0
0
34
61
87
92
0.78
191


416838
12
9
28
40
77
88
1.20
203


416850
26
38
51
73
90
95
0.30
215


416858
23
45
52
64
87
92
0.30
223


416864
4
3
6
35
75
87
2.20
229


416892
9
12
28
65
89
98
0.61
190


416925
27
39
50
73
88
96
0.20
114


416999
31
45
62
78
94
97
0.16
214


417002
19
0
31
47
86
93
1.20
114


417003
31
0
15
43
84
92
1.50
217









Example 8
Antisense Inhibition of Murine Factor 11 in Primary Mouse Hepatocytes
Chimeric antisense oligonucleotides targeting murine Factor 11 were designed as 5-10-5 MOE gapmers targeting murine Factor 11 (GENBANK Accession No. NM—028066.1, incorporated herein as SEQ ID NO: 6). The gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 10 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising 5 nucleotides each. Each nucleotide in each wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gaper are phosphorothioate (P═S) linkages. All cytidine residues throughout each gapmer are 5-methylcytidines. The antisense oligonucleotides were evaluated for their ability to reduce murine Factor 11 mRNA in primary mouse hepatocytes.
Primary mouse hepatocytes were treated with 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM, and 200 nM of antisense oligonucleotides for a period of approximately 24 hours. RNA was isolated from the cells and murine Factor 11 mRNA levels were measured by quantitative real-time PCR. Murine Factor 11 primer probe set RTS 2898 (forward sequence ACATGACAGGCGCGATCTCT, incorporated herein as SEQ ID NO: 7; reverse sequence TCTAGGTTCACGTACACATCTTTGC, incorporated herein as SEQ ID NO: 8; probe sequence TTCCTTCAAGCAATGCCCTCAGCAATX, incorporated herein as SEQ ID NO: 9) was used to measure mRNA levels. Factor 11 mRNA levels were adjusted according to total RNA content as measured by RIBOGREEN. Several of the murine antisense oligonucleotides reduced Factor 11 mRNA levels in a dose-dependent manner.
Example 9
Cross-Reactive Antisense Inhibition of Murine Factor 11 in Primary Mouse Hepatocytes
Antisense oligonucleotides targeted to a murine factor 11 nucleic acid were tested for their effects on Factor 11 mRNA in vitro. Cultured primary mouse hepatocytes at a density of 10,000 cells per well were treated with 100 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and mouse Factor 11 mRNA levels were measured by quantitative real-time PCR. Factor 11 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of Factor 11, relative to untreated control cells.
The chimeric antisense oligonucleotides in Tables 16 were designed as 5-10-5 MOE gapmers. The gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 10 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising 5 nucleotides each. Each nucleotide in the 5′ wing segment and each nucleotide in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytidine residues throughout each gapmer are 5-methylcytidines. “Mouse target start site” indicates the 5′-most nucleotide to which the gapmer is targeted. “Mouse target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted. All the mouse oligonucleotides listed show cross-reactivity between the mouse Factor 11 mRNA (GENBANK Accession No. NM—028066.1), incorporated herein as SEQ ID NO: 6 and the human Factor 11 mRNA (GENBANK Accession No. NM—000128.3), incorporated herein as SEQ ID NO: 1. “Human Target Start Site” indicates the 5′-most nucleotide in the human mRNA (GENBANK Accession No. NM—000128.3) to which the antisense oligonucleotide is targeted. “Human Target Stop Site” indicates the 3′-most nucleotide in the human mRNA (GENBANK Accession No. NM—000128.3) to which the antisense oligonucleotide is targeted. “Number of mismatches” indicates the mismatches between the mouse oligonucleotide and the human mRNA sequence.







TABLE 16







Inhibition of mouse Factor 11 mRNA levels by chimeric antisense


oligonucleotides having 5-10-5 MOE wings and deoxy gap


targeted to SEQ ID NO: 1 and SEQ ID NO: 6
















Mouse
Mouse



Human
Human




Target
Target


SEQ
Target
Target



ISIS
Start
Stop

%
ID
Start
Stop
No. of


No
Site
Site
Sequence (5′ to 3′)
Inhibition
No.
Site
Site
mismatches


















404050
379
398
TGCTTGAAGGAATATCCAGA
82
233
619
638
2


 


404054
448
467
TAGTTCATGCCCTTCATGTC
45
234
688
707
1


 


404055
453
472
TGTTATAGTTCATGCCCTTC
27
235
693
712
1


 


404066
686
705
AATGTCCCTGATACAAGCCA
37
236
926
945
1


 


404067
691
710
GGGAAAATGTCCCTGATACA
39
237
931
950
1


 


404083
1299
1318
TGTGCAGAGTCACCTGCCAT
47
238
1533
1552
2


 


404087
1466
1485
TTCTTGAACCCTGAAGAAAG
29
239
1709
1728
2


 


404089
1477
1496
TGAATTATCATTTCTTGAAC
 6
240
1720
1739
2


 


404090
1483
1502
TGATCATGAATTATCATTTC
42
241
1726
1745
2









Example 10
In Vivo Antisense Inhibition of Murine Factor 11
Several antisense oligonucleotides targeted to murine Factor 11 mRNA (GENBANK Accession No. NM—028066.1, incorporated herein as SEQ ID NO: 6) showing statistically significant dose-dependent inhibition were evaluated in vivo. BALB/c mice were treated with ISIS 404057 (TCCTGGCATTCTCGAGCATT, target start site 487, incorporated herein as SEQ ID NO: 10) and ISIS 404071 (TGGTAATCCACTTTCAGAGG, target start site 869, incorporated herein as SEQ ID NO: 11).
Treatment
BALB/c mice were injected with 5 mg/kg, 10 mg/kg, 25 mg/kg, or 50 mg/kg of ISIS 404057 or ISIS 404071 twice a week for 3 weeks. A control group of mice was injected with phosphate buffered saline (PBS) twice a week for 3 weeks. Mice were sacrificed 5 days after receiving the last dose. Whole liver was harvested for RNA analysis and plasma was collected for clotting analysis (PT and aPTT) and protein analysis.
RNA Analysis
RNA was extracted from liver tissue for real-time PCR analysis of Factor 11. As shown in Table 17, the antisense oligonucleotides achieved dose-dependent reduction of murine Factor 11 over the PBS control. Results are presented as percent inhibition of Factor 11, relative to control.







TABLE 17







Dose-dependent antisense inhibition of


murine Factor 11 mRNA in BALB/c mice










mg/kg
% inhibition













404057
5
40



10
64



25
85



50
95


404071
5
72



10
82



25
93



50
96









PT and aPTT Assay

Prothrombin Time (PT) and Activated Partial Thromboplastin Time (aPTT) were measured using platelet poor plasma (PPP) from mice treated with ISIS 404057 and ISIS 404071. PT and aPTT values provided in Table 18 are reported as International Normalized Ratio (INR) values. INR values for PT and aPTT were determined by dividing the PT or aPTT value for each experimental group (i.e. 5 mg/kg, 10 mg/kg, 25 mg/kg, and 50 mg/kg treatment with ISIS 404057 or ISIS 404071) by the PT or aPTT for the PBS treated group. This ratio was then raised to the power of the International Sensitivity Index (ISI) of the tissue factor used. As shown in Table 18, PT was not significantly prolonged in mice treated with ISIS 404057 or ISIS 404071. However, aPTT was prolonged in a dose-dependent manner in mice treated with ISIS 404057 and ISIS 404071. These data suggest that antisense reduction of Factor 11 affects the contact activation pathway, but not the extrinsic pathway of blood coagulation.







TABLE 18







Effect of ISIS 404071 and 404057


on PT and aPTT in BALB/c mice











Dose in





mg/kg
PT INR
aPTT INR
















ISIS 404057
5
1.00
1.07




10
0.94
1.19




25
1.02
1.27




50
1.00
1.37



ISIS 404071
5
1.06
1.09




10
1.08
1.13




25
1.06
1.35




50
1.02
2.08










Protein Analysis

Factor 11 proenzyme from the plasma of mice treated with ISIS 404071, was measured using a F11 assay based on clotting time. Clotting times were determined in duplicate with a ST4 semi-automated coagulation instrument (Diagnostica Stago, N.J.). Thirty μl of citrated sample plasma diluted 1/20 in HEPES-NaCl buffer with BSA was incubated with 30 μl aPTT reagent (Platelet Factor 3 reagent plus particulate activator) and 30 μl of citrated plasma deficient of Factor 11 (human congential, George King Bio-Medical Inc.) at 37° C. to initiate clotting. Results were interpolated on a standard curve of serially diluted citrated control murine plasma.
As shown in Table 19, treatment with ISIS 404071 resulted in a significant dose-dependent reduction of Factor 11 protein. Results are presented as percent inhibition of Factor 11, relative to PBS control.







TABLE 19







Dose-dependent inhibition of murine Factor


11 protein by ISIS 404071 in BALB/c mice










Dose in




mg/kg
% Inhibition














5
39



10
67



25
89



50
96










Example 11
In Vivo Effect of Antisense Inhibition of Murine Factor 11 in the FeCl3 Induced Venous Thrombosis (VT) Model as Compared to Warfarin
Treatment
ISIS 404071 and warfarin (COUMADIN) were evaluated in the FeCl3 induced VT mouse model. Six groups of BALB/c mice were treated with 1.25 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, or 40 mg/kg of ISIS 404071, administered subcutaneously twice a week for 3 weeks. Two days after receiving the last dose of ISIS 404071, mice were anesthetized with 150 mg/kg ketamine mixed with 10 mg/kg xylazine administered by intraperitoneal injection. An additional 6 groups of BALB/c mice were treated with 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, and 5 mg/kg of warfarin, administered intraperioneally daily for 6 days. Four hours after the last dose of warfarin, mice were anesthetized with 150 mg/kg ketamine mixed with 10 mg/kg xylazine administered by intraperitoneal injection. Two control groups of BALB/c mice were treated with PBS, administered subcutaneously twice a week for 3 weeks. Two days after the last dose of PBS, mice in both groups were anesthetized with 150 mg/kg ketamine mixed with 10 mg/kg xylazine administered by intraperitoneal injection. Thrombus formation was induced with FeCl3 in all groups of mice except the first control group.
In mice undergoing FeCl3 treatment, thrombus formation was induced by applying a piece of filter paper (2×4 mm) pre-saturated with 10% FeCl3 solution directly on the vena cava. After 3 minutes of exposure, the filter paper was removed. Thirty minutes after the filter paper application, a fixed length of the vein containing the thrombus was dissected out for platelet analysis. Liver was collected for RNA analysis.
RNA Analysis
RNA was extracted from liver tissue for real-time PCR analysis of Factor 11. Results are presented as percent inhibition of Factor 11, relative to PBS control. As shown in Table 20, treatment with ISIS 404071 resulted in significant dose-dependent reduction of Factor 11 mRNA in comparison to the PBS control. Conversely, treatment with warfarin did not result in significant reduction of Factor 11 as compared to the PBS control.







TABLE 20







Dose-dependent reduction of Factor 11 mRNA in


the FeCl3 induced venous thrombosis model












Dose in




Treatment
mg/kg
% inhibition















Warfarin
0.5
0




1
0




2
1




3
5




4
8




5
11



ISIS 404071
1.25
0




2.5
8




5
62




10
78




20
92




40
96










Quantification of Platelet Composition

Real-time PCR quantification of platelet factor-4 (PF-4) was used to quantify platelets in the vena cava as a measure of thrombus formation. Results are presented as a percentage of PF-4 in ISIS 404071 or warfarin treated mice, as compared to the two PBS-treated control groups. As shown in Table 21, treatment with ISIS 404071 resulted in a dose-dependent reduction of PF-4 in comparison to the PBS control for dosages of 5 mg/kg and higher. Treatment with warfarin resulted in a reduction of PF-4 in comparison to the PBS control for dosages of 2 mg/kg and higher. Therefore, reduction of Factor 11 by the compounds provided herein is useful for inhibiting thrombus and clot formation.







TABLE 21







Analysis of thrombus formation by real-time PCR quantification


of PF-4 in the FeCl3 induced venous thrombosis model










Dose in




mg/kg
PF-4















PBS − FeCl3

0



PBS + FeCl3

100



Warfarin
0.5
128




1
124




2
80




3
21




4
12




5
33



ISIS 404071
1.25
143




2.5
120




5
95




10
21




20
37




40
20










Example 12
In Vivo Effect of Antisense Inhibition of Murine Factor 11 Compared to Warfarin in a Tail Bleeding Assay
Treatment
Tail-bleeding was measured to observe whether treatment with ISIS 404071 or warfarin causes internal hemorrhage in mice. ISIS 404071 and warfarin (COUMADIN) were evaluated in the tail bleeding assay. Six groups of BALB/c mice were treated with 1.25 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, or 40 mg/kg of ISIS 404071, administered subcutaneously twice a week for 3 weeks. An additional 6 groups of BALB/c mice were treated with 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, and 5 mg/kg of warfarin, administered intraperioneally daily for 6 days. A separate control group of BALB/c mice was treated with PBS, administered subcutaneously twice a week for 3 weeks.
Tail-Bleeding Assay
Two days after the final treatment of ISIS 404071, warfarin, or PBS, mice were placed in a tail bleeding chamber. Mice were anesthetized in the chamber with isoflurane and a small piece of tail (approximately 4 mm from the tip) was cut with sterile scissors. The tail cut was immediately placed in a 15 mL Falcon tube filled with approximately 10 mL of 0.9% NaCl buffer solution warmed to 37° C. The blood was collected over the course of 40 minutes. The saline filled tubes were weighed both before and after bleeding. The results are provided in Table 22.
Treatment with ISIS 404071 did not affect bleeding as compared to PBS treated mice. However, warfarin did increase bleeding in mice as compared to the PBS control. Increased doses of warfarin correlated positively with increased blood loss. These data suggest that the hemorrhagic potential of the compounds provided herein is low, especially in comparison to warfarin. These data taken with the results provided in example 11 suggest inhibition of Factor 11 with the compounds described herein are useful for providing antithrombotic activity without associated bleeding risk.







TABLE 22







Tail bleeding assay in the FeCl3 induced venous thrombosis model












Dose in
Blood



Treatment
mg/kg
(g)















PBS
0
0.01



Warfarin
0.5
0.07




1
0.35




2
0.39




3
0.51




4
0.52




5
0.76



ISIS 404071
1.25
0.00




2.5
0.00




5
0.03




10
0.00




20
0.06




40
0.03










Example 13
In Vivo Effect of Antisense Inhibition of Murine Factor 11 Compared to Warfarin on PT and aPTT
Treatment
PT and aPTT were measured using PPP from mice treated with ISIS 404071 or warfarin. Six groups of BALB/c mice were treated with 1.25 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, or 40 mg/kg of ISIS 404071, administered subcutaneously twice a week for 3 weeks. An additional 6 groups of BALB/c mice were treated with 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, and 5 mg/kg of warfarin, administered intraperioneally daily for 6 days. In a control group, BALB/c mice were treated with PBS, administered subcutaneously mice twice a week for 3 weeks. Two days after the final dose was administered, PPP was collected and PT and aPTT assays were performed.
PT and aPTT Assay
PT and aPTT values provided in Table 16 are reported as International Normalized Ratio (INR) values. INR values for PT and aPTT were determined by dividing the PT or aPTT value for each experimental group (i.e. 5 mg/kg, 10 mg/kg, 25 mg/kg, and 50 mg/kg treatment with ISIS 404071) by the PT or aPTT for the PBS treated group. This ratio was then raised to the power of the International Sensitivity Index (ISI) of the tissue factor used. As shown in Table 23, PT in warfarin treated mice is significantly prolonged at every dosage. aPTT in warfarin treated mice was prolonged, particularly at dosages of 1 mg/kg and higher. ISIS 404071 did not significantly affect PT, but did prolong aPTT; however, not as significantly as in warfarin treated mice. These data suggest that ISIS 404071 affects the contact activation pathway, but not the extrinsic pathway of blood coagulation whereas warfarin affects both the contact activation pathway and the extrinsic pathway of blood coagulation.







TABLE 23







Effect of ISIS 404071 and warfarin


on PT and aPTT in BALB/c mice













Dose in





Treatment
mg/kg
PT INR
aPTT INR
















Warfarin
0.5
1.41
1.10




1
2.03
1.31




2
2.77
1.54




3
22.76
2.90




4
6.74
2.18




5
9.20
2.29



ISIS 404071
1.25
0.99
0.98




2.5
1.01
1.03




5
1.07
1.09




10
1.08
1.29




20
1.09
1.32




40
0.98
1.64










Example 14
In Vivo Effect of Antisense Inhibition of Murine Factor 11 in the FeCl3 Induced Venous Thrombosis (VT) Model as Compared to Apixaban
Treatment
ISIS 404071 and Apixaban were evaluated in the FeCl3 induced VT mouse model. Six groups of BALB/c mice were treated with 1.25 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, or 40 mg/kg of ISIS 404071, administered subcutaneously twice a week for 3 weeks. Two days after receiving the last dose of ISIS 404071, mice were anesthetized with 150 mg/kg ketamine mixed with 10 mg/kg xylazine administered by intraperitoneal injection. An additional 6 groups of BALB/c mice were treated with 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, and 5 mg/kg of Apixaban, administered subcutaneously one time. Twenty minutes after receiving Apixaban, mice were anesthetized with 150 mg/kg ketamine mixed with 10 mg/kg xylazine administered by intraperitoneal injection. Two control groups of BALB/c mice were treated with PBS, administered subcutaneously twice a week for 3 weeks. Two days after the last dose of PBS, mice in both groups were anesthetized with 150 mg/kg ketamine mixed with 10 mg/kg xylazine administered by intraperitoneal injection. Thrombus formation was induced with FeCl3 in all of the mice except the first control group.
In mice undergoing FeCl3 treatment, thrombus formation was induced by applying a piece of filter paper (2×4 mm) pre-saturated with 10% FeCl3 solution directly on the vena cava. After 3 minutes of exposure, the filter paper was removed. Thirty minutes after the filter paper application, a fixed length of the vein containing the thrombus was dissected out for platelet analysis. Liver was collected for RNA analysis.
RNA Analysis
RNA was extracted from liver tissue for real-time PCR analysis of Factor 11. Results are presented as percent inhibition of Factor 11, relative to PBS control. As shown in Table 24, treatment with ISIS 404071 resulted in significant dose-dependent reduction of Factor 11 mRNA in comparison to the PBS control. Conversely, treatment with Apixaban did not result in significant reduction of Factor 11 as compared to the PBS control.







TABLE 24







Dose-dependent reduction of Factor 11 mRNA in the


FeCl3 induced venous thrombosis model










Dose in




mg/kg
% inhibition















Apixaban
0.5
5




2
8




5
12




10
2




20
0



ISIS 404071
1.25
15




2.5
44




5
63




10
76




25
91




50
95










Quantification of Platelet Composition

Real-time PCR quantification of platelet factor-4 (PF-4) was used to quantify platelets in the vena cava as a measure of thrombus formation. As shown in Table 25, treatment with ISIS 404071 resulted in reduction of PF-4 in comparison to the PBS control. Treatment with Apixaban also resulted in reduction of PF-4, in comparison to the PBS control. Results are presented as a percentage of PF-4 in ISIS 404071 or Apixaban treated mice, as compared to the two PBS-treated control groups.







TABLE 25







Analysis of thrombus formation by real-time PCR quantification


of PF-4 in the FeCl3 induced venous thrombosis model












Dose in




Treatment
mg/kg
PF-4















PBS − FeCl3

0



PBS + FeCl3

100



Apixaban
0.5
67




2
46




5
15




10
5




20
26



ISIS 404071
1.25
42




2.5
87




5
60




10
28




25
14




50
4










Example 15
In Vivo Effect of Antisense Inhibition of Murine Factor 11 Compared to Apixaban in the Tail Bleeding Assay
Treatment
Tail bleeding was measured to observe whether treatment with ISIS 404071 or warfarin causes internal hemorrhage in mice. ISIS 404071 and Apixaban were evaluated in the tail bleeding model. Six groups of BALB/c mice were treated with 1.25 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, or 40 mg/kg of ISIS 404071, administered subcutaneously twice a week for 3 weeks. An additional 6 groups of BALB/c mice were treated with 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, and 5 mg/kg of Apixaban, administered in a single subcutaneous dose. A separate control group of BALB/c mice was treated with PBS, administered subcutaneously twice a week for 3 weeks.
Tail-Bleeding Assay
Two days after the final treatment of ISIS 404071, Apixaban, or PBS, mice were placed in a tail bleeding chamber. Mice were anesthetized in the chamber and a small piece of tail (approximately 4 mm from the tip) was cut with sterile scissors. The cut tail was immediately placed in a 15 mL Falcon tube filled with approximately 10 mL of 0.9% NaCl buffer solution warmed to 37° C. The blood was collected over the course of 40 minutes. The saline filled tubes were weighed before and after bleeding.
As shown in Table 26, treatment with ISIS 404071 did not affect bleeding as compared to PBS treated mice. However, Apixaban did increase bleeding in mice as compared to the PBS control. Increased doses of Apixaban correlated positively with increased blood loss. These data suggest that the hemorrhagic potential of the compounds provided herein is low, especially in comparison to Apixaban. These data taken with the results provided in example 14 suggest inhibition of Factor 11 with the compounds described herein are useful for providing antithrombotic without associated bleeding risk.







TABLE 26







Tail bleeding assay in BABL/c mice










mg/kg
Blood (g)















PBS
0
0.06



Apixaban
0.5
0.03




2
0.34




5
0.37




10
0.40




20
0.52



ISIS 404071
1.25
0.00




2.5
0.03




5
0.00




10
0.04




25
0.01




50
0.01










Example 16
Ex Vivo Effect of Antisense Inhibition of Murine Factor 11 in Combination with LOVENOX
Treatment
Three groups of BALB/c mice were treated with 10 mg/kg, 20 mg/kg, or 40 mg/kg of ISIS 404071, administered subcutaneously twice a week for 3 weeks. A control mouse group was treated with PBS, administered twice a week for 3 weeks. Five days after the last dose, the mice were sacrificed and plasma was collected. The low-molecular-weight (LMW) heparin, LOVENOX, was administered to the plasma ex vivo at varying concentrations of 0 μg/ml, 2.5 μg/ml, 5.0 μg/ml, and 7.5 μg/ml. PT and aPTT were measured 20 minutes after LOVENOX was administered.
PT and aPTT Assay
As shown in Table 27, treatment with LOVENOX increases PT in a dose-dependent manner. Treatment with ISIS 404071 does not significantly increase PT. PT is not significantly affected by treatment with ISIS 404071. There is no evidence of a combinational effect on PT in ISIS 404071 and LOVENOX treated plasma.







TABLE 27







Effect of combination of ISIS 404071 and


LOVENOX on PT INR in murine plasma









ISIS 404071
LOVENOX (mg/ml)












(mg/kg)
0
2.5
5.0
7.5














0
1.00
1.02
1.10
1.12


10
0.97
1.07
1.10
1.12


20
1.00
1.10
1.07
1.10


40
0.97
1.02
1.07
1.10









As shown in Table 28, treatment with LOVENOX increases aPTT in a dose-dependent manner. Treatment with ISIS 404071 also increases aPTT in a dose-dependent manner. Furthermore, the combined treatment of ISIS 404071 and LOVENOX appears to have a synergistic effect on aPTT.







TABLE 28







Effect of combination of ISIS 404071 and


LOVENOX on aPTT INR in murine plasma









ISIS 404071
LOVENOX (mg/ml)












mg/kg
0
2.5
5.0
7.5














0
1.00
1.53
2.10
2.70


10
1.14
1.76
2.39
3.20


20
1.28
1.95
2.83
3.65


40
1.52
2.66
n.d.
4.78





n.d. = no data






Example 17
In Vivo Effect of Antisense Inhibition of Murine Factor 11 in Combination with LOVENOX in the FeCl3 Induced Venous Thrombosis (VT) Model
Treatment
The combination of ISIS 404071 and LOVENOX were evaluated in the FeCl3 induced VT mouse model. Four groups of BALB/c mice were treated with 15 mg/kg, 30 mg/kg, 45 mg/kg, or 60 mg/kg of LOVENOX, administered subcutaneously once daily for 3 days. An additional 4 groups of BALB/c mice were treated with 20 mg/kg of ISIS 404071, administered subcutaneously twice weekly for 3 weeks. After the last dose of ISIS 404071, mice were treated with 15 mg/kg, 30 mg/kg, 45 mg/kg, or 60 mg/kg of LOVENOX, administered subcutaneously once daily for 3 days. Two control groups of BALB/c mice were treated with PBS, administered subcutaneously twice a week for 3 weeks. Thrombus formation was induced with FeCl3 in all of the mice except the first control group. All mice were anesthetized with 150 mg/kg of ketamine mixed with 10 mg/kg of xylazine administered by intraperitoneal injection.
In mice undergoing FeCl3 treatment, thrombus formation was induced by applying a piece of filter paper (2×4 mm) pre-saturated with 10% FeCl3 solution directly on the vena cava. After 3 minutes of exposure, the filter paper was removed. Thirty minutes after the filter paper application, a fixed length of the vein containing the thrombus was dissected out for platelet analysis.
Quantification of Platelet Composition
Real-time PCR quantification of PF-4 was used to quantify platelets in the vena cava as a measure of thrombus formation. As shown in Table 29, treatment with LOVENOX resulted in a reduction of PF-4 in comparison to the PBS control. Treatment with LOVENOX in combination with ISIS 404071 resulted in a higher reduction of PF-4 in comparison to LOVENOX alone.







TABLE 29







Analysis of thrombus formation by real-time PCR quantification


of PF-4 in the FeCl3 induced venous thrombosis model











Treatment
mg/kg
PF-4















PBS − FeCl3

0



PBS + FeCl3

100



LOVENOX
15
57




30
33




45
10




60
5



LOVENOX (+ISIS
15
0



404071)
30
0




45
11




60
5










Example 18
In Vivo Effect of Antisense Inhibition of Murine Factor 11 in Combination with LOVENOX on Bleeding
Treatment
Tail-bleeding was measured to observe whether treatment with ISIS 404071 and LOVENOX causes internal hemorrhage in mice. ISIS 404071 was administered subcutaneously at a dosage of 20 mg/kg twice a week for 3 weeks to 4 groups of BALB/c mice, and LOVENOX was administered subcutaneously at varying dosages of 15 mg/kg, 30 mg/kg, 45 mg/kg, and 60 mg/kg once daily on the last three days of ISIS 404071 treatment. In a fifth group, ISIS 404071 was administered subcutaneously to BALB/c mice at a dosage of 20 mg/kg twice a week for 3 weeks. In a sixth group, PBS was administered subcutaneously twice a week for three weeks to BALB/c mice, as a control.
Tail-Bleeding Assay
Two days after receiving their final treatment, mice were placed in a tail bleeding chamber. Mice were anesthetized in the chamber with isoflurane and a small piece of tail (approximately 4 mm from the tip) was cut with sterile scissors. The cut tail was immediately placed in a 15 mL Falcon tube filled with approximately 10 mL of 0.9% NaCl buffer solution warmed to 37° C. The blood was collected over the course of 40 minutes. The saline filled tubes were weighed both before and after bleeding.
As shown in Table 30, LOVENOX increased bleeding in mice compared to the PBS treated mice. Increased doses of LOVENOX correlated positively with increased blood loss. ISIS 404071 combined with LOVENOX did not significant increase bleeding beyond the increased blood loss shown in LOVENOX only treated mice.







TABLE 30







Tail bleeding assay comparing LOVENOX and


the combination of LOVENOX and ISIS 404071










Dose in
Blood



mg/kg
(g)















PBS

0.05



LOVENOX
15
0.11




30
0.20




45
0.27




60
0.47



LOVENOX (+ISIS
15
0.14



404071)
30
0.19




45
0.36




60
0.61










Example 19
In Vivo Effect of Antisense Inhibition of Murine Factor 11 in Combination with LOVENOX on PT and aPTT
Treatment
PT and aPTT were measured using PPP from mice treated with ISIS 404071 in combination with LOVENOX. In the first cohort, ISIS 404071 was administered subcutaneously to BALB/c mice at a dosage of 25 mg/kg twice a week for 3 weeks. Plasma was collected from these mice 5 days after receiving the last dose of ISIS 404071. In the second cohort, LOVENOX was administered subcutaneously to BALB/c mice at a dosage of 20 mg/kg once daily for three days. Plasma was collected from these mice 4 hours after receiving the last dose of LOVENOX. In the third cohort, ISIS 404071 was administered subcutaneously to BALB/c mice at a dosage of 20 mg/kg twice a week for 3 weeks, and 2 days after receiving the last dose of ISIS 404071, LOVENOX was administered subcutaneously at a dosage of 20 mg/kg once daily. Plasma was collected from these mice 4 hours after the last dose of LOVENOX. In a fourth cohort, PBS was administered subcutaneously twice a week for three weeks, as a control. Plasma was collected from these mice 5 days after the last dose.
PT and aPTT Assay
PT and aPTT values provided in Table 31 are reported as International Normalized Ratio (INR) values. As shown in Table 31, PT is not significantly affected by treatment with ISIS 404071, LOVENOX, or treatment with ISIS 40471 combined with LOVENOX. These data suggest that there is no combinational effect on PT by ISIS 404071 combined with LOVENOX. Also shown in Table 31, treatment with LOVENOX and treatment with ISIS 404071 combined with LOVENOX increase aPTT. These data suggest that the combined treatment of ISIS 404071 and LOVENOX has an additive effect on aPTT.







TABLE 31







Effect of combination of ISIS 404071 and


LOVENOX on PT and aPTT in murine plasma










PT INR
aPTT INR















ISIS 404071
0.95
1.31



LOVENOX
1.04
2.04



404071 + LOVENOX
1.04
2.58










Example 20
In Vivo Effect of Antisense Inhibition of Murine Factor 11 in Combination with Apixaban on PT and aPTT
Treatment
PT and aPTT were measured using PPP from mice treated with ISIS 404071 in combination with Apixaban. In the first cohort, ISIS 404071 was administered subcutaneously to BALB/c mice at a dosage of 25 mg/kg twice a week for 3 weeks. Plasma was collected from these mice 5 days after receiving the last dose of ISIS 404071. In the second cohort, Apixaban was administered subcutaneously to BALB/c mice at a dosage of 6 mg/kg twice daily for three days. Plasma was collected from these mice 20 minutes after receiving the last dose of Apixaban. In the third cohort, ISIS 404071 was administered subcutaneously to BALB/c mice at a dosage of 20 mg/kg twice a week for 3 weeks, and Apixaban was administered subcutaneously at a dosage of 6 mg/kg twice daily on the last three days of ISIS 404071 treatment. Plasma was collected from these mice 20 minutes after receiving the last dose of Apixaban. In a fourth cohort, PBS was administered subcutaneously twice a week for three weeks, as a control. Plasma was collected 5 days after the last dose of PBS.
PT and aPTT Assay
PT and aPTT values provided in Table 32 are reported as International Normalized Ratio (INR) values. As shown in Table 32, PT is not significantly affected by treatment with ISIS 404071. However, Apixaban and Apixaban combined with ISIS 404071 increased PT. Also shown in Table 32, Apixaban, ISIS 404071, and ISIS 404071 combined with Apixaban increase aPTT.







TABLE 32







Effect of combination of ISIS 404071 and


Apixaban on PT and aPTT in murine plasma










PT INR
aPTT INR















ISIS 404071
0.95
1.31



Apixaban
3.25
1.44



404071 + Apixaban
3.50
2.26










Example 21
In Vivo Effect of Antisense Inhibition of Murine Factor 11 in Combination with Warfarin on PT and aPTT
Treatment
PT and aPTT were measured using PPP from mice treated with ISIS 404071 in combination with warfarin. Two groups of BALB/c mice were treated with either 25 mg/kg or 50 mg/kg of ISIS 404071, administered subcutaneously twice a week for 3 weeks. Plasma was collected from each group 5 days after the last dose was administered. In a third group, BALB/c mice were treated with 2 mg/kg of warfarin once daily for 5 days. Plasma was collected 6 hours after the last dose of warfarin was administered. Two additional groups of BALB/c mice were treated with either 25 mg/kg or 50 mg/kg of ISIS 404071, administered subcutaneously twice a week for 3 weeks and warfarin was administered subcutaneously at a dosage of 2 mg/kg once daily on the last 5 days of ISIS 404071 treatment. Plasma was collected from each group 6 hours after the last warfarin treatment. In a final group of BALB/c mice, PBS was administered subcutaneously twice a week for three weeks, as a control. Plasma was collected 5 days after the last PBS treatment.
PT and aPTT Assay
PT and aPTT values provided in Table 33 are reported as International Normalized Ratio (INR) values. As shown in Table 33, PT is not affected by treatment with PBS or ISIS 404071 at either dosage. However, treatment with 2 mg/kg warfarin, 25 mg/kg ISIS 404071 in combination with 2 mg/kg warfarin, and 50 mg/kg ISIS 404071 in combination with 2 mg/kg warfarin increase PT. These data suggest that the combined treatment of ISIS 404071 and warfarin has an additive effect on PT. Also shown in Table 33, aPTT is affected by treatment with ISIS 404071 and warfarin. The combination of ISIS 404071 and warfarin show an increase in aPTT greater than either drug alone. These data suggest that the combined treatment of ISIS 404071 and warfarin has a synergistic effect on aPTT.







TABLE 33







Effect of combination of ISIS 404071 and


warfarin on PT and aPTT in murine plasma











Dose in





mg/kg
PT INR
aPTT INR
















ISIS 404071
25
0.98
1.37




50
0.93
1.49



Warfarin
2
21.33
2.52



ISIS 404071(+Warfarin)
25
25.77
4.45




50
36.33
4.75










Example 22
In Vivo Antithrombotic Effect of Antisense Inhibition of Murine FACTOR 11 on Mesenteric Vein Thrombosis in Mice
Treatment
In a first cohort, ISIS 404071 was administered subcutaneously to C57BL/6 mice twice a week for three weeks at a dose of 50 mg/kg. In a second cohort, a control oligonucleotide, ISIS 405277 (AAGGACCTACACTATGGAAT; antisense oligonucleotide for Factor 2), incorporated herein as SEQ ID NO: 12 was administered subcutaneously to C57B1/6 mice twice a week for three weeks at a dose of 50 mg/kg.
Platelet Preparation
Blood was collected from the retro-orbital venous plexus of naïve C57BL/6 mice by puncture and collected in polypropylene tubes containing 300 μl of heparin (30 U/ml). Platelet rich plasma (PRP) was obtained by centrifugation at 1000 rpm for 5 min. The PRP was transferred to fresh tubes containing 2 μl of Prostaglandin I2 (PGI2) (2 μg/ml) and incubated at 37° C. for 5 min. After centrifugation at 2600 rpm, pellets were resuspended in 1 ml modified Tyrode's-HEPES buffer (137 mM NaCl, 0.3 mM Na2HPO4, 2 mM KCl, 12 mM NaHCO3, 5 mM HEPES, 5 mM glucose, 0.35% BSA, pH 7.2) containing 2 μl of PGI2 and incubated at 37° C. for 5 min. The suspended pellet was centrifuged at 2600 rpm for 5 min. To remove PGI2, the washing step was repeated twice and platelets were fluorescently labeled with calcein AM 2.5 μg/mL (Molecular Probes, Eugene, Oreg.) for 10 min at room temperature.
Intravital Microscopy for Thrombosis
Fluorescently-labeled platelets were injected intravenously in ISIS 404071 treated and control oligonucleotide treated C57BL/6 mice. The mice were anaesthetized with 2.5% avertin, and an incision was made through the abdominal wall to expose mesenteric veins 250-300-μm in diameter and having a shear rate of approximately 150 s−1. The exposed mesentery was kept moist throughout the experiment by periodic superfusion with warmed (3TC) PBS. The mesentery was transluminated with a 12V, 100 W, DC stabilized source. Veins were visualized using a Zeiss (Germany) Axiovert 135 inverted microscope (Objective 32×) connected to an SVHS video recorder (AG-6730; Panasonic, Tokyo, Japan) using a CCD video camera (Hamamatsu Photonic Systems, Hamamatsu City, Japan). Centerline erythrocyte velocity (Vrbc) was measured using an optical Doppler velocimeter (Microcirculation Research Institute, Texas A&M College of Medicine, College Station, Tex.). Venular shear rate (τ) was calculated based on Poiseuille's Law for a newtonian fluid, τ=8(Vmean/Dv), where Dv is the diameter of the venule and Vmean is estimated from the measured Vrbc using the empirical correlation Vmean=Vrbc/1.6.
Results Analysis
Mesenteric vein thrombosis was performed two days after the last antisense oligonucleotide injection. Thrombosis was induced by applying Whatman paper soaked in a 10% FeCl3 solution for 5 minutes on the mesenteric vein. The vein was monitored for 40 minutes, or until occlusion. The elapsed time before the first thrombus 30-50 μm in diameter and the elapsed time before blood stopped flowing for 30 seconds were observed.
Thrombus formation (30 μm in diameter) occurred in mice treated with ISIS 404071 at 14.8±1.7 minutes. Thrombus formation (30 μm in diameter) occurred in control mice at 8.9±0.6 minutes. Occlusive thrombi formed in control mice at 19.3±0.8 min and all injured venules occluded. In contrast, the majority of the veins in ISIS 404071 treated mice did not occlude when observation was terminated 40 minutes after injury and those veins showing occlusion. The only vein showing occlusion in the ISIS 404071 treated mice occluded at 29.5 minutes and reopened after 5 minutes, prior to the end of the study.
Example 23
In Vivo Sense-Oligonucleotide-Antidote for Antisense Inhibition of Murine Factor 11 in BALB/c Mice
Treatment
The effect of the specific sense oligonucleotide to ISIS 404071 as an antidote was tested in BALB/c mice. In a first cohort, ISIS 404071 was administered subcutaneously to BALB/c mice twice a week for three weeks at a dose of 40 mg/kg. In a second cohort, ISIS 404057 was administered subcutaneously to BALB/c mice twice a week for three weeks at a dose of 40 mg/kg. The ISIS 404071 specific antidote, ISIS 418026 (CCTCTGAAAGTGGATTACCA; complementary to ISIS 404071), incorporated herein as SEQ ID NO: 13, was administered to both cohorts subcutaneously in a single injection of 90 mg/kg 48 hours after the final treatment of ISIS 404071 or 404057. In a third cohort, ISIS 404071 was administered subcutaneously to BALB/c mice twice a week for three weeks at a dose of 40 mg/kg. Following the last treatment of ISIS 404071, mice were injected subcutaneously injected with PBS. In a fourth cohort, ISIS 404057 was administered subcutaneously to BALB/c mice twice a week for three weeks at a dose of 40 mg/kg. Following the last treatment of ISIS 404057, mice were injected subcutaneously injected with PBS. Following antidote administration, a set of 4 mice from each cohort were sacrificed at 12 hours, 1 day, 2 days, 3 days, 7 days, and 14 days. Whole liver was collected for RNA analysis and PPP was collected for aPTT analysis.
RNA Analysis
RNA was extracted from liver tissue for real-time PCR analysis of Factor 11. Results are presented as percent inhibition of Factor 11, relative to PBS control. As shown in Table 34, mice treated with ISIS 404071 without antidote showed progressive decrease in inhibition over the 14 day observation period. However, mice treated with ISIS 404071 and antidote showed an accelerated decrease in inhibition over the 14 day observation period in comparison to mice which did not receive antidote. Also shown in Table 34, treatment with ISIS 418026 had no effect on inhibition of Factor 11 mRNA expression in ISIS 404057 treated mice.







TABLE 34







Percent inhibition of mouse Factor


11 mRNA compared to PBS control














12 hours
1 day
2 days
3 days
7 days
14 days

















ISIS 404071
93
90
89
88
81
67


ISIS 404071 +
90
87
72
66
57
31


ISIS 418026


ISIS 404057
n.d.
n.d.
n.d.
95
n.d.
n.d.


ISIS 404057 +
n.d.
n.d.
n.d.
97
n.d.
n.d.


ISIS 418026





n.d. = no data






aPTT Assay

As shown in Table 35, mice treated with ISIS 404071 and antidote (ISIS 418026) showed progressive decrease of aPTT over the 14 day observation period compared to mice treated with ISIS 404071 without antidote.







TABLE 35







Effect of antidote treatment on aPTT INR














12 hours
1 day
2 day
3 day
7 day
14 day

















ISIS 404071
1.51
1.30
1.35
1.27
1.18
1.05


ISIS 404071 +
1.45
1.23
1.16
1.15
1.10
0.95


ISIS 418026









Example 24
In Vivo Factor 7a Protein-Antidote for Antisense Inhibition of Murine Factor 11 in BALB/c Mice
Treatment
The effect of human Factor 7a (Factor VIIa) protein as an antidote for ISIS 404071 was tested in BALB/c mice. Two experimental groups of BALB/c mice were treated with 20 mg/kg of ISIS 404071, administered subcutaneously twice a week for 3 weeks. Two control groups of BALB/c mice were treated with PBS, administered subcutaneously twice a week for 3 weeks. Thrombus formation was induced with FeCl3 in all of the mice except the first control group. Fifteen minutes before FeCl3 treatment, the first experimental group was treated with 5 μg/kg of human Factor 7a protein antidote (product no. 407act, American Diagnostica Inc.). Two days after their last dose, all mice were anesthetized with 150 mg/kg of ketamine mixed with 10 mg/kg of xylazine administered by intraperitoneal injection.
In mice undergoing FeCl3 treatment, thrombus formation was induced by applying a piece of filter paper (2×4 mm) pre-saturated with 10% FeCl3 solution directly on the vena cava. After 3 minutes of exposure, the filter paper was removed. Thirty minutes after the filter paper application, a fixed length of the vein containing the thrombus was dissected out for platelet analysis.
Quantification of Platelet Composition
Real-time PCR quantification of platelet factor-4 (PF-4) was used to quantify platelets in the vena cava as a measure of thrombus formation. Results are presented as a percentage of PF-4 in antidote treated and untreated mice, as compared to the two PBS-treated control groups. As shown in Table 36, animals treated with human Factor 7a protein antidote expressed more PF-4 in comparison to animals treated with ISIS 404071 alone. These data indicate that human Factor 7a is successful in rescuing the effect of antisense oligonucleotide inhibition.







TABLE 36







Analysis of thrombus formation by real-time PCR quantification


of PF-4 in the FeCl3 induced venous thrombosis model










Treatment
PF-4














PBS − FeCl3
0



PBS + FeCl3
100



ISIS 404071
18



ISIS 404071 + hFV7a
68










Example 25
In Vivo Antisense Inhibition of Murine Factor 11 in the Collagenase-Induced Intracerebral Hemorrhage Model
Treatment
ISIS 404071 and warfarin (COUMADIN) were examined in the collegenase-induced intracerebral hemorrhage model. In a first cohort, ISIS 404071 was administered subcutaneously to BALB/c mice twice a week for two weeks at a dose 40 mg/kg. In a second cohort, warfarin was administered intraperioneally to mice twice a week for two weeks at a dose of 2 mg/kg. In a third cohort, ISIS 421208 (TCGGAAGCGACTCTTATATG, 8 mismatches to murine Factor 11, incorporated herein as SEQ ID NO: 14) was administered subcutaneously to BALB/c mice twice a week for two weeks at a dose 40 mg/kg. In a fourth cohort, PBS was administered to BALB/c mice twice a week for two weeks.
Two days after receiving their final dose, all mice in all cohorts were anesthetized with 5 μg/g of avertin. Next, the mice were injected at −1 mm AP, 1 mm R ML, −4 mm DV from bregma flat skull with a 10 μL Hamilton syringe containing 0.075 U collagenase (150 U/mL). Collagenase was delivered over 5 minutes and the needle was kept in place for an additional 5 minutes to prevent reflux. The mice were then analyzed for hemorrhagic size, neurologic deficit score, and mortality.
Table 37 presents the hemorrhage volume detected in mice after collagenase treatment, Table 38 presents the neurologic deficit score of the mice, and Table 39 presents the mortality rate of the mice. Neurological deficit is measured by a standard scoring system where no deficiency is zero and severe deficit is five. Collectively, the data suggest that ISIS 404071 did not have a significant effect on the hemorrhagic size, neurologic deficit score, or mortality of the mice. Thus, risk of intracerebral hemorrhage (a risk factor for warfarin treated individuals) is significantly reduced in ISIS 404071 treated mice in comparison to warfarin treated mice.







TABLE 37







Hemorrhagic volume after collagenase treatment









Volume



(mm3)














PBS
51



ISIS 421208
41



ISIS 404071
38

















TABLE 38







Neurologic Deficit Score after collagenase treatment









Score














PBS
2.4



ISIS 421208
2.0



ISIS 404071
3.8

















TABLE 39







Mortality after collagenase treatment









% mortality














PBS
0



ISIS 421208
0



ISIS 404071
20



Warfarin
80










Example 26
In Vivo Effect of Antisense Inhibition of Murine Factor 11 in Combination with PLAVIX in the FeCl3 Induced Venous Thrombosis (VT) Model
Treatment
The combination of ISIS 404071 and PLAVIX was evaluated in the FeCl3 induced VT mouse model. Four groups of eight BALB/c mice, weighing approximately 25 g each, were treated with 6.25 mg/kg, 12.50 mg/kg, 25.00 mg/kg, or 50.00 mg/kg of PLAVIX. Mice were given two doses of PLAVIX on day one and one dose of PLAVIX on day two, two hours before surgery.
An additional four groups of eight BALB/c mice, weighing approximately 25 g each, were treated with 20 mg/kg of ISIS 404071, administered subcutaneously twice a week for three weeks. After the last dose of ISIS 404071, mice were treated with 6.25 mg/kg, 12.50 mg/kg, 25.00 mg/kg, or 50.00 mg/kg of PLAVIX. Two doses of PLAVIX were administered to the mice on day one and one dose of PLAVIX was administered on day two, two hours before surgery.
Two control groups of eight BALB/c mice, weighing approximately 25 g each, were not treated with ISIS 404071 or PLAVIX. An additional two control groups of eight BALB/c mice, weighing approximately 25 g each, were treated with 20 mg/kg of ISIS 404071, administered subcutaneously twice a week for three weeks, but were not treated with PLAVIX. Thrombus formation was induced with FeCl3 in all of the mice except the first and third control groups. All mice were anesthetized with 150 mg/kg of ketamine mixed with 10 mg/kg of xylazine administered by intraperitoneal injection.
In mice undergoing FeCl3 treatment, thrombus formation was induced by applying a piece of filter paper (2×4 mm) pre-saturated with 10% FeCl3 solution directly on the inferior vena cava. After 3 minutes of exposure, the filter paper was removed. Thirty minutes after the filter paper application, a fixed length of the vein containing the thrombus was dissected out for platelet analysis.
Quantification of Platelet Composition
Real-time PCR quantification of PF-4 was used to quantify platelets in the vena cava as a measure of thrombus formation. As shown in Table 40, treatment with PLAVIX resulted in a reduction of PF-4 in comparison to the PBS control. Treatment with PLAVIX in combination with ISIS 404071 resulted in a higher reduction of PF-4 in comparison to PLAVIX alone. Therefore, the combination of anti-platelet therapy with Factor 11 ASO increases antithrombotic activity. Data is presented as percent of PF-4 mRNA as compared to the PBS+FeCl3 control.







TABLE 40







Analysis of thrombus formation by real-time PCR quantification


of PF-4 in the FeCl3 induced venous thrombosis model













ISIS 404071
PLAVIX




Treatment
mg/kg
mg/kg
PF-4
















PBS − FeCl3
0
0
29



PBS + FeCl3
0
0
100



PLAVIX only
0
6.25
59




0
12.50
37




0
25.00
30




0
50.00
30



ISIS 404071 − FeCl3
20
0
27



ISIS 404071 + FeCl3
20
0
40



PLAVIX (+ISIS
20
6.25
35



404071)
20
12.50
38




20
25.00
25




20
50.00
35










Example 27
In Vivo Effect of Antisense Inhibition of Murine Factor 11 in Combination with PLAVIX on Bleeding
Treatment
Tail-bleeding was measured to observe whether treatment with ISIS 404071 in combination with PLAVIX causes an increase in bleeding tendency. ISIS 404071 was administered subcutaneously at a dosage of 20 mg/kg twice a week for 3 weeks to 5 groups of eight BALB/c mice. After the last dose of ISIS 404071, mice were treated with 0 mg/kg, 6.25 mg/kg, 12.50 mg/kg, 25.00 mg/kg, or 50.00 mg/kg of PLAVIX. Two doses of PLAVIX were administered to the mice on day one and one dose of PLAVIX was administered on day two, two hours before bleeding.
An additional 5 groups of eight BABL/c mice were treated similarly, except they did not receive ISIS 404071 injections.
Tail-Bleeding Assay
Two hours after receiving their final treatment, mice were placed in a tail bleeding chamber. Mice were anesthetized in the chamber with isoflurane and a small piece of tail (approximately 4 mm from the tip) was cut with sterile scissors. The cut tail was immediately placed in a 15 mL Falcon tube filled with approximately 10 mL of 0.9% NaCl buffer solution warmed to 37° C. The blood was collected for the course of 40 minutes. The saline filled tubes were weighed both before and after bleeding.
Taken with the results of Example 26, these data show that the combination of anti-platelet therapy with Factor 11 ASO increases antithrombotic activity without increased bleeding risk.







TABLE 41







Tail bleeding assay comparing PLAVIX and


the combination of PLAVIX and ISIS 404071













ISIS 404071
PLAVIX
Blood



Treatment
mg/kg
mg/kg
(g)
















No treatment
0
0
0.040



PLAVIX only
0
 6.25 mg/kg
0.075




0
12.50 mg/kg
0.205




0
25.00 mg/kg
0.524




0
50.00 mg/kg
0.628



ISIS 404071 only
20 mg/kg
0
0



PLAVIX (+ISIS
20 mg/kg
 6.25 mg/kg
0.065



404071)
20 mg/kg
12.50 mg/kg
0.300




20 mg/kg
25.00 mg/kg
0.401




20 mg/kg
50.00 mg/kg
0.577










Example 28
In Vivo Effect of a Factor Xa Small Molecule Inhibitor in Combination with PLAVIX on Bleeding
Treatment
Tail-bleeding was measured to observe whether treatment with a Factor 10a small molecule in combination with PLAVIX causes an increase in bleeding tendency. Five groups of eight BALB/c mice were treated with 0 mg/kg, 6.25 mg/kg, 12.50 mg/kg, 25.00 mg/kg, or 50.00 mg/kg of PLAVIX. Mice were given two doses of PLAVIX on day one and one dose of PLAVIX on day two, two hours before bleeding.
An additional five groups of eight BALB/c mice were treated with 0 mg/kg, 6.25 mg/kg, 12.50 mg/kg, 25.00 mg/kg, or 50.00 mg/kg of PLAVIX. Mice were given two doses of PLAVIX on day one and one dose of PLAVIX on day two, two hours before bleeding. These mice were also treated with 0.5 mg/kg of Apixaban, a small molecule Factor 10a inhibitor, intraperitoneally one time 20 minutes before bleeding.
Tail-Bleeding Assay
Two hours after receiving their final treatment, mice were placed in a tail bleeding chamber. Mice were anesthetized in the chamber with isoflurane and a small piece of tail (approximately 4 mm from the tip) was cut with sterile scissors. The cut tail was immediately placed in a 15 mL Falcon tube filled with approximately 10 mL of 0.9% NaCl buffer solution warmed to 37° C. The blood was collected for the course of 40 minutes. The saline filled tubes were weighed both before and after bleeding.
As shown below in Table 42, these data show that the combination of anti-platelet therapy with a small molecule Factor 10a inhibitor, such as Apixaban, increases bleeding risk. Therefore, treatment with the combination of anti-platelet therapy with a Factor 11 ASO provides a better safety profile in comparison to the safety profile of a combination of anti-platelet therapy with a small molecule Factor 10a inhibitor.







TABLE 42







Tail bleeding assay comparing PLAVIX, Apixaban,


and the combination of PLAVIX and Apixaban













Apixaban
PLAVIX
Blood



Treatment
mg/kg
mg/kg
(g)







No treatment
0
0
0.002



PLAVIX only
0
 6.25 mg/kg
0.061




0
12.50 mg/kg
0.149




0
25.00 mg/kg
0.246




0
50.00 mg/kg
0.258



Apixaban only
0.5 mg/kg
0
0.004



PLAVIX (+Apixaban)
0.5 mg/kg
 6.25 mg/kg
0.258




0.5 mg/kg
12.50 mg/kg
0.252




0.5 mg/kg
25.00 mg/kg
0.361




0.5 mg/kg
50.00 mg/kg
0.363










Example 29
Time Course of In Vivo, Antisense-Mediated Reduction of Murine Factor 11 and Corresponding Anticoagulation in Blood
Treatment
The time course of antisense-mediated reduction of murine Factor 11 mRNA was observed in BALB/c mice. One dose of 50 mg/kg ISIS 404071 was administered subcutaneously to BALB/c mice. Following ISIS 404071 administration, mice were sacrificed at 12 hours, 1 day, 2 days, 3 days, 4 days, 7 days, 14 days, 28 days, and 56 days. Whole liver was collected for RNA analysis and PPP was collected for aPTT analysis. A control group of mice was treated with one subcutaneous dose of PBS.
RNA Analysis
RNA was extracted from liver tissue for real-time PCR analysis of Factor 11. Results are presented relative to PBS control. Mice treated with ISIS 404071 showed significant Factor 11 mRNA down-regulation by day 1. Mice began regaining Factor 11 mRNA expression by day 14. Mice regained full Factor 11 mRNA expression by day 28 and results from day 56 indicate that Factor mRNA was maintained at pre-treatment levels. Therefore, ISIS 404071 treated mice did not experience a rebound effect.
The rebound effect has been previously observed in antibody-mediated reduction of Factor 11 (Blood, First Edition Paper, prepublished online Oct. 22, 2008; Prevention of vascular graft occlusion and thrombus-associated thrombin generation by inhibition of factor XI). Because over expression of Factor 11 can be damaging by leading to increased coagulation, these data suggest that antisense-mediated inhibition of Factor 11 is safer than antibody-mediated inhibition of Factor 11 since antisense-mediated inhibition of Factor 11 does not rebound.
aPTT Assay
aPTT values provided in Table 43 are reported as International Normalized Ratio (INR) values. INR values for aPTT were determined by dividing the aPTT value for ISIS 404071 treated mice by the aPTT for the PBS treated group. This ratio was then raised to the power of the International Sensitivity Index (ISI) of the tissue factor used. As shown in Table 43, mice treated with ISIS 404071 showed progressive decrease of aPTT until day 4 and then progressive increase to pre-treatment levels from day 7 to day 28.







TABLE 43







Effect of ISIS 404071 treatment on aPTT INR*

















12 hours
day 1
day 2
day 3
day 4
day 7
day 14
day 28
day 56




















ISIS 404071
0
1.02
1.12
1.29
1.30
1.25
1.11
1.02
0





*values in Table 43 are approximate






Example 30
Antisense Inhibition of Human Factor 11 in HepG2 Cells by Oligonucleotides Designed by Microwalk
Additional gapmers were designed based on ISIS 416850 and ISIS 416858 (see Table 8 above). These gapmers were shifted slightly upstream and downstream (i.e. “microwalk”) of ISIS 416850 and ISIS 416858. The microwalk gapmers were designed with either 5-8-5 MOE or 6-8-6 MOE motifs.
These microwalk gapmers were tested in vitro. Cultured HepG2 cells at a density of 20,000 cells per well were transfected using electroporation with 8,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Factor 11 mRNA levels were measured by quantitative real-time PCR. Factor 11 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of Factor 11, relative to untreated control cells.
ISIS 416850 and ISIS 416858, as well as selected gapmers from Tables 1 and 8 (i.e., ISIS 412206, ISIS 412223, ISIS 412224, ISIS 412225, ISIS 413481, ISIS 413482, ISIS 416825, ISIS 416848, ISIS 416849, ISIS 416850, ISIS 416851, ISIS 416852, ISIS 416853, ISIS 416854, ISIS 416855, ISIS 416856, ISIS 416857, ISIS 416858, ISIS 416859, ISIS 416860, ISIS 416861, ISIS 416862, ISIS 416863, ISIS 416864, ISIS 416865, ISIS 416866, and ISIS 416867) were retested in vitro along with the microwalk gapmers under the same condition as described above.
The chimeric antisense oligonucleotides in Table 44 were designed as 5-10-5 MOE, 5-8-5 and 6-8-6 MOE gapmers. The first two listed gapmers in Table 44 are the original gapmers (ISIS 416850 and ISIS 416858) from which ISIS 445493-445543 were designed via microwalk, and are designated by an asterisk. The 5-10-5 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of ten 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising five nucleotides each. The 5-8-5 gapmers are 18 nucleotides in length, wherein the central gap segment is comprised of eight 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising five nucleotides each. The 6-8-6 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of eight 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising six nucleotides each. For each of the motifs (5-10-5, 5-8-5 and 6-8-6), each nucleotide in the 5′ wing segment and each nucleotide in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytidine residues throughout each gapmer are 5-methylcytidines. “Human Target start site” indicates the 5′-most nucleotide to which the gapmer is targeted in the human sequence. “Human Target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted in the human sequence. Each gapmer listed in Table 44 is targeted to SEQ ID NO: 1 (GENBANK Accession No. NM—000128.3). Each gapmer is Table 44 is also fully cross-reactive with the rhesus monkey Factor 11 gene sequence, designated herein as SEQ ID NO: 274 (exons 1-15 GENBANK Accession No. NW—001118167.1). ‘Rhesus monkey start site’ indicates the 5′-most nucleotide to which the gapmer is targeted in the rhesus monkey sequence. ‘Rhesus monkey stop site’ indicates the 3′-most nucleotide to which the gapmer is targeted to the rhesus monkey sequence.
As shown in Table 44, all of the microwalk designed gapmers targeted to the target region beginning at the target start site 1275 and ending at the target stop site 1317 (i.e. nucleobases 1275-1317) of SEQ ID NO: 1 exhibited at least 60% inhibition of Factor 11 mRNA. Similarly, all of the re-tested gapmers from Tables 1 and 8 exhibited at least 60% inhibition.
Several of the gapmers exhibited at least 70% inhibition, including ISIS numbers: ISIS 412206, 412224, 412225, 413481, 413482, 416825, 416848, 416849, 416850, 416851, 416852, 416853, 416854, 416855, 416856, 416857, 416858, 416859, 416860, 416861, 416862, 416863, 416864, 416865, 416866, 416867, 445494, 445495, 445496, 445497, 445498, 445499, 445500, 445501, 445502, 445503, 445504, 445505, 445506, 445507, 445508, 445509, 445510, 445511, 445512, 445513, 445514, 445515, 445516, 445517, 445518, 445519, 445520, 445521, 445522, 445523, 445524, 445525, 445526, 445527, 445528, 445529, 445530, 445531, 445532, 445533, 445534, 445535, 445536, 445537, 455538, 445539, 445540, 445541, 445542, and 445543.
Several of the gapmers exhibited at least 80% inhibition, including ISIS numbers: ISIS 412206, 412224, 412225, 413481, 413482, 416825, 416848, 416849, 416850, 416851, 416852, 416853, 416854, 416855, 416856, 416857, 416858, 416859, 416860, 416861, 416862, 416863, 416864, 416865, 416866, 416867, 445494, 445495, 445496, 445497, 445498, 445500, 445501, 445502, 445503, 445504, 445505, 445506, 445507, 445508, 445509, 445510, 445513, 445514, 445519, 445520, 445521, 445522, 445525, 445526, 445529, 445530, 445531, 445532, 445533, 445534, 445535, 445536, 455538, 445541, and 445542.
Several of the gapmers exhibited at least 90% inhibition, including ISIS numbers: ISIS 412206, 416825, 416850, 416857, 416858, 416861, 445522, and 445531.







TABLE 44







Inhibition of human Factor 11 mRNA levels by chimeric antisense


oligonucleotides targeted to SEQ ID NO: 1


(GENBANK Accession No. NM_000128.3)






















Rhesus
Rhesus



Human
Human



SEQ
monkey
monkey



Start
Stop
Sequence
Percent

ID
Start
Stop


ISIS No.
Site
Site
(5′ to 3′)
inhibition
Motif
No.
Site
Site


















*416850
1278
1297
TGCACAGTTT
91
5-10-5
215
1277
1296





CTGGCAGGCC







 


*416858
1288
1307
ACGGCATTGG
90
5-10-5
223
1287
1306





TGCACAGTTT







 


416825
680
699
GCCCTTCATGT
90
5-10-5
190
679
698





CTAGGTCCA







 


412206
738
757
CCGTGCATCTT
91
5-10-5
34
737
756





TCTTGGCAT







 


412223
1275
1294
ACAGTTTCTG
62
5-10-5
51
1274
1293





GCAGGCCTCG







 


445493
1275
1294
ACAGTTTCTG
69
6-8-6
51
1274
1293





GCAGGCCTCG







 


445518
1275
1292
AGTTTCTGGC
75
5-8-5
242
1274
1291





AGGCCTCG







 


416848
1276
1295
CACAGTTTCT
87
5-10-5
213
1275
1294





GGCAGGCCTC







 


445494
1276
1295
CACAGTTTCT
85
6-8-6
213
1275
1294





GGCAGGCCTC







 


445519
1276
1293
CAGTTTCTGG
81
5-8-5
243
1275
1292





CAGGCCTC







 


416849
1277
1296
GCACAGTTTC
88
5-10-5
214
1276
1295





TGGCAGGCCT







 


445495
1277
1296
GCACAGTTTC
89
6-8-6
214
1276
1295





TGGCAGGCCT







 


445520
1277
1294
ACAGTTTCTG
82
5-8-5
244
1276
1293





GCAGGCCT







 


445496
1278
1297
TGCACAGTTT
87
6-8-6
215
1277
1296





CTGGCAGGCC







 


445521
1278
1295
CACAGTTTCT
87
5-8-5
245
1277
1294





GGCAGGCC







 


416851
1279
1298
GTGCACAGTT
89
5-10-5
216
1278
1297





TCTGGCAGGC







 


445497
1279
1298
GTGCACAGTT
81
6-8-6
216
1278
1297





TCTGGCAGGC







 


445522
1279
1296
GCACAGTTTC
91
5-8-5
246
1278
1295





TGGCAGGC







 


413481
1280
1299
GGTGCACAGT
82
5-10-5
114
1279
1298





TTCTGGCAGG







 


445498
1280
1299
GGTGCACAGT
83
6-8-6
114
1279
1298





TTCTGGCAGG







 


445523
1280
1297
TGCACAGTTT
73
5-8-5
267
1279
1296





CTGGCAGG







 


416852
1281
1300
TGGTGCACAG
87
5-10-5
217
1280
1299





TTTCTGGCAG







 


445499
1281
1300
TGGTGCACAG
75
6-8-6
217
1280
1299





TTTCTGGCAG







 


445524
1281
1298
GTGCACAGTT
75
5-8-5
247
1280
1297





TCTGGCAG







 


416853
1282
1301
TTGGTGCACA
84
5-10-5
218
1281
1300





GTTTCTGGCA







 


445500
1282
1301
TTGGTGCACA
81
6-8-6
218
1281
1300





GTTTCTGGCA







 


445525
1282
1299
GGTGCACAGT
85
5-8-5
248
1281
1298





TTCTGGCA







 


416854
1283
1302
ATTGGTGCAC
86
5-10-5
219
1282
1301





AGTTTCTGGC







 


445501
1283
1302
ATTGGTGCAC
83
6-8-6
219
1282
1301





AGTTTCTGGC







 


445526
1283
1300
TGGTGCACAG
81
5-8-5
249
1282
1299





TTTCTGGC







 


416855
1284
1303
CATTGGTGCA
85
5-10-5
220
1283
1302





CAGTTTCTGG







 


445502
1284
1303
CATTGGTGCA
83
6-8-6
220
1283
1302





CAGTTTCTGG







 


445527
1284
1301
TTGGTGCACA
70
5-8-5
250
1283
1300





GTTTCTGG







 


412224
1285
1304
GCATTGGTGC
84
5-10-5
52
1284
1303





ACAGTTTCTG







 


445503
1285
1304
GCATTGGTGC
89
6-8-6
52
1284
1303





ACAGTTTCTG







 


445528
1285
1302
ATTGGTGCAC
73
5-8-5
251
1284
1301





AGTTTCTG







 


416856
1286
1305
GGCATTGGTG
84
5-10-5
221
1285
1304





CACAGTTTCT







 


445504
1286
1305
GGCATTGGTG
87
6-8-6
221
1285
1304





CACAGTTTCT







 


445529
1286
1303
CATTGGTGCA
85
5-8-5
252
1285
1302





CAGTTTCT







 


416857
1287
1306
CGGCATTGGT
91
5-10-5
222
1286
1305





GCACAGTTTC







 


445505
1287
1306
CGGCATTGGT
89
6-8-6
222
1286
1305





GCACAGTTTC







 


445530
1287
1304
GCATTGGTGC
83
5-8-5
253
1286
1303





ACAGTTTC







 


445506
1288
1307
ACGGCATTGG
86
6-8-6
223
1287
1306





TGCACAGTTT







 


445531
1288
1305
GGCATTGGTG
90
5-8-5
254
1287
1304





CACAGTTT







 


416859
1289
1308
GACGGCATTG
85
5-10-5
224
1288
1307





GTGCACAGTT







 


445507
1289
1308
GACGGCATTG
85
6-8-6
224
1288
1307





GTGCACAGTT







 


445532
1289
1306
CGGCATTGGT
89
5-8-5
255
1288
1305





GCACAGTT







 


413482
1290
1309
GGACGGCATT
88
5-10-5
115
1289
1308





GGTGCACAGT







 


445508
1290
1309
GGACGGCATT
81
6-8-6
115
1289
1308





GGTGCACAGT







 


445533
1290
1307
ACGGCATTGG
87
5-8-5
256
1289
1306





TGCACAGT







 


416860
1291
1310
CGGACGGCAT
89
5-10-5
225
1290
1309





TGGTGCACAG







 


445509
1291
1310
CGGACGGCAT
84
6-8-6
225
1290
1309





TGGTGCACAG







 


445534
1291
1308
GACGGCATTG
82
5-8-5
257
1290
1307





GTGCACAG







 


416861
1292
1311
GCGGACGGCA
90
5-10-5
226
1291
1310





TTGGTGCACA







 


445510
1292
1311
GCGGACGGCA
88
6-8-6
226
1291
1310





TTGGTGCACA







 


445535
1292
1309
GGACGGCATT
83
5-8-5
258
1291
1308





GGTGCACA







 


416862
1293
1312
AGCGGACGGC
89
5-10-5
227
1292
1311





ATTGGTGCAC







 


445511
1293
1312
AGCGGACGGC
77
6-8-6
227
1292
1311





ATTGGTGCAC







 


445536
1293
1310
CGGACGGCAT
82
5-8-5
259
1292
1309





TGGTGCAC







 


416863
1294
1313
CAGCGGACGG
86
5-10-5
228
1293
1312





CATTGGTGCA







 


445512
1294
1313
CAGCGGACGG
79
6-8-6
228
1293
1312





CATTGGTGCA







 


445537
1294
1311
GCGGACGGCA
78
5-8-5
260
1293
1310





TTGGTGCA







 


412225
1295
1314
GCAGCGGACG
86
5-10-5
53
1294
1313





GCATTGGTGC







 


445513
1295
1314
GCAGCGGACG
85
6-8-6
53
1294
1313





GCATTGGTGC







 


445538
1295
1312
AGCGGACGGC
80
5-8-5
261
1294
1311





ATTGGTGC







 


416864
1296
1315
GGCAGCGGAC
88
5-10-5
229
1295
1314





GGCATTGGTG







 


445514
1296
1315
GGCAGCGGAC
81
6-8-6
229
1295
1314





GGCATTGGTG







 


445539
1296
1313
CAGCGGACGG
79
5-8-5
262
1295
1312





CATTGGTG







 


416865
1297
1316
TGGCAGCGGA
86
5-10-5
230
1296
1315





CGGCATTGGT







 


445515
1297
1316
TGGCAGCGGA
75
6-8-6
230
1296
1315





CGGCATTGGT







 


445540
1297
1314
GCAGCGGACG
74
5-8-5
263
1296
1313





GCATTGGT







 


416866
1298
1317
CTGGCAGCGG
84
5-10-5
231
1297
1316





ACGGCATTGG







 


445516
1298
1317
CTGGCAGCGG
79
6-8-6
231
1297
1316





ACGGCATTGG







 


445541
1298
1315
GGCAGCGGAC
80
5-8-5
264
1297
1314





GGCATTGG







 


416867
1299
1318
ACTGGCAGCG
85
5-10-5
232
1298
1317





GACGGCATTG







 


445517
1299
1318
ACTGGCAGCG
74
6-8-6
232
1298
1317





GACGGCATTG







 


445542
1299
1316
TGGCAGCGGA
83
5-8-5
265
1298
1315





CGGCATTG







 


445543
1300
1317
CTGGCAGCGG
74
5-8-5
266
1299
1316





ACGGCATT









Example 31
Dose-Dependent Antisense Inhibition of Human Factor 11 in HepG2 Cells
Gapmers from Example 30 exhibiting in vitro inhibition of human Factor 11 were tested at various doses in HepG2 cells. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 123.46 nM, 370.37 nM, 1,111.11 nM, 3,333.33 nM and 10,000 nM concentrations of antisense oligonucleotide, as specified in Table 45. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Factor 11 mRNA levels were measured by quantitative real-time PCR. Human Factor 11 primer probe set RTS 2966 was used to measure mRNA levels. Factor 11 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of Factor 11, relative to untreated control cells. As illustrated in Table 45, Factor 11 mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells.
The half maximal inhibitory concentration (IC50) of each oligonucleotide was calculated by plotting the concentrations of antisense oligonucleotides used versus the percent inhibition of Factor 11 mRNA expression achieved at each concentration, and noting the concentration of antisense oligonucleotide at which 50% inhibition of Factor 11 mRNA expression was achieved compared to the PBS control. IC50 values are presented in Table 45.







TABLE 45







Dose-dependent antisense inhibition of human Factor 11 in HepG2


cells via transfection of oligonucleotides using electroporation














123.47
370.37
1,111.11
3,333.33
10,000.0
IC50


ISIS No.
nM
nM
nM
nM
nM
(μM)
















416849
5
5
26
57
68
2.7


416850
0
12
36
74
73
2.8


416851
13
35
36
64
72
1.5


416856
12
23
35
59
83
1.6


416857
2
20
35
62
72
2.3


416858
0
27
36
64
70
2.2


416860
0
28
39
41
40
n.d.


416861
0
15
27
66
80
2.0


445498
3
1
27
50
58
4.8


445503
0
0
22
36
60
5.9


445504
8
20
38
53
68
2.7


445505
12
30
39
59
77
1.8


445522
0
0
44
63
74
2.9


445531
8
16
52
61
77
1.8


445532
5
12
39
60
70
2.0





n.d. = no data






Example 32
Dose-Dependent Antisense Inhibition of Human Factor 11 in HepG2 Cells by Oligonucleotides Designed by Microwalk
Additional gapmers were designed based on ISIS 416850 and ISIS 416858 (see Table 8 above). These gapmers are shifted slightly upstream and downstream (i.e. microwalk) of ISIS 416850 and ISIS 416858. Gapmers designed by microwalk have 3-8-3 MOE, 4-8-4 MOE, 2-10-2 MOE, 3-10-3 MOE, or 4-10-4 MOE motifs.
These gapmers were tested at various doses in HepG2 cells. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 375 nM, 750 nM, 1,500 nM, 3,000 nM, 6,000 nM and 12,000 nM concentrations of antisense oligonucleotide, as specified in Table 47. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Factor 11 mRNA levels were measured by quantitative real-time PCR. Human Factor 11 primer probe set RTS 2966 was used to measure mRNA levels. Factor 11 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of Factor 11, relative to untreated control cells.
ISIS 416850, ISIS 416858, ISIS 445522, and ISIS 445531 (see Table 45 above) were re-tested in vitro along with the microwalk gapmers under the same conditions described above.
The chimeric antisense oligonucleotides in Table 46 were designed as 3-8-3 MOE, 4-8-4 MOE, 2-10-2 MOE, 3-10-3 MOE, or 4-10-4 MOE gapmers. The 3-8-3 gapmer is 14 nucleotides in length, wherein the central gap segment is comprised of eight 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising three nucleotides each. The 4-8-4 gapmer is 16 nucleotides in length, wherein the central gap segment is comprised of eight 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising four nucleotides each. The 2-10-2 gapmer is 14 nucleotides in length, wherein the central gap segment is comprised of ten 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising two nucleotides each. The 3-10-3 gapmer is 16 nucleotides in length, wherein the central gap segment is comprised of ten 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising three nucleotides each. The 4-10-4 gapmer is 18 nucleotides in length, wherein the central gap segment is comprised of ten 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising four nucleotides each. For each of the motifs (3-8-3, 4-8-4, 2-10-2, 3-10-3, and 4-10-4), each nucleotide in the 5′ wing segment and each nucleotide in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytidine residues throughout each gapmer are 5-methylcytidines. “Human Target start site” indicates the 5′-most nucleotide to which the gapmer is targeted in the human sequence. “Human Target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted in the human sequence. Each gapmer listed in Table 46 is targeted to SEQ ID NO: 1 (GENBANK Accession No. NM—000128.3). Each gapmer is Table 46 is also fully cross-reactive with the rhesus monkey Factor 11 gene sequence, designated herein as SEQ ID NO: 274 (exons 1-15 GENBANK Accession No. NW—001118167.1). ‘Rhesus monkey start site’ indicates the 5′-most nucleotide to which the gapmer is targeted in the rhesus monkey sequence. ‘Rhesus monkey stop site’ indicates the 3′-most nucleotide to which the gapmer is targeted to the rhesus monkey sequence.







TABLE 46







Chimeric antisense oligonucleotides targeted to SEQ ID


NO: 1 (GENBANK Accession No. NM_000128.3)


and designed by microwalk of ISIS 416850 and ISIS 416858















Human
Human



Rhesus
Rhesus



Target
Target


SEQ
monkey
monkey


ISIS
Start
Stop


ID
Start
Stop


No.
Site
Site
Sequence (5′ to 3′)
Motif
No.
Site
Site





449707
1280
1295
CACAGTTT
4-8-4
268
1279
1294





CTGGCAGG






 


449708
1281
1294
ACAGTTT
3-8-3
269
1280
1293





CTGGCAG






 


449709
1279
1296
GCACAGTT
4-10-4
246
1278
1295





TCTGGCAGGC






 


449710
1280
1295
CACAGTTT
3-10-3
268
1279
1294





CTGGCAGG






 


449711
1281
1294
ACAGTTT
2-10-2
269
1280
1293





CTGGCAG









Dose-response inhibition data is given in Table 47. As illustrated in Table 47, Factor 11 mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells. The IC50 of each antisense oligonucleotide was also calculated and presented in Table 47. The first two listed gapmers in Table 47 are the original gapmers (ISIS 416850 and ISIS 416858) from which the remaining gapmers were designed via microwalk and are designated by an asterisk.







TABLE 47







Dose-dependent antisense inhibition of human Factor 11 in HepG2


cells via transfection of oligonucleotides using electroporation















375
750
1,500
3,000
6,000
12,000
IC50


ISIS No.
nM
nM
nM
nM
nM
nM
(μM)

















*416850 
40
59
69
87
90
95
0.56


*416858 
31
35
78
85
90
93
0.83


445522
59
71
83
82
81
92
n.d.


445531
44
64
78
86
91
93
0.44


449707
7
35
63
73
85
91
1.26


449708
0
0
22
33
61
85
4.46


449709
52
71
80
87
92
95
0.38


449710
2
21
52
70
82
87
1.59


449711
6
14
1
7
32
52
11.04





n.d. = no data






Example 33
Tolerability of Antisense Oligonucleotides Targeting Human Factor 11 in CD1 Mice
CD1 mice were treated with ISIS antisense oligonucleotides targeting human Factor 11 and evaluated for changes in the levels of various metabolic markers.
Treatment
Groups of five CD1 mice each were injected subcutaneously twice a week for 2, 4, or 6 weeks with 50 mg/kg of ISIS 416825, ISIS 416826, ISIS 416838, ISIS 416850, ISIS 416858, ISIS 416864, ISIS 416892, ISIS 416925, ISIS 416999, ISIS 417002, or ISIS 417003. A control group of five mice was injected subcutaneously with PBS for 2 weeks. All experimental groups (i.e. ASO treated mice at 2, 4, 6 weeks) were compared to the control group (i.e. PBS, 2 weeks).
Three days after the last dose was administered to all groups, the mice were sacrificed. Organ weights were measured and blood was collected for further analysis.
Organ Weight
Liver, spleen, and kidney weights were measured at the end of the study, and are presented in Tables 48, 49, and 50 as a percent of the PBS control, normalized to body weight. Those antisense oligonucleotides which did not affect more than six-fold increases in liver and spleen weight above the PBS controls were selected for further studies.







TABLE 48







Percent change in liver weight of CD1 mice


after antisense oligonucleotide treatment












ISIS No.
2 weeks
4 weeks
6 weeks
















416825
+5
+22
+13



416826
+10
+32
+33



416838
+8
−6
0



416850
+5
+3
+6



416858
+7
+1
+10



416864
−2
+2
−5



416925
+14
+14
+33



416999
+13
+30
+47



417002
+14
+8
+35



416892
+35
+88
+95



417003
+8
+42
+32

















TABLE 49







Percent change in spleen weight of CD1 mice


after antisense oligonucleotide treatment












ISIS No.
2 weeks
4 weeks
6 weeks
















416825
−12
+19
+21



416826
−12
−5
+22



416838
+21
−8
+9



416850
−4
+6
+48



416858
−2
+8
+28



416864
−10
−2
−6



416925
−7
+33
+78



416999
+7
+22
+38



417002
+29
+26
+108



416892
+24
+30
+65



417003
+12
+101
+98

















TABLE 50







Percent change in kidney weight of CD1 mice


after antisense oligonucleotide treatment












ISIS No.
2 weeks
4 weeks
6 weeks
















416825
−12
−12
−11



416826
−13
−7
−22



416838
−2
−12
−8



416850
−10
−12
−11



416858
+1
−18
−10



416864
−4
−9
−15



416925
−4
−14
−2



416999
−9
−6
−7



417002
+3
−5
−2



416892
+2
−3
+19



417003
−9
−2
−1










Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma concentrations of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Measurements of alanine transaminase (ALT) and aspartate transaminase (AST) are expressed in IU/L and the results are presented in Tables 51 and 52. Plasma levels of bilirubin and albumin were also measured using the same clinical chemistry analyzer and expressed in mg/dL. The results are presented in Tables 53 and 54. Those antisense oligonucleotides which did not affect an increase in ALT/AST levels above seven-fold of control levels were selected for further studies. Those antisense oligonucleotides which did not increase levels of bilirubin more than two-fold of the control levels were selected for further studies.







TABLE 51







Effect of antisense oligonucleotide


treatment on ALT (IU/L) in CD1 mice











2 weeks
4 weeks
6 weeks
















PBS
36
n.d.
n.d.



ISIS 416825
64
314
507



ISIS 416826
182
126
1954



ISIS 416838
61
41
141



ISIS 416850
67
58
102



ISIS 416858
190
57
216



ISIS 416864
44
33
92



ISIS 416925
160
284
1284



ISIS 416999
61
160
1302



ISIS 417002
71
138
2579



ISIS 416892
66
1526
1939



ISIS 417003
192
362
2214







n.d. = no data













TABLE 52







Effect of antisense oligonucleotide


treatment on AST (IU/L) in CD1 mice











2 weeks
4 weeks
6 weeks
















PBS
68
n.d.
n.d.



ISIS 416825
82
239
301



ISIS 416826
274
156
1411



ISIS 416838
106
73
107



ISIS 416850
72
88
97



ISIS 416858
236
108
178



ISIS 416864
58
46
101



ISIS 416925
144
206
712



ISIS 416999
113
130
671



ISIS 417002
96
87
1166



ISIS 416892
121
1347
1443



ISIS 417003
152
249
839







n.d. = no data













TABLE 53







Effect of antisense oligonucleotide treatment


on bilirubin (mg/dL) in CD1 mice











2 weeks
4 weeks
6 weeks
















PBS
0.28
n.d.
n.d.



ISIS 416825
0.41
0.69
0.29



ISIS 416826
0.39
0.20
0.37



ISIS 416838
0.57
0.24
0.20



ISIS 416850
0.46
0.23
0.22



ISIS 416858
0.57
0.24
0.16



ISIS 416864
0.40
0.26
0.22



ISIS 416925
0.45
0.25
0.25



ISIS 416999
0.48
0.18
0.28



ISIS 417002
0.50
0.25
0.29



ISIS 416892
0.38
2.99
0.50



ISIS 417003
0.33
0.15
0.24







n.d. = no data













TABLE 54







Effect of antisense oligonucleotide treatment


on albumin (mg/dL) in CD1 mice











2 weeks
4 weeks
6 weeks
















PBS
3.7
n.d.
n.d.



ISIS 416825
3.6
3.4
3.5



ISIS 416826
3.3
3.4
3.4



ISIS 416838
3.5
3.8
3.6



ISIS 416850
3.6
3.5
3.1



ISIS 416858
3.4
3.5
2.8



ISIS 416864
3.5
3.6
3.5



ISIS 416925
3.5
3.5
3.2



ISIS 416999
3.4
3.3
3.2



ISIS 417002
3.2
3.4
3.4



ISIS 416892
3.2
4.0
4.4



ISIS 417003
3.4
3.4
3.2







n.d. = no data






Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function, plasma concentrations of blood urea nitrogen (BUN) and creatinine were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Results are presented in Tables 55 and 56, expressed in mg/dL. Those antisense oligonucleotides which did not affect more than a two-fold increase in BUN levels compared to the PBS control were selected for further studies.







TABLE 55







Effect of antisense oligonucleotide


treatment on BUN (mg/dL) in CD1 mice











2 weeks
4 weeks
6 weeks
















PBS
30
n.d.
n.d.



ISIS 416825
29
35
31



ISIS 416826
24
34
27



ISIS 416838
25
38
30



ISIS 416850
25
30
23



ISIS 416858
21
29
19



ISIS 416864
22
31
28



ISIS 416925
21
30
17



ISIS 416999
22
27
22



ISIS 417002
19
23
19



ISIS 416892
19
28
23



ISIS 417003
23
26
24







n.d. = no data













TABLE 56







Effect of antisense oligonucleotide treatment


on creatinine (mg/dL) in CD1 mice











2 weeks
4 weeks
6 weeks
















PBS
0.14
n.d.
n.d.



ISIS 416825
0.14
0.21
0.17



ISIS 416826
0.15
0.20
0.15



ISIS 416838
0.09
0.27
0.14



ISIS 416850
0.13
0.22
0.19



ISIS 416858
0.13
0.23
0.10



ISIS 416864
0.11
0.22
0.16



ISIS 416925
0.12
0.25
0.13



ISIS 416999
0.07
0.18
0.13



ISIS 417002
0.06
0.16
0.10



ISIS 416892
0.11
0.20
0.17



ISIS 417003
0.17
0.24
0.18







n.d. = no data






Hematology Assays

Blood obtained from all mice groups were sent to Antech Diagnostics for hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) measurements and analyses, as well as measurements of the various blood cells, such as WBC (neutrophils, lymphocytes, and monocytes), RBC, and platelets, and total hemoglobin content. The results are presented in Tables 57-67. Percentages given in the tables indicate the percent of total blood cell count. Those antisense oligonucleotides which did not affect a decrease in platelet count of more than 50% and/or an increase in monocyte count of more than three-fold were selected for further studies.







TABLE 57







Effect of antisense oligonucleotide


treatment on HCT (%) in CD1 mice











2 weeks
4 weeks
6 weeks
















PBS
50
n.d.
n.d.



ISIS 416825
49
46
40



ISIS 416826
47
41
37



ISIS 416838
42
44
39



ISIS 416850
44
44
38



ISIS 416858
50
45
46



ISIS 416864
50
45
42



ISIS 416925
51
47
47



ISIS 416999
51
42
40



ISIS 417002
44
44
51



ISIS 416892
48
42
45



ISIS 417003
48
41
43







n.d. = no data













TABLE 58







Effect of antisense oligonucleotide


treatment on MCV (fL) in CD1 mice











2 weeks
4 weeks
6 weeks
















PBS
61
n.d.
n.d.



ISIS 416825
58
53
51



ISIS 416826
56
52
53



ISIS 416838
56
54
48



ISIS 416850
57
51
50



ISIS 416858
59
51
50



ISIS 416864
57
52
51



ISIS 416925
61
52
47



ISIS 416999
60
49
48



ISIS 417002
61
50
52



ISIS 416892
59
49
53



ISIS 417003
60
48
45







n.d. = no data













TABLE 59







Effect of antisense oligonucleotide


treatment on MCH (pg) in CD1 mice












ISIS No.
2 weeks
4 weeks
6 weeks







PBS
18
n.d.
n.d.



ISIS 416825
17
16
15



ISIS 416826
17
16
16



ISIS 416838
17
17
15



ISIS 416850
17
16
15



ISIS 416858
17
16
15



ISIS 416864
18
16
16



ISIS 416925
17
16
15



ISIS 416999
17
16
15



ISIS 417002
17
16
16



ISIS 416892
18
16
16



ISIS 417003
17
16
16







n.d. = no data













TABLE 60







Effect of antisense oligonucleotide


treatment on MCHC (%) in CD1 mice











2 weeks
4 weeks
6 weeks
















PBS
30
n.d.
n.d.



ISIS 416825
29
31
31



ISIS 416826
29
31
30



ISIS 416838
30
31
32



ISIS 416850
30
31
31



ISIS 416858
30
32
31



ISIS 416864
31
31
31



ISIS 416925
30
32
32



ISIS 416999
27
32
31



ISIS 417002
29
32
31



ISIS 416892
30
32
30



ISIS 417003
29
32
33







n.d. = no data













TABLE 61







Effect of antisense oligonucleotide treatment


on WBC count (cells/nL) in CD1 mice











2 weeks
4 weeks
6 weeks
















PBS
6
n.d.
n.d.



ISIS 416825
8
8
6



ISIS 416826
5
6
8



ISIS 416838
4
6
5



ISIS 416850
4
5
5



ISIS 416858
6
7
4



ISIS 416864
7
6
5



ISIS 416925
6
6
11



ISIS 416999
4
9
7



ISIS 417002
8
8
16



ISIS 416892
5
8
9



ISIS 417003
7
9
10







n.d. = no data













TABLE 62







Effect of antisense oligonucleotide treatment


on RBC count (cells/pL) in CD1 mice











2 weeks
4 weeks
6 weeks
















PBS
8
n.d.
n.d.



ISIS 416825
9
9
8



ISIS 416826
8
8
7



ISIS 416838
8
8
8



ISIS 416850
8
9
8



ISIS 416858
9
9
9



ISIS 416864
9
9
8



ISIS 416925
9
9
10



ISIS 416999
9
9
8



ISIS 417002
9
9
10



ISIS 416892
7
9
9



ISIS 417003
8
9
10







n.d. = no data













TABLE 63







Effect of antisense oligonucleotide treatment


on neutrophil count (%) in CD1 mice











2 weeks
4 weeks
6 weeks
















PBS
16
n.d.
n.d.



ISIS 416825
15
43
23



ISIS 416826
26
33
23



ISIS 416838
19
33
31



ISIS 416850
15
21
16



ISIS 416858
14
24
27



ISIS 416864
13
27
20



ISIS 416925
12
39
33



ISIS 416999
12
25
22



ISIS 417002
14
31
36



ISIS 416892
19
43
28



ISIS 417003
10
39
24







n.d. = no data













TABLE 64







Effect of antisense oligonucleotide treatment


on lymphocyte count (%) in CD1 mice











2 weeks
4 weeks
6 weeks
















PBS
81
n.d.
n.d.



ISIS 416825
82
53
71



ISIS 416826
70
61
67



ISIS 416838
76
64
60



ISIS 416850
82
73
76



ISIS 416858
83
73
65



ISIS 416864
84
71
74



ISIS 416925
86
58
57



ISIS 416999
86
72
69



ISIS 417002
83
64
51



ISIS 416892
79
52
64



ISIS 417003
86
54
66







n.d. = no data













TABLE 65







Effect of antisense oligonucleotide treatment


on monocyte count (%) in CD1 mice











2 weeks
4 weeks
6 weeks
















PBS
3
n.d.
n.d.



ISIS 416825
2
5
4



ISIS 416826
3
5
8



ISIS 416838
2
2
6



ISIS 416850
3
6
6



ISIS 416858
2
3
7



ISIS 416864
2
2
5



ISIS 416925
2
4
8



ISIS 416999
2
4
8



ISIS 417002
3
4
12



ISIS 416892
3
6
7



ISIS 417003
2
6
8







n.d. = no data













TABLE 66







Effect of antisense oligonucleotide treatment


on platelet count (cells/nL) in CD1 mice











2 weeks
4 weeks
6 weeks
















PBS
2126
n.d.
n.d.



ISIS 416825
1689
1229
942



ISIS 416826
1498
970
645



ISIS 416838
1376
1547
1229



ISIS 416850
1264
1302
1211



ISIS 416858
2480
1364
1371



ISIS 416864
1924
1556
933



ISIS 416925
1509
1359
1211



ISIS 416999
1621
1219
1057



ISIS 417002
1864
1245
1211



ISIS 416892
1687
636
1004



ISIS 417003
1309
773
922







n.d. = no data













TABLE 67







Effect of antisense oligonucleotide treatment


on hemoglobin content (g/dL) in CD1 mice











2 weeks
4 weeks
6 weeks
















PBS
15.1
n.d.
n.d.



ISIS 416825
14.5
14.1
12.1



ISIS 416826
13.4
12.8
11.0



ISIS 416838
12.4
13.6
12.6



ISIS 416850
13.1
13.5
11.6



ISIS 416858
14.8
14.2
14.1



ISIS 416864
15.2
13.9
13.0



ISIS 416925
14.9
14.8
15.3



ISIS 416999
14.2
13.3
12.8



ISIS 417002
14.7
14.0
15.7



ISIS 416892
13.0
13.5
13.1



ISIS 417003
13.7
13.4
14.0







n.d. = no data






Example 34
Measurement of Half-Life of Antisense Oligonucleotide in CD1 Mice Liver
CD1 mice were treated with ISIS antisense oligonucleotides targeting human Factor 11 and the oligonucleotide half-life as well as the elapsed time for oligonucleotide degradation and elimination from the liver was evaluated.
Treatment
Groups of fifteen CD1 mice each were injected subcutaneously twice per week for 2 weeks with 50 mg/kg of ISIS 416825, ISIS 416826, ISIS 416838, ISIS 416850, ISIS 416858, ISIS 416864, ISIS 416892, ISIS 416925, ISIS 416999, ISIS 417002, or ISIS 417003. Five mice from each group were sacrificed 3 days, 28 days and 56 days following the final dose. Livers were harvested for analysis.
Measurement of Oligonucleotide Concentration
The concentration of the full-length oligonucleotide as well as the total oligonucleotide concentration (including the degraded form) was measured. The method used is a modification of previously published methods (Leeds et al., 1996; Geary et al., 1999) which consist of a phenol-chloroform (liquid-liquid) extraction followed by a solid phase extraction. An internal standard (ISIS 355868, a 27-mer 2′-O-methoxyethyl modified phosphorothioate oligonucleotide, GCGTTTGCTCTTCTTCTTGCGTTTTTT, designated herein as SEQ ID NO: 270) was added prior to extraction. Tissue sample concentrations were calculated using calibration curves, with a lower limit of quantitation (LLOQ) of approximately 1.14 μg/g. Half-lives were then calculated using WinNonlin software (PHARSIGHT).
The results are presented in Tables 68 and 69, expressed as μg/g liver tissue. The half-life of each oligonucleotide is presented in Table 70.







TABLE 68







Full-length oligonucleotide concentration


(μg/g) in the liver of CD1 mice











ISIS No.
Motif
day 3
day 28
day 56














416825
5-10-5
151
52
7


416826
5-10-5
186
48
8


416838
5-10-5
170
46
10


416850
5-10-5
238
93
51


416858
5-10-5
199
102
18


416864
5-10-5
146
38
25


416999
2-13-5
175
26
0


417002
2-13-5
119
24
1


417003
2-13-5
245
42
4


416925
3-14-3
167
39
5


416892
3-14-3
135
31
6
















TABLE 69







Total oligonucleotide concentration (μg/g) in the liver of CD1 mice











ISIS No.
Motif
day 3
day 28
day 56














416825
5-10-5
187
90
39


416826
5-10-5
212
61
12


416838
5-10-5
216
98
56


416850
5-10-5
295
157
143


416858
5-10-5
273
185
56


416864
5-10-5
216
86
112


416999
2-13-5
232
51
0


417002
2-13-5
206
36
1


417003
2-13-5
353
74
4


416925
3-14-3
280
72
8


416892
3-14-3
195
54
6
















TABLE 70







Half-life of antisense oligonucleotides in the liver of CD1 mice











Half-life


ISIS No.
Motif
(days)












416825
5-10-5
16


416826
5-10-5
13


416838
5-10-5
13


416850
5-10-5
18


416858
5-10-5
26


416864
5-10-5
13


416999
2-13-5
9


417002
2-13-5
11


417003
2-13-5
10


416925
3-14-3
12


416892
3-14-3
12









Example 35
Tolerability of Antisense Oligonucleotides Targeting Human Factor 11 in Sprague-Dawley Rats
Sprague-Dawley rats were treated with ISIS antisense oligonucleotides targeting human Factor 11 and evaluated for changes in the levels of various metabolic markers.
Treatment
Groups of four Sprague Dawley rats each were injected subcutaneously twice per week for 6 weeks with 50 mg/kg of ISIS 416825, ISIS 416826, ISIS 416838, ISIS 416850, ISIS 416858, ISIS 416848, ISIS 416864, ISIS 416892, ISIS 416925, ISIS 416999, ISIS 417002, or ISIS 417003. A control group of four Sprague Dawley rats was injected subcutaneously with PBS twice per week for 6 weeks. Body weight measurements were taken before and throughout the treatment period. Urine samples were taken before the start of treatment. Three days after the last dose, urine samples were taken and the rats were sacrificed. Organ weights were measured and blood was collected for further analysis.
Body Weight and Organ Weight
Body weights of the rats were measured at the onset of the study and subsequently twice per week. The body weights are presented in Table 71 and are expressed as a percent change over the weights taken at the start of the study. Liver, spleen, and kidney weights were measured at the end of the study and are presented in Table 71 as a percent of the saline control normalized to body weight. Those antisense oligonucleotides which did not affect more than a six-fold increase in liver and spleen weight above the PBS control were selected for further studies.







TABLE 71







Percent change in organ weight of Sprague Dawley


rats after antisense oligonucleotide treatment











ISIS



Body


No.
Liver
Spleen
Kidney
weight














416825
+20
+245
+25
−18


416826
+81
+537
+44
−40


416838
+8
+212
−0.5
−23


416850
+23
+354
+47
−33


416858
+8
+187
+5
−21


416864
+16
+204
+16
−24


416925
+44
+371
+48
−32


416999
+51
+405
+71
−37


417002
+27
+446
+63
−29


416892
+38
+151
+32
−39


417003
+51
+522
+25
−40









Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma concentrations of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Measurements of alanine transaminase (ALT) and aspartate transaminase (AST) are expressed in IU/L and the results are presented in Table 72. Those antisense oligonucleotides which did not affect an increase in ALT/AST levels above seven-fold of control levels were selected for further studies. Plasma levels of bilirubin and albumin were also measured with the same clinical analyzer and the results are also presented in Table 72, expressed in mg/dL. Those antisense oligonucleotides which did not affect an increase in levels of bilirubin more than two-fold of the control levels by antisense oligonucleotide treatment were selected for further studies.







TABLE 72







Effect of antisense oligonucleotide treatment on metabolic


markers in the liver of Sprague-Dawley rats












ALT
AST
Bilirubin
Albumin



(IU/L)
(IU/L)
(mg/dL)
(mg/dL)















PBS
9
5
20
2


ISIS 416825
89
17
4
2


ISIS 416826
611
104
115
6


ISIS 416838
5
2
4
2


ISIS 416850
80
5
1
4


ISIS 416858
13
4
4
2


ISIS 416864
471
68
3
4


ISIS 416925
102
20
13
5


ISIS 416999
92
28
54
5


ISIS 417002
44
11
12
3


ISIS 416892
113
183
1
8


ISIS 417003
138
23
50
6









Kidney Function

To evaluate the effect of kidney function, plasma concentrations of blood urea nitrogen (BUN) and creatinine were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Results are presented in Table 73, expressed in mg/dL. Those antisense oligonucleotides which did not affect more than a two-fold increase in BUN levels compared to the PBS control were selected for further studies. The ratio of urine protein to creatinine in total urine samples was also calculated before and after antisense oligonucleotide treatment and is presented in Table 74. Those antisense oligonucleotides which did not affect more than a five-fold increase in urine protein/creatinine ratios compared to the PBS control were selected for further studies.







TABLE 73







Effect of antisense oligonucleotide treatment on metabolic


markers in the kidney of Sprague-Dawley rats










BUN
Creatinine













PBS
4
8


ISIS 416825
7
17


ISIS 416826
25
6


ISIS 416838
4
5


ISIS 416850
5
7


ISIS 416858
8
4


ISIS 416864
5
6


ISIS 416925
7
5


ISIS 416999
2
4


ISIS 417002
11
1


ISIS 416892
188
1


ISIS 417003
9
9
















TABLE 74







Effect of antisense oligonucleotide treatment on urine


protein/creatinine ratio in Sprague Dawley rats










Before
After













PBS
1.2
1.3


416825
1.1
5.4


416826
1.0
11.4


416838
1.2
3.7


416850
1.0
4.0


416858
0.9
4.4


416864
1.2
4.0


416925
1.0
4.3


416999
1.3
9.1


417002
1.0
2.4


416892
0.8
21.3


417003
0.9
4.8









Hematology Assays

Blood obtained from all rat groups were sent to Antech Diagnostics for hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCV), and mean corpuscular hemoglobin concentration (MCHC) measurements and analyses, as well as measurements of various blood cells, such as WBC (neutrophils, lymphocytes and monocytes), RBC, and platelets as well as hemoglobin content. The results are presented in Tables 75 and 76. Those antisense oligonucleotides which did not, affect a decrease in platelet count of more than 50% and an increase in monocyte count of more than three-fold were selected for further studies.







TABLE 75







Effect of antisense oligonucleotide treatment


on blood cell count in Sprague-Dawley rats














WBC
RBC
Neutrophils
Lymphocytes
Monocytes
Platelets



(/nL)
(/pL)
(%)
(%)
(%)
(103/μL)

















PBS
21
6
37
7
26
18


ISIS 416825
22
2
25
3
15
6


ISIS 416826
7
5
30
5
7
11


ISIS 416838
13
4
17
3
6
27


ISIS 416850
16
7
48
8
11
26


ISIS 416858
28
2
20
3
10
19


ISIS 416864
15
4
26
2
29
12


ISIS 416925
24
6
20
4
23
8


ISIS 416999
12
5
23
3
20
12


ISIS 417002
23
5
22
4
25
7


ISIS 416892
68
12
92
18
58
66


ISIS 417003
83
11
17
3
6
19
















TABLE 76







Effect of antisense oligonucleotide treatment on hematologic


factors (% control) in Sprague-Dawley rats













Hemoglobin
HCT
MCV
MCH
MCHC



(g/dL)
(%)
(fL)
(pg)
(%)
















PBS
6
4
6
2
4


ISIS 416825
2
2
4
2
4


ISIS 416826
7
7
6
3
4


ISIS 416838
2
5
4
2
5


ISIS 416850
4
5
3
4
2


ISIS 416858
2
3
2
2
1


ISIS 416864
4
2
4
2
4


ISIS 416925
6
8
5
2
4


ISIS 416999
6
5
2
3
1


ISIS 417002
5
7
7
3
5


ISIS 416892
14
13
1
2
0


ISIS 417003
11
8
6
4
4









Example 36
Measurement of Half-Life of Antisense Oligonucleotide in Sprague-Dawley Rat Liver and Kidney
Sprague Dawley rats were treated with ISIS antisense oligonucleotides targeting human Factor 11 and the oligonucleotide half-life as well as the elapsed time for oligonucleotide degradation and elimination from the liver and kidney was evaluated.
Treatment
Groups of four Sprague Dawley rats each were injected subcutaneously twice a week for 2 weeks with 20 mg/kg of ISIS416825, ISIS 416826, ISIS 416838, ISIS 416850, ISIS 416858, ISIS 416864, ISIS 416892, ISIS 416925, ISIS 416999, ISIS 417002, or ISIS 417003. Three days after the last dose, the rats were sacrificed and livers and kidneys were collected for analysis.
Measurement of Oligonucleotide Concentration
The concentration of the full-length oligonucleotide as well as the total oligonucleotide concentration (including the degraded form) was measured. The method used is a modification of previously published methods (Leeds et al., 1996; Geary et al., 1999) which consist of a phenol-chloroform (liquid-liquid) extraction followed by a solid phase extraction. An internal standard (ISIS 355868, a 27-mer 2′-O-methoxyethyl modified phosphorothioate oligonucleotide, GCGTTTGCTCTTCTTCTTGCGTTTTTT, designated herein as SEQ ID NO: 270) was added prior to extraction. Tissue sample concentrations were calculated using calibration curves, with a lower limit of quantitation (LLOQ) of approximately 1.14 μg/g. The results are presented in Tables 77 and 78, expressed as μg/g liver or kidney tissue. Half-lives were then calculated using WinNonlin software (PHARSIGHT).







TABLE 77







Full-length oligonucleotide concentration (μg/g)


in the liver and kidney of Sprague-Dawley rats












ISIS No.
Motif
Kidney
Liver







416825
5-10-5
632
236



416826
5-10-5
641
178



416838
5-10-5
439
171



416850
5-10-5
259
292



416858
5-10-5
575
255



416864
5-10-5
317
130



416999
2-13-5
358
267



417002
2-13-5
291
118



417003
2-13-5
355
199



416925
3-14-3
318
165



416892
3-14-3
351
215

















TABLE 78







Total oligonucleotide concentration (μg/g)


in the liver and kidney of Sprague-Dawley rats












ISIS No.
Motif
Kidney
Liver







416825
5-10-5
845
278



416826
5-10-5
775
214



416838
5-10-5
623
207



416850
5-10-5
352
346



416858
5-10-5
818
308



416864
5-10-5
516
209



416999
2-13-5
524
329



417002
2-13-5
490
183



417003
2-13-5
504
248



416925
3-14-3
642
267



416892
3-14-3
608
316

















TABLE 79







Half-life (days) of ISIS oligonucleotides in


the liver and kidney of Sprague-Dawley rats









ISIS No.
Motif
Half-life












416825
5-10-5
16


416826
5-10-5
13


416838
5-10-5
13


416850
5-10-5
18


416858
5-10-5
26


416864
5-10-5
13


416999
2-13-5
9


417002
2-13-5
11


417003
2-13-5
10


416925
3-14-3
12


416892
3-14-3
12









Example 37
Tolerability of Antisense Oligonucleotides Targeting Human Factor 11 in CD1 Mice
CD1 mice were treated with ISIS antisense oligonucleotides targeting human Factor 11 and evaluated for changes in the levels of various metabolic markers.
Treatment
Groups of five CD1 mice each were injected subcutaneously twice per week for 6 weeks with 50 mg/kg of ISIS 412223, ISIS 412224, ISIS 412225, ISIS 413481, ISIS 413482, ISIS 416848, ISIS 416849, ISIS 416850, ISIS 416851, ISIS 416852, ISIS 416853, ISIS 416854, ISIS 416855, ISIS 416856, ISIS 416857, ISIS 416858, ISIS 416859, ISIS 416860, ISIS 416861, ISIS 416862, ISIS 416863, ISIS 416864, ISIS 416865, ISIS 416866, or ISIS 416867, or. A control group of ten CD1 mice was injected subcutaneously with PBS twice per week for 6 weeks. Body weight measurements were taken before and throughout the treatment period. Three days after the last dose, the mice were sacrificed, organ weights were measured, and blood was collected for further analysis.
Body Weight and Organ Weights
Body weight was measured at the onset of the study and subsequently twice per week. The body weights of the mice are presented in Table 80 and are expressed increase in grams over the PBS control weight taken before the start of treatment. Liver, spleen, and kidney weights were measured at the end of the study, and are also presented in Table 80 as percentage of the body weight. Those antisense oligonucleotides which did not affect more than six-fold increases in liver and spleen weight above the PBS control were selected for further studies.







TABLE 80







Change in body and organ weights of CD1 mice


after antisense oligonucleotide treatment















body



Liver
Kidney
Spleen
weight



(%)
(%)
(%)
(g)















PBS
5
1.5
0.3
7


ISIS 416850
6
1.6
0.4
12


ISIS 416858
7
1.6
0.6
12


ISIS 416864
5
1.6
0.3
12


ISIS 412223
6
1.5
0.4
12


ISIS 412224
6
1.6
0.5
10


ISIS 412225
6
1.5
0.4
10


ISIS 413481
6
1.5
0.5
9


ISIS 413482
6
1.6
0.5
11


ISIS 416848
6
1.5
0.4
11


ISIS 416849
8
1.5
0.4
8


ISIS 416851
7
1.5
0.5
11


ISIS 416852
6
1.5
0.4
10


ISIS 416853
8
1.5
0.7
13


ISIS 416854
7
1.2
0.4
13


ISIS 416855
8
1.4
0.6
12


ISIS 416856
6
1.4
0.4
10


ISIS 416857
7
1.6
0.5
10


ISIS 416859
6
1.5
0.4
10


ISIS 416860
6
1.4
0.4
10


ISIS 416861
5
1.3
0.4
9


ISIS 416862
6
1.5
0.4
10


ISIS 416863
5
1.5
0.4
9


ISIS 416865
6
1.5
0.4
8


ISIS 416866
5
1.6
0.4
10


ISIS 416867
5
1.4
0.4
9









Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma concentrations of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Measurements of alanine transaminase (ALT) and aspartate transaminase (AST) are expressed in IU/L and the results are presented in Table 81. Those antisense oligonucleotides which did not affect an increase in ALT/AST levels above seven-fold of control levels were selected for further studies. Plasma levels of bilirubin, cholesterol and albumin were also measured using the same clinical chemistry analyzer and are presented in Table 81 expressed in mg/dL. Those antisense oligonucleotides which did not affect an increase in levels of bilirubin more than two-fold of the control levels by antisense oligonucleotide treatment were selected for further studies.







TABLE 81







Effect of antisense oligonucleotide treatment on


metabolic markers in the liver of CD1 mice













ALT
AST
Bilirubin
Albumin
Cholesterol



(IU/L)
(IU/L)
(mg/dL)
(mg/dL)
(mg/dL)















PBS
32
68
0.25
3.7
135


ISIS 416850
75
99
0.21
3.5
142


ISIS 416858
640
547
0.28
4.4
181


ISIS 416864
36
67
0.19
2.6
152


ISIS 412223
60
125
0.20
3.0
117


ISIS 412224
214
183
0.19
3.4
114


ISIS 412225
40
69
0.23
3.3
128


ISIS 413481
85
143
0.18
3.2
153


ISIS 413482
54
77
0.24
3.0
138


ISIS 416848
153
153
0.19
3.1
151


ISIS 416849
1056
582
0.22
2.5
109


ISIS 416851
47
76
0.19
3.1
106


ISIS 416852
49
91
0.16
4.9
125


ISIS 416853
1023
1087
0.25
3.1
164


ISIS 416854
1613
1140
0.21
5.5
199


ISIS 416855
786
580
0.25
4.2
162


ISIS 416856
130
129
0.23
5.2
109


ISIS 416857
370
269
0.22
3.7
94


ISIS 416859
214
293
0.20
4.2
160


ISIS 416860
189
160
0.23
3.5
152


ISIS 416861
38
85
0.27
4.3
133


ISIS 416862
225
172
0.36
3.9
103


ISIS 416863
41
101
0.24
3.6
118


ISIS 416865
383
262
0.27
4.1
95


ISIS 416866
36
120
0.29
4.3
113


ISIS 416867
45
82
0.21
3.3
144









Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function, plasma concentrations of blood urea nitrogen (BUN) were measured using an automated clinical chemistry analyzer and results are presented in Table 82 expressed in mg/dL. Those antisense oligonucleotides which did not affect more than a two-fold increase in BUN levels compared to the PBS control were selected for further studies.







TABLE 82







Effect of antisense oligonucleotide treatment


on BUN levels (mg/dL) in the kidney of CD1 mice









BUN














PBS
22



ISIS 416850
24



ISIS 416858
23



ISIS 416864
24



ISIS 412223
28



ISIS 412224
29



ISIS 412225
23



ISIS 413481
23



ISIS 413482
27



ISIS 416848
23



ISIS 416849
23



ISIS 416851
21



ISIS 416852
21



ISIS 416853
22



ISIS 416854
27



ISIS 416855
23



ISIS 416856
21



ISIS 416857
17



ISIS 416859
18



ISIS 416860
25



ISIS 416861
23



ISIS 416862
21



ISIS 416863
22



ISIS 416865
20



ISIS 416866
22



ISIS 416867
20










Hematology Assays

Blood obtained from all the mice groups were sent to Antech Diagnostics for hematocrit (HCT) measurements, as well as measurements of various blood cells, such as WBC (neutrophils, lymphocytes, and monocytes), RBC, and platelets, as well as total hemoglobin content analysis. The results are presented in Tables 83 and 84. Those antisense oligonucleotides which did not affect a decrease in platelet count of more than 50% and an increase in monocyte count of more than three-fold were selected for further studies.







TABLE 83







Effect of antisense oligonucleotide treatment


on hematologic factors in CD1 mice












RBC
Hemoglobin
HCT
WBC



(106/μL)
(g/dL)
(%)
(103/μL)















PBS
10
15
51
7


ISIS 416850
10
15
49
5


ISIS 416858
9
14
50
8


ISIS 416864
10
15
52
5


ISIS 412223
9
15
48
7


ISIS 412224
10
15
50
9


ISIS 412225
9
15
50
7


ISIS 413481
9
13
45
7


ISIS 413482
10
15
50
8


ISIS 416848
9
14
47
7


ISIS 416849
9
14
48
9


ISIS 416851
9
14
47
6


ISIS 416852
9
14
49
5


ISIS 416853
11
17
56
8


ISIS 416854
9
13
43
12


ISIS 416855
9
14
50
6


ISIS 416856
9
14
47
5


ISIS 416857
10
15
53
6


ISIS 416859
10
15
49
6


ISIS 416860
10
15
51
7


ISIS 416861
9
14
48
7


ISIS 416862
9
14
49
6


ISIS 416863
9
14
48
7


ISIS 416865
9
14
50
7


ISIS 416866
9
15
51
6


ISIS 416867
10
14
47
8
















TABLE 84







Effect of antisense oligonucleotide treatment on blood


cell count in CD1 mice












Neutrophil
Lymphocyte
Monocytes
Platelets



(cells/μL)
(cells/μL)
(cells/μL)
(103/μL)















PBS
1023
6082
205
940


ISIS 416850
1144
4004
156
916


ISIS 416858
2229
5480
248
782


ISIS 416864
973
3921
141
750


ISIS 412223
1756
4599
200
862


ISIS 412224
2107
6284
195
647


ISIS 412225
1547
4969
293
574


ISIS 413481
1904
4329
204
841


ISIS 413482
1958
5584
275
818


ISIS 416848
1264
5268
180
953


ISIS 416849
1522
6967
253
744


ISIS 416851
1619
4162
194
984


ISIS 416852
1241
3646
189
903


ISIS 416853
2040
5184
225
801


ISIS 416854
2082
9375
455
1060


ISIS 416855
1443
4236
263
784


ISIS 416856
1292
3622
151
753


ISIS 416857
1334
3697
215
603


ISIS 416859
1561
4363
229
826


ISIS 416860
1291
4889
161
937


ISIS 416861
1122
5119
219
836


ISIS 416862
1118
4445
174
1007


ISIS 416863
1330
5617
226
1131


ISIS 416865
1227
5148
315
872


ISIS 416866
1201
4621
211
1045


ISIS 416867
1404
6078
188
1006









Example 38
Measurement of Half-Life of Antisense Oligonucleotide in CD1 Mouse Liver
Fifteen antisense oligonucleotides which had been evaluated in CD1 mice (Example 37) were further evaluated. CD1 mice were treated with ISIS antisense oligonucleotides and the oligonucleotide half-life as well the elapsed time for oligonucleotide degradation and elimination in the liver was evaluated.
Treatment
Groups of fifteen CD1 mice each were injected subcutaneously twice per week for 2 weeks with 50 mg/kg of ISIS 412223, ISIS 412225, ISIS 413481, ISIS 413482, ISIS 416851, ISIS 416852, ISIS 416856, ISIS 416860, ISIS 416861, ISIS 416863, ISIS 416866, ISIS 416867, ISIS 412224, ISIS 416848 or ISIS 416859. Five mice from each group were sacrificed 3 days, 28 days, and 56 days after the last dose, livers were collected for analysis.
Measurement of Oligonucleotide Concentration
The concentration of the full-length oligonucleotide was measured. The method used is a modification of previously published methods (Leeds et al., 1996; Geary et al., 1999) which consist of a phenol-chloroform (liquid-liquid) extraction followed by a solid phase extraction. An internal standard (ISIS 355868, a 27-mer 2′-O-methoxyethyl modified phosphorothioate oligonucleotide, GCGTTTGCTCTTCTTCTTGCGTTTTTT, designated herein as SEQ ID NO: 270) was added prior to extraction. Tissue sample concentrations were calculated using calibration curves, with a lower limit of quantitation (LLOQ) of approximately 1.14 μg/g. The results are presented in Table 85 expressed as μg/g liver tissue. The half-life of each oligonucleotide was also presented in Table 85.







TABLE 85







Full-length oligonucleotide concentration and


half-life in the liver of CD1 mice

















Half-Life


ISIS No
Motif
day 3
day 28
day 56
(days)















412223
5-10-5
276
127
52
21.9


412224
5-10-5
287
111
31
16.6


412225
5-10-5
279
91
47
20.7


413481
5-10-5
185
94
31
20.6


413482
5-10-5
262
95
40
19.5


416848
5-10-5
326
147
68
23.5


416851
5-10-5
319
147
68
23.8


416852
5-10-5
306
145
83
28.4


416856
5-10-5
313
115
46
19.2


416859
5-10-5
380
156
55
19.0


416860
5-10-5
216
96
36
20.6


416861
5-10-5
175
59
39
24.5


416863
5-10-5
311
101
48
19.8


416866
5-10-5
246
87
25
16.0


416867
5-10-5
246
87
35
18.9









Example 39
Tolerability of Antisense Oligonucleotides Targeting Human Factor 11 in Sprague-Dawley Rats
Fifteen antisense oligonucleotides which had been evaluated in CD1 mice (Example 37) were further evaluated in Sprague-Dawley rats for changes in the levels of various metabolic markers.
Treatment
Groups of four Sprague Dawley rats each were injected subcutaneously twice per week for 6 weeks with 50 mg/kg of ISIS 412223, ISIS 412224, ISIS 412225, ISIS 413481, ISIS 413482, ISIS 416848, ISIS 416851, ISIS 416852, ISIS 416856, ISIS 416859, ISIS 416860, ISIS 416861, ISIS 416863, ISIS 416866, or ISIS 416867. A control group of four Sprague Dawley rats was injected subcutaneously with PBS twice per week for 6 weeks. Body weight measurements were taken before and throughout the treatment period. Three days after the last dose, urine samples were collected and the rats were then sacrificed, organ weights were measured, and blood was collected for further analysis.
Body Weight and Organ Weights
The body weights of the rats were measured at the onset of the study and subsequently twice per week. The body weights are presented in Table 86 and are expressed as increase in grams over the PBS control weight taken before the start of treatment. Liver, spleen and kidney weights were measured at the end of the study, and are also presented in Table 86 as a percentage of the body weight. Those antisense oligonucleotides which did not affect more than six-fold increases in liver and spleen weight above the PBS control were selected for further studies.







TABLE 86







Change in body and organ weights of Sprague Dawley


rats after antisense oligonucleotide treatment












Body






weight
Liver
Kidney
Spleen



(g)
(%)
(%)
(%)















PBS
179
4
0.9
0.2


ISIS 412223
126
5
1.0
0.5


ISIS 412224
165
5
1.0
0.5


ISIS 412225
184
4
1.0
0.5


ISIS 413481
147
5
0.9
0.3


ISIS 413482
158
5
1.0
0.6


ISIS 416848
117
5
1.1
0.8


ISIS 416851
169
5
0.9
0.3


ISIS 416852
152
5
1.0
0.4


ISIS 416856
156
5
1.0
0.4


ISIS 416859
128
4
1.0
0.4


ISIS 416860
123
5
1.0
0.5


ISIS 416861
182
5
0.9
0.3


ISIS 416863
197
5
1.0
0.4


ISIS 416866
171
5
1.0
0.5


ISIS 416867
129
5
1.0
0.5









Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma concentrations of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Measurements of alanine transaminase (ALT) and aspartate transaminase (AST) are expressed in IU/L and the results are presented in Table 87. Those antisense oligonucleotides which did not affect an increase in ALT/AST levels above seven-fold of control levels were selected for further studies. Plasma levels of bilirubin and albumin were also measured using the same clinical chemistry analyzer and results are presented in Table 87 and expressed in mg/dL. Those antisense oligonucleotides which did not affect an increase in levels of bilirubin more than two-fold of the control levels by antisense oligonucleotide treatment were selected for further studies.







TABLE 87







Effect of antisense oligonucleotide treatment on metabolic


markers in the liver of Sprague-Dawley rats












ALT
AST
Bilirubin
Albumin



(IU/L)
(IU/L)
(mg/dL)
(mg/dL)















PBS
42
71
0.13
4


ISIS 412223
85
180
0.14
5


ISIS 412224
84
132
0.12
4


ISIS 412225
48
108
0.15
5


ISIS 413481
54
80
0.22
4


ISIS 413482
59
157
0.14
4


ISIS 416848
89
236
0.14
3


ISIS 416851
64
91
0.14
4


ISIS 416852
49
87
0.15
4


ISIS 416856
123
222
0.13
4


ISIS 416859
114
206
0.21
5


ISIS 416860
70
157
0.15
4


ISIS 416861
89
154
0.15
5


ISIS 416863
47
78
0.13
4


ISIS 416866
41
78
0.16
4


ISIS 416867
47
126
0.17
4









Kidney Function

To evaluate the effect of ISIS oligonucleotides on the kidney function, plasma concentrations of blood urea nitrogen (BUN) and creatinine were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Results are presented in Table 88, expressed in mg/dL. Those antisense oligonucleotides which did not affect more than a two-fold increase in BUN levels compared to the PBS control were selected for further studies. The total urine protein and ratio of urine protein to creatinine in total urine samples after antisense oligonucleotide treatment was calculated and is also presented in Table 88. Those antisense oligonucleotides which did not affect more than a five-fold increase in urine protein/creatinine ratios compared to the PBS control were selected for further studies.







TABLE 88







Effect of antisense oligonucleotide treatment on metabolic


markers in the kidney of Sprague-Dawley rats














Total urine




BUN
Creatinine
protein
Urine protein/



(mg/dL)
(mg/dL)
(mg/dL)
creatinine ratio















PBS
19
38
60
1.7


ISIS 412223
24
46
224
4.6


ISIS 412224
24
44
171
3.8


ISIS 412225
23
58
209
4.0


ISIS 413481
26
45
148
3.6


ISIS 413482
23
34
157
4.8


ISIS 416848
26
64
231
3.9


ISIS 416851
24
70
286
4.0


ISIS 416852
25
60
189
3.0


ISIS 416856
23
48
128
2.7


ISIS 416859
24
44
144
3.3


ISIS 416860
23
58
242
4.6


ISIS 416861
22
39
205
5.1


ISIS 416863
29
73
269
3.8


ISIS 416866
22
85
486
6.2


ISIS 416867
22
70
217
3.1









Hematology Assays

Blood obtained from all rat groups were sent to Antech Diagnostics for hematocrit (HCT) measurements, as well as measurements of the various blood cells, such as WBC (neutrophils and lymphocytes), RBC, and platelets, and total hemoglobin content. The results are presented in Tables 89 and 90. Those antisense oligonucleotides which did not affect a decrease in platelet count of more than 50% and an increase in monocyte count of more than three-fold were selected for further studies.







TABLE 89







Effect of antisense oligonucleotide treatment


on hematologic factors in Sprague-Dawley rats












RBC
Hemoglobin
HCT
WBC



(106/mL)
(g/dL)
(%)
(103/mL)















PBS
6.9
13.2
42
9


ISIS 412223
7.2
13.1
41
20


ISIS 412224
7.4
13.4
42
20


ISIS 412225
7.4
13.4
42
15


ISIS 413481
7.5
14.2
43
14


ISIS 413482
7.1
13.2
40
13


ISIS 416848
6.0
11.1
35
17


ISIS 416851
7.4
13.7
42
11


ISIS 416852
7.2
13.4
42
13


ISIS 416856
7.7
14.1
43
19


ISIS 416859
7.8
14.0
45
16


ISIS 416860
7.8
14.1
45
17


ISIS 416861
7.7
14.6
45
15


ISIS 416863
7.6
14.1
45
17


ISIS 416866
7.8
14.0
44
20


ISIS 416867
7.8
14.0
45
14
















TABLE 90







Effect of antisense oligonucleotide treatment


on blood cell count in Sprague-Dawley rats











Neutrophil
Lymphocyte
Platelets



(/mL)
(/mL)
(103/mL)
















PBS
988
7307
485



ISIS 412223
1826
16990
567



ISIS 412224
1865
16807
685



ISIS 412225
1499
13204
673



ISIS 413481
1046
12707
552



ISIS 413482
1125
11430
641



ISIS 416848
1874
14316
384



ISIS 416851
1001
9911
734



ISIS 416852
836
11956
632



ISIS 416856
3280
14328
740



ISIS 416859
1414
14323
853



ISIS 416860
1841
13986
669



ISIS 416861
1813
12865
1008



ISIS 416863
1720
14669
674



ISIS 416866
1916
16834
900



ISIS 416867
3044
10405
705










Example 40
Measurement of Half-Life of Antisense Oligonucleotide in the Liver and Kidney of Sprague-Dawley Rats
Sprague Dawley rats were treated with ISIS antisense oligonucleotides targeting human Factor 11 and the oligonucleotide half-life as well as the elapsed time for oligonucleotide degradation and elimination from the liver and kidney was evaluated.
Treatment
Groups of four Sprague Dawley rats each were injected subcutaneously twice per week for 2 weeks with 20 mg/kg of ISIS 412223, ISIS 412224, ISIS 412225, ISIS 413481, ISIS 413482, ISIS 416848, ISIS 416851, ISIS 416852, ISIS 416856, ISIS 416859, ISIS 416860, ISIS 416861, ISIS 416863, ISIS 416866, or ISIS 416867. Three days after the last dose, the rats were sacrificed, and livers and kidneys were harvested.
Measurement of Oligonucleotide Concentration
The concentration of the full-length oligonucleotide as well as the total oligonucleotide concentration (including the degraded form) was measured. The method used is a modification of previously published methods (Leeds et al., 1996; Geary et al., 1999) which consist of a phenol-chloroform (liquid-liquid) extraction followed by a solid phase extraction. An internal standard (ISIS 355868, a 27-mer 2′-O-methoxyethyl modified phosphorothioate oligonucleotide, GCGTTTGCTCTTCTTCTTGCGTTTTTT, designated herein as SEQ ID NO: 270) was added prior to extraction. Tissue sample concentrations were calculated using calibration curves, with a lower limit of quantitation (LLOQ) of approximately 1.14 μg/g. The results are presented in Tables 91 and 92, expressed as μg/g liver or kidney tissue. Half-lives were then calculated using WinNonlin software (PHARSIGHT).







TABLE 91







Full-length oligonucleotide concentration (μg/g)


in the liver and kidney of Sprague-Dawley rats












ISIS No
Motif
Kidney
Liver
















412223
5-10-5
551
97



412224
5-10-5
487
107



412225
5-10-5
202
119



413481
5-10-5
594
135



413482
5-10-5
241
95



416848
5-10-5
488
130



416851
5-10-5
264
193



416852
5-10-5
399
108



416856
5-10-5
378
84



416859
5-10-5
253
117



416860
5-10-5
247
94



416861
5-10-5
187
159



416863
5-10-5
239
82



416866
5-10-5
210
98



416867
5-10-5
201
112

















TABLE 92







Total oligonucleotide concentration (μg/g)


in the liver and kidney of Sprague-Dawley rats












ISIS No
Motif
Kidney
Liver
















412223
5-10-5
395
86



412224
5-10-5
292
78



412225
5-10-5
189
117



413481
5-10-5
366
96



413482
5-10-5
217
91



416848
5-10-5
414
115



416851
5-10-5
204
178



416852
5-10-5
304
87



416856
5-10-5
313
80



416859
5-10-5
209
112



416860
5-10-5
151
76



416861
5-10-5
165
144



416863
5-10-5
203
79



416866
5-10-5
145
85



416867
5-10-5
157
98

















TABLE 93







Half-life (days) of ISIS oligonucleotides in


the liver and kidney of Sprague-Dawley rats









ISIS No
Motif
Half-life





412223
5-10-5
22


412224
5-10-5
17


412225
5-10-5
21


413481
5-10-5
21


413482
5-10-5
20


416848
5-10-5
24


416851
5-10-5
24


416852
5-10-5
28


416856
5-10-5
19


416859
5-10-5
19


416860
5-10-5
21


416861
5-10-5
25


416863
5-10-5
20


416866
5-10-5
16


416867
5-10-5
19









Example 41
Tolerability of Antisense Oligonucleotides Targeting Human Factor 11 in CD1 Mice
ISIS oligonucleotides with 6-8-6 MOE and 5-8-5 MOE motifs targeting human Factor 11 were administered in CD1 mice evaluated for changes in the levels of various metabolic markers.
Treatment
Groups of five CD1 mice each were injected subcutaneously twice per week for 6 weeks with 50 mg/kg of ISIS 416850, ISIS 445498, ISIS 445503, ISIS 445504, ISIS 445505, ISIS 445509, ISIS 445513, ISIS 445522, ISIS 445530, ISIS 445531, or ISIS 445532. A control group of five CD1 mice was injected subcutaneously with PBS twice per week for 6 weeks. Body weight measurements were taken before and at the end of the treatment period. Three days after the last dose, the mice were sacrificed, organ weights were measured, and blood was collected for further analysis.
Body Weight and Organ Weight
The body weight changes in the mice are presented in Table 94 and are expressed increase in grams over the PBS control weight taken before the start of treatment. Liver, spleen and kidney weights were measured at the end of the study, and are also presented in Table 94 as percentage of the body weight. Those antisense oligonucleotides which did not affect more than six-fold increases in liver and spleen weight above the PBS control were selected for further studies.







TABLE 94







Change in body and organ weights of CD1 mice


after antisense oligonucleotide treatment












Body






weight
Liver
Kidney
Spleen



(g)
(%)
(%)
(%)















PBS
10
5
1.6
0.3


ISIS 416850
11
6
1.5
0.4


ISIS 445498
10
6
1.6
0.5


ISIS 445503
9
8
1.4
0.6


ISIS 445504
11
6
1.6
0.4


ISIS 445505
12
6
1.5
0.5


ISIS 445509
10
6
1.6
0.5


ISIS 445513
9
5
1.6
0.4


ISIS 445522
11
6
1.7
0.4


ISIS 445530
11
6
1.5
0.5


ISIS 445531
10
6
1.5
0.5


ISIS 445532
10
6
1.6
0.4









Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma concentrations of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Measurements of alanine transaminase (ALT) and aspartate transaminase (AST) are expressed in IU/L and the results are presented in Table 95. Those antisense oligonucleotides which did not affect an increase in ALT/AST levels above seven-fold of control levels were selected for further studies. Plasma levels of bilirubin and albumin were also measured and results are also presented in Table 95 and expressed in mg/dL. Those antisense oligonucleotides which did not affect an increase in levels of bilirubin more than two-fold of the control levels by antisense oligonucleotide treatment were selected for further studies.







TABLE 95







Effect of antisense oligonucleotide treatment


on metabolic markers in the liver of CD1 mice












ALT
AST
Bilirubin
Albumin



(IU/L)
(IU/L)
(mg/dL)
(mg/dL)















PBS
34
49
0.23
3.6


ISIS 416850
90
115
0.20
3.2


ISIS 445498
66
102
0.24
3.4


ISIS 445503
1314
852
0.28
3.4


ISIS 445504
71
107
0.17
3.4


ISIS 445505
116
153
0.18
3.2


ISIS 445509
80
117
0.17
3.1


ISIS 445513
37
84
0.22
3.1


ISIS 445522
51
110
0.19
3.4


ISIS 445530
104
136
0.18
3.2


ISIS 445531
60
127
0.16
3.2


ISIS 445532
395
360
0.20
2.9









Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function, plasma concentrations of blood urea nitrogen (BUN) were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Results are presented in Table 96, expressed in mg/dL. Those antisense oligonucleotides which did not affect more than a two-fold increase in BUN levels compared to the PBS control were selected for further studies.







TABLE 96







Effect of antisense oligonucleotide treatment


on BUN levels (mg/dL) in the kidney of CD1 mice









BUN














PBS
29



ISIS 416850
28



ISIS 445498
28



ISIS 445503
29



ISIS 445504
29



ISIS 445505
29



ISIS 445509
29



ISIS 445513
27



ISIS 445522
28



ISIS 445530
26



ISIS 445531
27



ISIS 445532
23










Hematology Assays

Blood obtained from all mice groups were sent to Antech Diagnostics for hematocrit (HCT) measurements, as well as measurements of the various blood cells, such as WBC (neutrophils and lymphocytes), RBC, and platelets, and total hemoglobin content. The results are presented in Tables 97 and 98. Those antisense oligonucleotides which did not affect a decrease in platelet count of more than 50% and an increase in monocyte count of more than three-fold were selected for further studies.







TABLE 97







Effect of antisense oligonucleotide treatment


on hematologic factors in CD1 mice












RBC
Hemoglobin
HCT
WBC



(106/mL)
(g/dL)
(%)
(103/mL)















PBS
9.6
15.0
51
6


ISIS 416850
9.8
14.8
50
6


ISIS 445498
9.4
13.9
47
5


ISIS 445503
9.2
13.6
46
8


ISIS 445504
9.6
14.7
49
5


ISIS 445505
9.6
14.6
49
5


ISIS 445509
10.2
15.3
51
5


 ISIS 445513,
9.8
15.0
50
7


ISIS 445522
9.7
14.6
49
5


ISIS 445530
10.0
15.1
50
7


ISIS 445531
9.4
14.5
48
9


ISIS 445532
9.7
14.8
48
7
















TABLE 98







Effect of antisense oligonucleotide treatment


on blood cell count in CD1 mice











Neutrophil
Lymphocyte
Platelets



(/mL)
(/mL)
(103/mL)
















PBS
1356
4166
749



ISIS 416850
1314
4710
614



ISIS 445498
1197
3241
802



ISIS 445503
1475
6436
309



ISIS 445504
959
3578
826



ISIS 445505
818
3447
725



ISIS 445509
1104
3758
1085



ISIS 445513
959
5523
942



ISIS 445522
698
3997
1005



ISIS 445530
930
5488
849



ISIS 445531
2341
6125
996



ISIS 445532
1116
5490
689










Example 42
Tolerability of Antisense Oligonucleotides Targeting Human Factor 11 in Sprague-Dawley Rats
Eight antisense oligonucleotides which had been evaluated in CD1 mice (Example 41) were further evaluated in Sprague-Dawley rats for changes in the levels of various metabolic markers.
Treatment
Groups of four Sprague Dawley rats each were injected subcutaneously twice per week for 6 weeks with 50 mg/kg of ISIS 445498, ISIS 445504, ISIS 445505, ISIS 445509, ISIS 445513, ISIS 445522, ISIS 445530, or ISIS 445531. A control group of Sprague Dawley rats was injected subcutaneously with PBS twice per week for 6 weeks. Body weight measurements were taken before and throughout the treatment period. Three days after the last dose, urine samples were collected and the rats were then sacrificed, organ weights were measured, and blood was collected for further analysis.
Body Weight and Organ Weight
The body weights of the rats were measured at the onset of the study and subsequently twice per week. The body weights are presented in Table 99 and are expressed as percent increase over the PBS control weight taken before the start of treatment. Liver, spleen and kidney weights were measured at the end of the study, and are also presented in Table 99 as a percentage of the body weight. Those antisense oligonucleotides which did not affect more than six-fold increases in liver and spleen weight above the PBS control were selected for further studies.







TABLE 99







Change in body and organ weights of Sprague Dawley rats


after antisense oligonucleotide treatment (%)












Body weight
Liver
Spleen
Kidney

















ISIS 445498
−17
+26
+107
−10



ISIS 445504
−15
+22
+116
+6



ISIS 445505
−21
+12
+146
+2



ISIS 445509
−17
+16
+252
+3



ISIS 445513
−13
+25
+194
+15



ISIS 445522
−13
+26
+184
+19



ISIS 445530
−7
+24
+99
+4



ISIS 445531
−10
+17
+89
+4










Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma concentrations of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma concentrations of ALT (alanine transaminase) and AST (aspartate transaminase) were measured and the results are presented in Table 100 expressed in IU/L. Those antisense oligonucleotides which did not affect an increase in ALT/AST levels above seven-fold of control levels were selected for further studies. Plasma levels of bilirubin and albumin were also measured using the same clinical chemistry analyzer; results are presented in Table 100 and expressed in mg/dL. Those antisense oligonucleotides which did not affect an increase in levels of bilirubin more than two-fold of the control levels by antisense oligonucleotide treatment were selected for further studies.







TABLE 100







Effect of antisense oligonucleotide treatment on metabolic


markers in the liver of Sprague-Dawley rats












ALT
AST
Bilirubin
Albumin



(IU/L)
(IU/L)
(mg/dL)
(mg/dL)















PBS
102
36
0.13
3.7


ISIS 445498
417
124
0.14
3.7


ISIS 445504
206
86
0.11
3.5


ISIS 445505
356
243
0.15
3.6


ISIS 445509
676
291
0.14
3.5


ISIS 445513
214
91
0.15
3.5


ISIS 445522
240
138
0.47
3.6


ISIS 445530
116
56
0.11
3.7


ISIS 445531
272
137
0.12
3.7









Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function, plasma concentrations of blood urea nitrogen (BUN) and creatinine were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Results are presented in Table 101, expressed in mg/dL. Those antisense oligonucleotides which did not affect more than a two-fold increase in BUN levels compared to the PBS control were selected for further studies. The total urine protein and ratio of urine protein to creatinine in total urine samples after antisense oligonucleotide treatment was calculated and is also presented in Table 101. Those antisense oligonucleotides which did not affect more than a five-fold increase in urine protein/creatinine ratios compared to the PBS control were selected for further studies.







TABLE 101







Effect of antisense oligonucleotide treatment on metabolic


markers in the kidney of Sprague-Dawley rats











BUN
Creatinine
Urine protein/



(mg/dL)
(mg/dL)
creatinine ratio
















PBS
18
0.4
1.4



ISIS 445498
25
0.5
3.1



ISIS 445504
26
0.4
4.3



ISIS 445505
24
0.4
3.8



ISIS 445509
27
0.5
4.0



ISIS 445513
24
0.4
4.6



ISIS 445522
25
0.4
6.4



ISIS 445530
22
0.4
4.2



ISIS 445531
23
0.4
3.4










Hematology Assays

Blood obtained from all rat groups were sent to Antech Diagnostics for hematocrit (HCT) measurements, as well as measurements of the various blood cells, such as WBC (neutrophils, lymphocytes, and monocytes), RBC, and platelets, and total hemoglobin content. The results are presented in Tables 102 and 103. Those antisense oligonucleotides which did not affect a decrease in platelet count of more than 50% and an increase in monocyte count of more than three-fold were selected for further studies.







TABLE 102







Effect of antisense oligonucleotide treatment


on hematologic factors in Sprague-Dawley rats












RBC
Hemoglobin
HCT
WBC



(/pL)
(g/dL)
(%)
(/nL)















PBS
8.8
16.0
55
13


ISIS 445498
8.5
14.7
49
13


ISIS 445504
8.9
14.7
50
16


ISIS 445505
9.1
15.0
50
21


ISIS 445509
8.4
14.1
47
17


ISIS 445513
7.8
13.0
44
17


ISIS 445522
7.7
13.6
47
18


ISIS 445530
8.9
14.7
50
12


ISIS 445531
8.8
14.8
50
13
















TABLE 103







Effect of antisense oligonucleotide treatment


on blood cell count in Sprague-Dawley rats












Neutrophil
Lymphocyte
Monocytes
Platelets



(%)
(%)
(%)
(/nL)















PBS
14
82
2.0
1007


ISIS 445498
9
89
2.0
1061


ISIS 445504
10
87
2.0
776


ISIS 445505
10
87
2.5
1089


ISIS 445509
11
84
3.8
1115


ISIS 445513
14
82
3.5
1051


ISIS 445522
13
84
2.8
1334


ISIS 445530
11
87
2.0
1249


ISIS 445531
10
86
2.8
1023









Example 43
Tolerability of Antisense Oligonucleotides Targeting Human Factor 11 in CD1 Mice
ISIS oligonucleotides with 4-8-4 MOE, 3-8-3 MOE, 2-10-2 MOE, 3-10-3 MOE, and 4-10-4 MOE motifs targeting human Factor 11 were administered in CD1 mice evaluated for changes in the levels of various metabolic markers.
Treatment
Groups of five CD1 mice each were injected subcutaneously twice per week for 6 weeks with 50 mg/kg of ISIS 449707, ISIS 449708, ISIS 449409, ISIS 449710, or ISIS 449711. A control group of five CD1 mice was injected subcutaneously with PBS twice per week for 6 weeks. Body weight measurements were taken before and at the end of the treatment period. Three days after the last dose, the mice were sacrificed, organ weights were measured, and blood was collected for further analysis.
Body Weight and Organ Weight
The body weights of the mice taken at the end of the study are presented in Table 104 and are expressed in grams. Liver, spleen and kidney weights were also measured at the end of the study and are also presented in Table 104 as percentage of the body weight. Those antisense oligonucleotides which did not affect more than six-fold increases in liver and spleen weight above the PBS control were selected for further studies.







TABLE 104







Change in body and organ weights of CD1 mice


after antisense oligonucleotide treatment












Body






weight
Liver
Spleen
Kidney



(g)
(%)
(%)
(%)















PBS
39
—
—
—


ISIS 449707
42
+11
+63
−5


ISIS 449708
40
+17
+66
0


ISIS 449709
40
+15
+62
−14


ISIS 449710
42
+6
+43
−7


ISIS 449711
42
+18
+63
−12









Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma concentrations of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma concentrations of ALT (alanine transaminase) and AST (aspartate transaminase) were measured and the results are presented in Table 105 expressed in IU/L. Those antisense oligonucleotides which did not affect an increase in ALT/AST levels above seven-fold of control levels were selected for further studies. Plasma levels of bilirubin and albumin were also measured using the same clinical chemistry analyzer and results are presented in Table 105 and expressed in mg/dL. Those antisense oligonucleotides which did not affect an increase in levels of bilirubin more than two-fold of the control levels by antisense oligonucleotide treatment were selected for further studies.







TABLE 105







Effect of antisense oligonucleotide treatment


on metabolic markers in the liver of CD1 mice












ALT
AST
Bilirubin
Albumin



(IU/L)
(IU/L)
(mg/dL)
(mg/dL)















PBS
39
52
0.22
3.2


ISIS 449707
41
62
0.19
2.3


ISIS 449708
66
103
0.17
2.8


ISIS 449709
62
83
0.18
2.8


ISIS 449710
43
95
0.18
2.8


ISIS 449711
52
83
0.22
2.8









Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function, plasma concentrations of blood urea nitrogen (BUN) and creatinine were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Results are presented in Table 106, expressed in mg/dL. Those antisense oligonucleotides which did not affect more than a two-fold increase in BUN levels compared to the PBS control were selected for further studies.







TABLE 106







Effect of antisense oligonucleotide treatment on metabolic


markers (mg/dL) in the kidney of CD1 mice










BUN
Creatinine













PBS
28
0.3


ISIS 449707
27
0.2


ISIS 449708
28
0.2


ISIS 449709
34
0.3


ISIS 449710
29
0.2


ISIS 449711
26
0.2









Hematology Assays

Blood obtained from all mice groups were sent to Antech Diagnostics for hematocrit (HCT), measurements, as well as measurements of the various blood cells, such as WBC (neutrophils, lymphocytes, and monocytes), RBC, and platelets, and total hemoglobin content. The results are presented in Tables 107 and 108. Those antisense oligonucleotides which did not affect a decrease in platelet count of more than 50% and an increase in monocyte count of more than three-fold were selected for further studies.







TABLE 107







Effect of antisense oligonucleotide treatment


on hematologic factors in CD1 mice












RBC
Hemoglobin
Hematocrit
WBC



(/pL)
(g/dL)
(%)
(/nL)















PBS
9.8
14.6
54
6


ISIS 449707
8.4
12.4
45
6


ISIS 449708
9.2
13.2
48
7


ISIS 449709
9.2
13.2
49
5


ISIS 449710
9.1
13.5
48
7


ISIS 449711
9.0
13.3
48
6
















TABLE 108







Effect of antisense oligonucleotide treatment


on blood cell count in CD1 mice












Neutrophils
Lymphocytes
Monocytes
Platelets



(%)
(%)
(%)
(/nL)















PBS
15
80
3
1383


ISIS 449707
11
85
3
1386


ISIS 449708
17
77
5
1395


ISIS 449709
19
76
4
1447


ISIS 449710
15
81
3
1245


ISIS 449711
15
79
6
1225









Example 44
Tolerability of Antisense Oligonucleotides Targeting Human Factor 11 in Sprague-Dawley Rats
Five antisense oligonucleotides which had been evaluated in CD1 mice (Example 43) were further evaluated in Sprague-Dawley rats for changes in the levels of various metabolic markers.
Treatment
Groups of four Sprague Dawley rats each were injected subcutaneously twice per week for 6 weeks with 50 mg/kg of ISIS 449707, ISIS 449708, ISIS 449709, ISIS 449710, or ISIS 449711. A control group of four Sprague Dawley rats was injected subcutaneously with PBS twice per week for 6 weeks. Body weight measurements were taken before and throughout the treatment period. Three days after the last dose, urine samples were collected and the rats were then sacrificed, organ weights were measured, and blood was collected for further analysis.
Body Weight and Organ Weight
The body weights of the rats were measured at the onset of the study and at the end of the study. The body weight changes are presented in Table 109 and are expressed as increase in grams over the PBS control weight taken before the start of treatment. Liver, spleen and kidney weights were measured at the end of the study, and are also presented in Table 109 as a percentage of the body weight. Those antisense oligonucleotides which did not affect more than six-fold increases in liver and spleen weight above the PBS control were selected for further studies.







TABLE 109







Change in body and organ weights of Sprague Dawley


rats after antisense oligonucleotide treatment












Body






weight
Liver
Spleen
Kidney



(g)
(%)
(%)
(%)















PBS
478
—
—
—


ISIS 449707
352
+41
+400
+80


ISIS 449708
382
+31
+259
+40


ISIS 449709
376
+8
+231
+19


ISIS 449710
344
+82
+302
+50


ISIS 449711
362
+52
+327
+72









Liver Function

To evaluate the impact of ISIS oligonucleotides on hepatic function, plasma concentrations of ALT and AST were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma concentrations of alanine transaminase (ALT) and aspartate transaminase (AST) were measured and the results are presented in Table 110 expressed in IU/L. Those antisense oligonucleotides which did not affect an increase in ALT/AST levels above seven-fold of control levels were selected for further studies. Plasma levels of bilirubin and albumin were also measured and results are presented in Table 110 and expressed in mg/dL. Those antisense oligonucleotides which did not affect an increase in levels of bilirubin more than two-fold of the control levels by antisense oligonucleotide treatment were selected for further studies.







TABLE 110







Effect of antisense oligonucleotide treatment on metabolic


markers in the liver of Sprague-Dawley rats












ALT
AST
Bilirubin
Albumin



(IU/L)
(IU/L)
(mg/dL)
(mg/dL)















PBS
41
107
0.1
3.4


ISIS 449707
61
199
0.2
3.1


ISIS 449708
25
90
0.1
3.2


ISIS 449709
63
126
0.2
3.1


ISIS 449710
36
211
0.1
2.9


ISIS 449711
32
163
0.1
2.9









Kidney Function

To evaluate the impact of ISIS oligonucleotides on kidney function, plasma concentrations of BUN and creatinine were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Results are presented in Table 111, expressed in mg/dL. Those antisense oligonucleotides which did not affect more than a two-fold increase in BUN levels compared to the PBS control were selected for further studies. The total urine protein and ratio of urine protein to creatinine in total urine samples after antisense oligonucleotide treatment was calculated and is also presented in Table 111. Those antisense oligonucleotides which did not affect more than a five-fold increase in urine protein/creatinine ratios compared to the PBS control were selected for further studies.







TABLE 111







Effect of antisense oligonucleotide treatment on metabolic


markers in the kidney of Sprague-Dawley rats











BUN
Creatinine
Urine protein/



(mg/dL)
(mg/dL)
creatinine ratio
















PBS
22
0.4
1.5



ISIS 449707
24
0.4
3.2



ISIS 449708
24
0.4
5.7



ISIS 449709
24
0.4
3.4



ISIS 449710
29
0.3
5.9



ISIS 449711
28
0.4
7.3










Hematology Assays

Blood obtained from all rat groups were sent to Antech Diagnostics for hematocrit (HCT) measurements, as well as measurements of the various blood cells, such as WBC (neutrophils, lymphocytes, and monocytes), RBC, and platelets, and total hemoglobin content. The results are presented in Tables 112 and 113. Those antisense oligonucleotides which did not affect a decrease in platelet count of more than 50% and an increase in monocyte count of more than three-fold were selected for further studies.







TABLE 112







Effect of antisense oligonucleotide treatment


on hematologic factors in Sprague-Dawley rats












RBC
Hemoglobin
Hematocrit
WBC



(/pL)
(g/dL)
(%)
(/nL)















PBS
8.2
15.1
50
16


ISIS 449707
6.0
12.0
40
20


ISIS 449708
6.6
12.2
40
22


ISIS 449709
6.9
12.6
41
14


ISIS 449710
6.3
12.5
41
13


ISIS 449711
6.4
12.6
43
13
















TABLE 113







Effect of antisense oligonucleotide treatment


on blood cell count in Sprague-Dawley rats












Neutrophils
Lymphocytes
Monocytes
Platelets



(%)
(%)
(%)
(/nL)















PBS
12
84
2
1004


ISIS 449707
6
91
2
722


ISIS 449708
6
92
2
925


ISIS 449709
5
91
3
631


ISIS 449710
6
91
2
509


ISIS 449711
7
90
2
919









Example 45
Dose-Dependent Pharmacologic Effect of Antisense Oligonucleotides Targeting Human Factor 11 in Cynomolgus Monkeys
Several antisense oligonucleotides were tested in cynomolgus monkeys to determine the pharmacologic effects of the oligonucleotides on Factor 11 activity, anticoagulation and bleeding times, liver and kidney distributions, and tolerability. All the ISIS oligonucleotides used in this study target human Factor 11 mRNA and are also fully cross-reactive with the rhesus monkey gene sequence (see Table 44). It is expected that the rhesus monkey ISIS oligonucleotides are fully cross-reactive with the cynomolgus monkey gene sequence as well. At the time the study was undertaken, the cynomolgus monkey genomic sequence was not available in the National Center for Biotechnology Information (NCBI) database; therefore, cross-reactivity with the cynomolgus monkey gene sequence could not be confirmed.
Treatment
Groups, each consisting of two male and three female monkeys, were injected subcutaneously with ISIS 416838, ISIS 416850, ISIS 416858, ISIS 416864, or ISIS 417002 in escalating doses. Antisense oligonucleotide was administered to the monkeys at 5 mg/kg three times per a week for week 1; 5 mg/kg twice per week for weeks 2 and 3; 10 mg/kg three times per week for week 4; 10 mg/kg twice per week for weeks 5 and 6; 25 mg/kg three times per week for week 7; and 25 mg/kg twice per week for weeks 8, 9, 10, 11, and 12. One control group, consisting of two male and three female monkeys, was injected subcutaneously with PBS according to the same dosing regimen. An additional experimental group, consisting of two male and three female monkeys, was injected subcutaneously with ISIS 416850 in a chronic, lower dose regimen. Antisense oligonucleotide was administered to the monkeys at 5 mg/kg three times per week for week 1; 5 mg/kg twice per week for week 2 and 3; 10 mg/kg three times per week for week 4; and 10 mg/kg twice per week for weeks 5 to 12. Body weights were measured weekly. Blood samples were collected 14 days and 5 days before the start of treatment and subsequently once per week for Factor 11 protein activity analysis in plasma, fibrinogen measurement, PT and aPTT measurements, bleeding times, and measurement of various hematologic factors. On day 85, the monkeys were euthanized by exsanguination while under deep anesthesia, and organs harvested for further analysis.
RNA Analysis
On day 85, RNA was extracted from liver tissue for real-time PCR analysis of Factor 11 using primer probe set LTS00301 (forward primer sequence ACACGCATTAAAAAGAGCAAAGC, designated herein as SEQ ID NO 271; reverse primer sequence CAGTGTCATGGTAAAATGAAGAATGG, designated herein as SEQ ID NO: 272; and probe sequence TGCAGGCACAGCATCCCAGTGTTCTX, designated herein as SEQ ID NO. 273). Results are presented as percent inhibition of Factor 11, relative to PBS control. As shown in Table 114, treatment with ISIS oligonucleotides resulted in significant reduction of Factor 11 mRNA in comparison to the PBS control.







TABLE 114







Inhibition of Factor 11 mRNA in the cynomolgus


monkey liver relative to the PBS control










ISIS No
% inhibition







416838
37



416850
84



416858
90



416864
44



417002
57










Protein Analysis

Plasma samples from all monkey groups taken on different days were analyzed by a sandwich-style ELISA assay (Affinity Biologicals Inc.) using an affinity-purified polyclonal anti-Factor 11 antibody as the capture antibody and a peroxidase-conjugated polyclonal anti-Factor 11 antibody as the detecting antibody. Monkey plasma was diluted 1:50 for the assay. Peroxidase activity was expressed by incubation with the substrate o-phenylenediamine. The color produced was quantified using a microplate reader at 490 nm and was considered to be proportional to the concentration of Factor 11 in the samples.
The results are presented in Table 115, expressed as percentage reduction relative to that of the PBS control. Treatment with ISIS 416850 and ISIS 416858 resulted in a time-dependent decrease in protein levels.







TABLE 115







Inhibition of Factor 11 protein in the cynomolgus


monkey liver relative to the PBS control














ISIS
ISIS
ISIS
ISIS
ISIS
ISIS


Days
416838
416850
416858
416864
417002
416850*
















−14
0
0
0
0
0
0


−5
0
0
0
5
0
1


8
3
8
6
7
0
6


15
4
4
16
9
4
13


22
5
11
23
7
2
12


29
8
15
28
10
8
20


36
11
17
35
9
8
22


43
5
23
39
9
9
24


50
8
42
49
10
13
30


57
10
49
60
7
24
34


64
11
55
68
5
26
37


71
12
57
71
10
30
41


78
10
63
73
9
22
42


85
10
64
78
8
23
34









PT and aPTT Assay

Blood samples were collected in tubes containing sodium citrate. PT and aPTT were determined in duplicate with an ACL 9000 coagulation instrument (Instrumentation Laboratory, Italy). The results were interpolated on a standard curve of serial dilutions citrated control monkey plasma tested to give a reported result in percent normal.
Prothrombin Time (PT) and Activated Partial Thromboplastin Time (aPTT) were measured using platelet poor plasma (PPP) from monkeys treated with ISIS oligonucleotides. PT and aPTT values are provided in Tables 116 and 117 and are reported as International Normalized Ratio (INR) values. INR values for PT and aPTT were determined by dividing the PT or aPTT value for each experimental group by the PT or aPTT for the PBS treated group. This ratio was then raised to the power of the International Sensitivity Index (ISI) of the tissue factor used. The ISIS oligonucleotide, ISIS 416850, given with the chronic dose regimen is distinguished from the other oligonucleotides with an asterisk (*).
As shown in Table 116, PT was not significantly prolonged in monkeys treated with ISIS oligonucleotides either in the escalating dose regimen or the chronic dose regimen. However, aPTT was prolonged in a dose-dependent manner, as presented in Table 117. These data suggest that antisense reduction of Factor 11 affects the contact activation pathway, but not the extrinsic pathway of blood coagulation. Therefore, antisense reduction of Factor 11 is useful for inhibiting the formation of a thrombus or clot in response to an abnormal vessel wall, but not in response to tissue injury.







TABLE 116







Effect of ISIS antisense oligonucleotides on


PT ratio in cynomolgus monkeys














ISIS
ISIS
ISIS
ISIS
ISIS
ISIS


day
416838
416850
416858
416864
417002
416850*
















−14
1.00
1.00
1.00
1.00
1.00
1.00


−5
1.00
1.00
1.00
1.00
1.00
1.00


8
1.03
1.00
1.05
1.02
1.02
1.03


15
1.03
1.02
1.07
1.07
1.04
1.06


22
1.07
1.02
1.06
1.03
1.04
1.06


29
1.03
1.03
1.08
1.06
1.01
1.00


36
1.05
1.02
1.07
1.06
1.05
1.06


43
1.03
1.01
1.08
1.04
1.03
1.02


50
1.02
1.02
1.03
1.01
0.99
0.98


57
1.04
1.04
1.09
1.08
1.03
n.d.


64
1.04
1.03
1.09
1.10
1.03
n.d.


71
1.02
1.03
1.07
1.07
0.99
n.d.


78
1.04
1.05
1.10
1.08
1.02
n.d.


85
1.05
1.04
1.07
1.13
1.02
n.d.





n.d. = no data













TABLE 117







Effect of ISIS antisense oligonucleotides


on aPTT ratio in cynomolgus monkeys














ISIS
ISIS
ISIS
ISIS
ISIS
ISIS


day
416838
416850
416858
416864
417002
416850*
















−14
1.00
1.00
1.00
1.00
1.00
1.00


−5
1.00
1.00
1.00
1.00
1.00
1.00


8
1.07
1.05
1.03
1.05
1.05
1.12


15
1.05
1.05
1.07
1.03
1.03
1.07


22
1.20
1.13
1.18
1.11
1.16
1.21


29
1.19
1.13
1.20
1.13
1.11
1.26


36
1.20
1.26
1.36
1.19
1.18
1.34


43
1.18
1.17
1.28
1.07
1.06
1.22


50
1.25
1.68
1.55
1.26
1.18
1.35


57
1.21
1.59
1.59
1.19
1.22
n.d.


64
1.18
1.64
1.60
1.12
1.11
n.d.


71
1.15
1.76
1.70
1.18
1.16
n.d.


78
1.19
1.88
1.79
1.18
1.18
n.d.


85
1.22
1.99
1.76
1.25
1.20
n.d.





n.d. = no data






Protein Activity Analysis

Blood samples were collected at various time points and Factor 11 proenzyme was measured using a F11 assay based on clotting time. Clotting times were determined in duplicate with a ST4 semi-automated coagulation instrument (Diagnostica Stago, N.J.). Thirty μl of citrated sample plasma diluted 1/20 in HEPES-NaCl buffer with BSA was incubated with 30 μl aPTT reagent (Automated aPTT, Organon Technika, N.C.) and 30 μl of citrated plasma deficient of Factor 11 (George King Bio-Medical Inc.) at 37° C. for 5 min, followed by the addition of 30 μl of 25 mM CaCl2 to initiate clotting. Results were interpolated on a standard curve of serially diluted citrated control plasma.
Results are presented in Table 118 as percent inhibition of Factor 11 activity, relative to PBS control. The ISIS oligonucleotide, ISIS 416850, given with the chronic dose regimen is distinguished from the other oligonucleotides with an asterisk (*).







TABLE 118







Inhibition of Factor 11 protein by ISIS antisense


oligonucleotides given in escalating dose/chronic


dose regimen in cynomolgus monkeys













Days








before/after
ISIS
ISIS
ISIS
ISIS
ISIS
ISIS


treatment
416838
416850
416858
416864
417002
416850*
















−14
0
0
0
0
0
0


−5
0
0
0
5
0
1


8
3
8
6
7
0
6


15
4
4
16
9
4
13


22
5
11
23
7
2
12


29
8
15
28
10
8
20


36
11
17
35
9
8
24


43
5
23
39
9
9
24


50
8
42
49
10
13
30


57
10
49
60
7
24
n.d.


64
11
55
68
5
26
n.d.


71
12
57
71
10
30
n.d.


78
10
63
73
9
22
n.d.


85
10
64
78
8
23
n.d.





n.d. = no data






Fibrinogen Assay

Nine parts of fresh monkey plasma was collected into one part of trisodium citrate. The samples were evaluated of fibrinogen content using an ACL 9000 coagulation instrument (Instrumentation Laboratory, Italy). Results are presented in Table 119 expressed in mg/dL. The ISIS oligonucleotide, ISIS 416850, given with the chronic dose regimen is distinguished from the other oligonucleotides with an asterisk (*).







TABLE 119







Effect of ISIS antisense oligonucleotides on fibrinogen


levels in cynomolgus monkeys














Days before/after


ISIS
ISIS
ISIS
ISIS
ISIS


treatment
PBS
ISIS 416838
416850
416858
416864
417002
416850*

















−14
296
251
310
277
300
291
274


−5
246
205
261
246
243
222
227


8
245
209
281
246
227
221
232


15
207
198
270
219
210
195
174


22
219
183
243
222
184
199
192


29
231
184
234
220
205
199
192


36
235
182
232
225
202
191
185


43
231
186
219
229
198
187
194


50
251
216
215
259
233
236
204


57
235
190
186
225
200
201
n.d.


64
240
190
190
236
218
236
n.d.


71
233
199
178
239
245
228
n.d.


78
234
189
177
234
250
221
n.d.


85
246
196
187
243
240
224
n.d.





n.d. = no data






Bleeding Assay

On different days during the treatment period, bleeding assay was performed using a Surgicutt Jr. device (ITC, New Jersey). Monkeys were placed in monkey chair with their arm placed in a steady support. The arm was lightly shaved and a sphygmomanometer was placed on the upper arm. The cuff of the sphygmomanometer was inflated to 40 mm Hg and this pressure was maintained throughout the procedure. The area on the upper arm to be incised was cleansed with an antiseptic swab and the Surgicutt Jr device was used to make an incision over the lateral aspect, volar surface of the forearm, parallel to and 5 cm below the antecubital crease. At the exact moment the incision was made, a stopwatch was started. Every 30 seconds, blood from the incision was blotted out using a blotting paper without directly touching the incision, so that formation of the platelet plug was not disturbed. Blood was blotted out every 30 seconds until blood no longer stained the paper. The stopwatch was then stopped and the bleeding time determined. The sphygmomanometer was removed from the animal's arm, the incision site was antiseptically swabbed and a wound closure strip applied. The results are provided in Table X, expressed in seconds. The results are provided in Table 120. The ISIS oligonucleotide, ISIS 416850, given with the chronic dose regimen is distinguished from the other oligonucleotides with an asterisk (*).
These data suggest that the hemorrhagic potential of the compounds provided herein is low.







TABLE 120







Bleeding assay in cynomolgus monkeys














Days before/after


ISIS
ISIS
ISIS
ISIS
ISIS


treatment
PBS
ISIS 416838
416850
416858
416864
417002
416850*

















−14
147
200
172
154
166
185
177


−5
153
150
127
149
111
175
93


15
111
167
165
146
153
174
149


22
113
165
151
100
133
194
143


36
174
166
137
206
205
186
221


43
157
120
216
111
146
120
156


57
147
238
195
138
216
206
n.d.


64
113
131
201
113
218
146
n.d.


78
114
145
203
186
170
163
n.d.


85
147
201
201
191
203
182
n.d.









Platelet Aggregation Assay

Platelet aggregation was initiated by adding 1 mmol/L ADP and/or 3 μg collagen (depending on the collection day, as outlined in Table 121) to plasma samples, and was allowed to proceed for 10 minutes. Aggregation was characterized by recording the change in the electrical resistance or impedance and the change in the initial slope of aggregation after platelet shape change. The aggregation test was performed twice per sample on each collection day and the average value was taken. The ISIS oligonucleotide, ISIS 416850, given with the chronic dose regimen is distinguished from the other oligonucleotides with an asterisk (*).







TABLE 121







Effect of antisense oligonucleotide treatment on platelet


aggregation in cynomolgus monkeys in Ohms

















day −5
day 15
day 36
day 43
day 57
day 64
day 78
day 85
day 85



(with
(with
(with
(with
(with
(with
(with
(with
(with



collagen)
ADP)
ADP)
collagen)
ADP)
collagen)
ADP)
ADP)
collagen)



















PBS
17
15
7
14
16
13
12
16
17


ISIS
15
15
8
16
7
13
11
15
24


416838











ISIS
23
12
16
16
18
17
9
22
26


416850











ISIS
22
19
17
16
11
14
8
18
23


416858











ISIS
27
20
17.8
20
18
17
13
22
28


416864











ISIS
21
16
13.9
19
18
18
18
22
24


417002











ISIS
21
14
11.6
21
n.d.
n.d.
n.d.
n.d.
n.d.


416850*





n.d. = no data






Body and Organ Weights

Body weights were taken once weekly throughout the dosing regimen. The measurements of each group are given in Table 122 expressed in grams. The results indicate that treatment with the antisense oligonucleotides did not cause any adverse changes in the health of the animals, which may have resulted in a significant alteration in weight compared to the PBS control. Organ weights were taken after the animals were euthanized and livers, kidneys and spleens were harvested and weighed. The results are presented in Table 123 and also show no significant alteration in weights compared to the PBS control, except for ISIS 416858, which shows increase in spleen weight. The ISIS oligonucleotide, ISIS 416850, given with the chronic dose regimen is distinguished from the other oligonucleotides with an asterisk (*).







TABLE 122







Weekly measurements of body weights (g) of cynomolgus monkeys
















ISIS
ISIS
ISIS
ISIS
ISIS
ISIS


day
PBS
416838
416850
416858
416864
417002
416850*

















1
2780
2720
2572
2912
2890
2640
2665


8
2615
2592
2430
2740
2784
2523
2579


15
2678
2642
2474
2760
2817
2571
2607


22
2715
2702
2514
2800
2857
2617
2661


29
2717
2689
2515
2763
2863
2622
2667


36
2738
2708
2545
2584
3327
2631
2656


43
2742
2700
2544
2607
3355
2630
2670


50
2764
2731
2613
2646
3408
2652
2679


57
2763
2737
2629
2617
3387
2654
n.d.


64
2781
2746
2642
2618
3384
2598
n.d.


71
2945
2869
2769
2865
2942
2727
n.d.


78
2815
2766
2660
2713
2822
2570
n.d.





n.d. = no data













TABLE 123







Organ weights (g) of cynomolgus monkeys


after antisense oligonucleotide treatment











Liver
Spleen
Kidney
















PBS
46
4
11



ISIS 416838
63
5
12



ISIS 416580
64
4
16



ISIS 416858
60
12
13



ISIS 416864
53
5
14



ISIS 417002
51
5
15










Liver Function

To evaluate the impact of ISIS oligonucleotides on hepatic function, plasma concentrations of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma concentrations of ALT (alanine transaminase) and AST (aspartate transaminase) were measured and the results are presented in Tables 124 and 125 expressed in IU/L. Those antisense oligonucleotides which did not affect an increase in ALT/AST levels above seven-fold of control levels were selected for further studies. Plasma levels of bilirubin were also measured and results are presented in Table 126 expressed in mg/dL. Those antisense oligonucleotides which did not affect an increase in levels of bilirubin more than two-fold of the control levels by antisense oligonucleotide treatment were selected for further studies. The ISIS oligonucleotide, ISIS 416850, given with the chronic dose regimen is distinguished from the other oligonucleotides with an asterisk (*).







TABLE 124







Effect of antisense oligonucleotide treatment on ALT


(IU/L) in the liver of cynomolgus monkeys














Days before/after


ISIS
ISIS
ISIS
ISIS
ISIS


treatment
PBS
ISIS 416838
416850
416858
416864
417002
416850*

















−14
57
76
54
47
54
61
80


22
39
36
41
28
37
36
42


43
36
35
43
36
36
35
41


64
38
40
60
47
43
42
n.d.


85
34
41
75
50
43
116
n.d.





n.d. = no data













TABLE 125







Effect of antisense oligonucleotide treatment on AST


(IU/L) in the liver of cynomolgus monkeys














Days before/after


ISIS
ISIS
ISIS
ISIS
ISIS


treatment
PBS
ISIS 416838
416850
416858
416864
417002
416850*

















−14
71
139
81
58
76
114
100


22
43
39
45
38
41
44
39


43
38
32
50
39
40
42
40


64
35
33
56
50
46
37
n.d.


85
41
30
82.
49
56
50
n.d.





n.d. = no data













TABLE 126







Effect of antisense oligonucleotide treatment on


bilirubin (mg/dL) in the liver of cynomolgus monkeys














Days before/after


ISIS
ISIS
ISIS
ISIS
ISIS


treatment
PBS
ISIS 416838
416850
416858
416864
417002
416850*

















−14
0.24
0.26
0.21
0.27
0.31
0.26
0.28


22
0.16
0.17
0.13
0.18
0.22
0.20
0.19


43
0.17
0.17
0.13
0.14
0.17
0.21
0.18


64
0.19
0.15
0.14
0.12
0.16
0.14
n.d.


85
0.20
0.13
0.14
0.14
0.17
0.12
n.d.





n.d. = no data






Kidney Function

To evaluate the impact of ISIS oligonucleotides on kidney function, urine samples were collected. The ratio of urine protein to creatinine in urine samples after antisense oligonucleotide treatment was calculated and is presented in Table 127. Those antisense oligonucleotides which did not affect more than a five-fold increase in urine protein/creatinine ratios compared to the PBS control were selected for further studies.







TABLE 127







Effect of antisense oligonucleotide treatment on urine


protein to creatinine ratio in cynomolgus monkeys










Day 80
Day 84













PBS
0.09
0.10


ISIS 416838
0.13
0.13


ISIS 416850
0.09
0.12


ISIS 416858
0.10
0.07


ISIS 416864
0.36
0.34


ISIS 417002
0.18
0.24









Measurement of Oligonucleotide Concentration

The concentration of the full-length oligonucleotide as well as the elapsed time oligonucleotide degradation and elimination from the liver and kidney were evaluated. The method used is a modification of previously published methods (Leeds et al., 1996; Geary et al., 1999) which consist of a phenol-chloroform (liquid-liquid) extraction followed by a solid phase extraction. An internal standard (ISIS 355868, a 27-mer 2′-O-methoxyethyl modified phosphorothioate oligonucleotide, GCGTTTGCTCTTCTTCTTGCGTTTTTT, designated herein as SEQ ID NO: 270) was added prior to extraction. Tissue sample concentrations were calculated using calibration curves, with a lower limit of quantitation (LLOQ) of approximately 1.14 μg/g. Half-lives were then calculated using WinNonlin software (PHARSIGHT). The results are presented in Tables 128 and 129, expressed as μg/g liver or kidney tissue.








TABLE 128







Full-length oligonucleotide concentration (μg/g)


in the liver and kidney of cynomolgus monkeys









ISIS No.
Kidney
Liver





416838
1339
1087


416850
2845
1225


416858
1772
1061


416864
2093
1275


417002
2162
1248
















TABLE 129







Total oligonucleotide concentration (μg/g)


in the liver and kidney of cynomolgus monkeys









ISIS No.
Kidney
Liver





416838
1980
1544


416850
3988
1558


416858
2483
1504


416864
3522
1967


417002
3462
1757









Hematology Assays

Blood obtained from all monkey groups were sent to Korea Institute of Toxicology (KIT) for HCT, MCV, MCH, and MCHC analysis, as well as measurements of the various blood cells, such as WBC (neutrophils, lymphocytes, monocytes, eosinophils, basophils, reticulocytes), RBC, platelets and total hemoglobin content. The results are presented in Tables 130-143. Those antisense oligonucleotides which did not affect a decrease in platelet count of more than 50% and an increase in monocyte count of more than three-fold were selected for further studies. The ISIS oligonucleotide, ISIS 416850, given with the chronic dose regimen is distinguished from the other oligonucleotides with an asterisk (*).







TABLE 130







Effect of antisense oligonucleotide treatment on WBC


count (×103/μL) in cynomolgus monkeys



















ISIS
ISIS
ISIS



PBS
ISIS 416838
ISIS 416850
ISIS 416858
416864
417002
416850*

















day −14
14
12
13
14
13
13
15


day −5
13
12
13
14
13
14
15


day 8
10
10
10
12
11
10
13


day 15
10
10
9
11
10
10
16


day 22
12
11
10
11
10
10
15


day 29
11
11
11
12
10
10
14


day 36
10
10
10
12
10
11
16


day 43
10
10
9
11
10
10
15


day 50
12
11
11
13
12
13
15


day 57
11
12
11
13
12
12
n.d.


day 64
11
13
11
12
11
11
n.d.


day 71
15
15
15
13
14
12
n.d.


day 78
10
11
12
11
11
9
n.d.


day 85
10
12
15
11
12
10
n.d.





n.d. = no data













TABLE 131







Effect of antisense oligonucleotide treatment on RBC


count (×106/μL) in cynomolgus monkeys



















ISIS
ISIS
ISIS



PBS
ISIS 416838
ISIS 416850
ISIS 416858
416864
417002
416850*

















day −14
5.7
5.6
5.3
5.6
5.5
5.6
5.5


day −5
5.7
5.6
5.5
5.6
5.6
5.6
5.5


day 8
5.7
5.7
5.4
5.6
5.7
5.6
5.5


day 15
5.6
5.6
5.3
5.4
5.7
5.4
5.3


day 22
5.5
5.4
5
5.3
5.3
5.2
5.1


day 29
5.6
5.3
4.9
5.3
5.3
5.2
5.2


day 36
5.7
5.5
5.3
5.5
5.6
5.4
5.3


day 43
5.7
5.6
5.2
5.5
5.5
5.4
5.2


day 50
5.8
5.5
5.2
5.5
5.6
5.4
5.3


day 57
5.7
5.5
5.2
5.6
5.5
4.9
n.d.


day 64
5.8
5.6
5.4
5.7
5.6
5.4
n.d.


day 71
5.6
5.5
5.4
5.6
5.6
5.5
n.d.


day 78
5.6
5.4
5.3
5.4
5.3
5.4
n.d.


day 85
5.6
5.5
5.5
5.5
5.4
5.4
n.d.





n.d. = no data













TABLE 132







Effect of antisense oligonucleotide treatment


on hemoglobin (g/dL) in cynomolgus monkeys
















ISIS
ISIS
ISIS
ISIS
ISIS
ISIS



PBS
416838
416850
416858
416864
417002
416850*


















day −14
13.2
12.9
12.4
13.2
12.7
13.0
12.8


day −5
13.1
13.1
12.7
13.2
13.0
13.2
12.8


day 8
13.1
12.9
12.4
12.8
12.7
12.8
12.5


day 15
12.9
12.9
12.1
12.6
12.8
12.3
12.2


day 22
12.7
12.5
11.6
12.4
12.1
12.1
11.7


day 29
12.8
12.4
11.5
12.3
12.1
12.0
12.0


day 36
13.0
12.8
12.2
12.6
12.5
12.5
12.3


day 43
12.9
12.7
11.8
12.4
12.2
12.3
11.8


day 50
12.6
12.3
11.8
12.2
12.1
12.3
11.9


day 57
13.1
12.6
12.1
12.7
12.3
11.3
n.d.


day 64
13.1
12.6
12.3
12.8
12.1
12.2
n.d.


day 71
12.9
12.7
12.3
12.7
12.2
12.5
n.d.


day 78
13.0
12.5
12.2
12.4
11.9
12.4
n.d.


day 85
13.2
12.4
12.7
11.9
12.3
12.2
n.d.





n.d. = no data













TABLE 133







Effect of antisense oligonucleotide treatment


on hematocrit (%) in cynomolgus monkeys
















ISIS
ISIS
ISIS
ISIS
ISIS
ISIS



PBS
416838
416850
416858
416864
417002
416850*


















day −14
46
42
41
43
43
44
44


day −5
44
42
43
42
44
45
43


day 8
44
43
43
43
44
44
43


day 15
44
42
40
40
42
40
40


day 22
45
43
41
41
42
41
40


day 29
46
43
41
41
43
42
42


day 36
46
43
42
40
42
42
41


day 43
46
43
40
40
42
41
40


day 50
48
44
42
41
44
43
42


day 57
46
43
42
41
42
38
n.d.


day 64
47
44
43
42
42
41
n.d.


day 71
46
44
43
42
44
43
n.d.


day 78
43
41
41
39
39
40
n.d.


day 85
43
42
42
39
40
41
n.d.





n.d. = no data













TABLE 134







Effect of antisense oligonucleotide treatment


on MCV (fL) in cynomolgus monkeys
















ISIS
ISIS
ISIS
ISIS
ISIS
ISIS



PBS
416838
416850
416858
416864
417002
416850*


















day −14
81
77
78
77
79
79
81


day −5
78
76
77
75
79
80
78


day 8
77
77
80
77
78
79
79


day 15
78
75
76
74
74
76
75


day 22
84
80
83
77
79
79
79


day 29
83
81
83
78
80
81
82


day 36
81
78
80
75
76
78
76


day 43
80
78
79
74
77
77
77


day 50
84
80
83
76
79
80
80


day 57
82
79
80
74
77
80
n.d.


day 64
81
79
79
73
75
76
n.d.


day 71
84
80
80
75
79
78
n.d.


day 78
78
76
79
72
74
75
n.d.


day 85
77
77
77
72
74
76
n.d.





n.d. = no data













TABLE 135







Effect of antisense oligonucleotide treatment


on MCH (pg) in cynomolgus monkeys
















ISIS
ISIS
ISIS
ISIS
ISIS
ISIS



PBS
416838
416850
416858
416864
417002
416850*


















day −14
23
23
23
24
23
24
24


day −5
23
23
23
23
23
24
23


day 8
23
23
23
23
23
23
23


day 15
23
23
23
23
23
23
23


day 22
23
23
24
24
23
23
23


day 29
23
23
23
23
23
23
23


day 36
23
23
23
23
23
23
23


day 43
23
23
23
23
22
23
23


day 50
22
23
23
23
22
23
23


day 57
23
23
23
22
23
23
n.d.


Day 64
23
23
22
22
23
22
n.d.


Day 71
23
23
23
22
23
23
n.d.


Day 78
23
23
23
23
23
23
n.d.


Day 85
23
23
22
22
23
23
n.d.





n.d. = no data













TABLE 136







Effect of antisense oligonucleotide treatment


on MCHC (g/dL) in cynomolgus monkeys
















ISIS
ISIS
ISIS
ISIS
ISIS
ISIS



PBS
416838
416850
416858
416864
417002
416850*


















day −14
29
30
30
31
29
30
29


day −5
30
31
30
31
29
30
30


day 8
30
30
29
30
29
29
29


day 15
30
31
30
31
30
31
30


day 22
28
29
28
30
29
29
29


day 29
28
29
28
30
29
29
28


day 36
28
30
29
31
30
30
30


day 43
28
30
29
31
29
30
30


day 50
26
28
28
30
28
29
29


day 57
29
29
29
31
29
29
n.d.


day 64
28
29
29
30
29
30
n.d.


day 71
28
29
28
30
28
29
n.d.


day 78
30
30
29
32
30
31
n.d.


day 85
31
30
30
31
30
30
n.d.





n.d. = no data













TABLE 137







Effect of antisense oligonucleotide treatment on platelet


count (×103/μL) in cynomolgus monkeys
















ISIS
ISIS
ISIS
ISIS
ISIS
ISIS



PBS
416838
416850
416858
416864
417002
416850*


















day −14
349
377
528
419
434
442
387


day −5
405
425
573
463
456
466
434


day 8
365
387
548
391
438
435
401


day 15
375
387
559
400
439
410
396


day 22
294
319
466
316
364
377
347


day 29
311
337
475
336
397
410
370


day 36
326
370
505
371
428
415
379


day 43
336
365
490
342
351
393
391


day 50
379
372
487
331
419
389
351


day 57
345
371
528
333
409
403
n.d.


day 64
329
358
496
295
383
436
n.d.


day 71
322
365
465
286
394
490
n.d.


day 78
309
348
449
262
366
432
n.d.


day 85
356
344
458
267
387
418
n.d.





n.d. = no data













TABLE 138







Effect of antisense oligonucleotide treatment


on reticulocytes (%) in cynomolgus monkeys
















ISIS
ISIS
ISIS
ISIS
ISIS
ISIS



PBS
416838
416850
416858
416864
417002
416850*


















day −14
1.4
1.0
1.7
1.0
0.9
0.9
1.1


day −5
1.0
0.9
1.2
0.9
0.9
0.8
0.8


day 8
1.0
1.2
1.2
1.2
0.8
1.1
1.1


day 15
1.5
1.2
1.9
1.6
0.8
1.1
1.0


day 22
1.2
1.2
1.9
1.3
0.9
1.2
1.0


day 29
1.6
1.6
2.5
1.5
1.3
1.6
1.4


day 36
1.7
1.6
2.2
1.6
1.3
1.3
1.3


day 43
1.3
1.2
1.6
1.3
1.1
1.1
1.0


day 50
1.6
1.6
2.7
1.5
1.3
1.6
1.2


day 57
1.8
1.5
2.0
1.4
1.0
4.6
n.d.


day 64
1.3
1.3
1.7
1.0
0.8
1.3
n.d.


day 71
1.6
1.3
1.8
1.3
1.0
1.3
n.d.


day 78
1.5
1.4
1.8
1.2
1.2
1.3
n.d.


day 85
1.5
1.5
2.3
1.3
1.5
1.4
n.d.





n.d. = no data













TABLE 139







Effect of antisense oligonucleotide treatment


on neutrophils (%) in cynomolgus monkeys
















ISIS
ISIS
ISIS
ISIS
ISIS
ISIS



PBS
416838
416850
416858
416864
417002
416850*


















day −14
40
36
49
37
53
43
48


day −5
37
35
52
46
51
43
53


day 8
54
42
57
51
52
46
53


day 15
49
43
58
54
59
57
73


day 22
41
37
57
47
59
55
64


day 29
44
36
53
43
44
45
42


day 36
37
39
57
47
58
61
72


day 43
40
30
50
45
57
57
61


day 50
36
31
45
46
49
61
62


day 57
41
32
49
44
57
54
n.d.


day 64
40
30
41
37
49
55
n.d.


day 71
38
28
27
26
42
34
n.d.


day 78
42
35
42
39
48
51
n.d.


day 85
30
22
60
40
39
36
n.d.





n.d. = no data













TABLE 140







Effect of antisense oligonucleotide treatment


on lymphocytes (%) in cynomolgus monkeys
















ISIS
ISIS
ISIS
ISIS
ISIS
ISIS



PBS
416838
416850
416858
416864
417002
416850*


















day −14
54
59
47
58
42
53
47


day −5
56
59
43
49
44
53
43


day 8
43
54
39
45
45
50
44


day 15
47
53
38
43
38
40
24


day 22
54
59
39
49
37
41
33


day 29
51
59
43
51
51
50
53


day 36
58
57
39
49
38
35
26


day 43
55
65
45
51
39
39
36


day 50
59
64
49
48
46
34
35


day 57
55
63
45
51
39
40
n.d.


day 64
56
64
53
56
46
39
n.d.


day 71
56
65
61
66
52
59
n.d.


day 78
53
60
51
54
46
41
n.d.


day 85
63
72
34
52
54
56
n.d.





n.d. = no data













TABLE 141







Effect of antisense oligonucleotide treatment


on eosinophils (%) in cynomolgus monkeys
















ISIS
ISIS
ISIS
ISIS
ISIS
ISIS



PBS
416838
416850
416858
416864
417002
416850*


















day −14
1.3
0.6
1.0
0.7
1.0
0.3
0.5


day −5
1.5
0.6
1.6
1.3
0.9
0.3
0.7


day 8
0.9
0.4
1.1
0.3
0.7
0.2
0.5


day 15
0.7
0.3
1.0
0.3
0.5
0.1
0.2


day 22
0.9
0.5
0.7
0.6
0.9
0.3
0.5


day 29
0.9
0.3
1.2
0.6
0.9
0.3
0.8


day 36
0.9
0.5
1.7
0.4
0.6
0.2
0.4


day 43
0.9
0.6
1.2
0.3
0.6
0.2
0.4


day 50
1.2
0.8
1.2
0.4
0.7
0.1
0.3


day 57
0.7
0.6
1.0
0.3
0.4
0.2
n.d.


day 64
1.0
0.7
1.3
0.4
0.7
0.2
n.d.


day 71
1.6
0.8
1.8
0.9
1.1
0.3
n.d.


day 78
1.0
0.9
1.0
0.5
1.2
0.1
n.d.


day 85
1.3
1.5
1.2
0.6
1.6
0.2
n.d.





n.d. = no data













TABLE 142







Effect of antisense oligonucleotide treatment


on monocytes (%) in cynomolgus monkeys
















ISIS
ISIS
ISIS
ISIS
ISIS
ISIS



PBS
416838
416850
416858
416864
417002
416850*


















day −14
3.3
3.1
2.3
2.8
2.8
3.0
2.9


day −5
3.8
3.6
2.8
2.8
3.3
3.2
2.4


day 8
2.3
2.5
1.8
2.7
2.1
3.3
1.8


day 15
2.7
2.4
2.0
2.2
2.4
2.3
1.5


day 22
3.4
2.9
2.4
2.8
2.8
3.1
1.9


day 29
3.3
3.2
2.7
3.8
3.4
3.5
2.7


day 36
3.1
2.5
2.1
2.9
2.3
2.6
1.5


day 43
3.5
3.3
2.6
3.1
2.1
2.8
1.8


day 50
2.6
3.2
3.7
4.6
2.9
3.1
1.8


day 57
2.6
3.2
n.d. 3.2
3.8
2.4
3.6
n.d.


day 64
2.6
3.5
n.d. 3.5
4.4
2.8
4.0
n.d.


day 71
3.4
4.3
n.d. 4.7
4.9
3.7
4.7
n.d.


day 78
3.3
3.6
n.d. 4.5
4.9
3.7
4.7
n.d.


day 85
4.4
3.7
n.d. 3.5
6.1
3.7
5.3
n.d.





n.d. = no data













TABLE 143







Effect of antisense oligonucleotide treatment


on basophils (%) in cynomolgus monkeys
















ISIS
ISIS
ISIS
ISIS
ISIS
ISIS



PBS
416838
416850
416858
416864
417002
416850*


















day −14
0.3
0.2
0.2
0.3
0.2
0.3
0.2


day −5
0.3
0.3
0.2
0.3
0.2
0.3
0.3


day 8
0.2
0.2
0.2
0.3
0.2
0.3
0.3


day 15
0.3
0.3
0.2
0.2
0.2
0.2
0.2


day 22
0.2
0.2
0.2
0.2
0.2
0.2
0.1


day 29
0.3
0.2
0.2
0.2
0.3
0.2
0.3


day 36
0.3
0.4
0.3
0.3
0.3
0.2
0.1


day 43
0.3
0.4
0.3
0.3
0.4
0.3
0.2


day 50
0.4
0.3
0.3
0.4
0.4
0.3
0.2


day 57
0.2
0.3
0.4
0.2
0.3
0.3
n.d.


day 64
0.3
0.4
0.4
0.4
0.4
0.2
n.d.


day 71
0.2
0.5
0.3
0.4
0.4
0.3
n.d.


day 78
0.2
0.4
0.3
0.4
0.3
0.3
n.d.


day 85
0.3
0.3
0.3
0.3
0.4
0.3
n.d.





n.d. = no data






Cytokine and Chemokine Assays

Blood samples obtained from the monkey groups treated with PBS, ISIS 416850 and ISIS 416858 administered in the escalating dose regimen were sent to Pierce Biotechnology (Woburn, Mass.) for measurement of chemokine and cytokine levels. Levels of IL-1β, IL-6, IFN-γ, and TNF-α were measured using the respective primate antibodies and levels of IL-8, MIP-1α, MCP-1, MIP-1β and RANTES were measured using the respective cross-reacting human antibodies. Measurements were taken 14 days before the start of treatment and on day 85, when the monkeys were euthanized. The results are presented in Tables 144 and 145.







TABLE 144







Effect of antisense oligonucleotide treatment on cytokine/chemokine


levels (pg/mL) in cynomolgus monkeys on day −14

















IL-1β
IL-6
IFN-γ
TNF-α
IL-8
MIP-1α
MCP-1
MIP-1β
RANTES




















PBS
16
10
114
7
816
54
1015
118
72423


ISIS 416850
3
30
126
14
1659
28
1384
137
75335


ISIS 416858
5
9
60
9
1552
36
1252
122
112253
















TABLE 145







Effect of antisense oligonucleotide treatment on cytokine/chemokine


levels (pg/mL) in cynomolgus monkeys on day 85

















IL-1β
IL-6
IFN-γ
TNF-α
IL-8
MIP-1α
MCP-1
MIP-1β
RANTES




















PBS
7
4
102
34
87
23
442
74
84430


ISIS 416850
13
17
18
27
172
41
2330
216
83981


ISIS 416858
5
25
18
45
303
41
1752
221
125511









Example 46
Pharmacologic Effect of Antisense Oligonucleotides Targeting Human Factor 11 in Cynomolgus Monkeys
Several antisense oligonucleotides chosen from the rodent tolerability studies (Examples 41-44) were tested in cynomolgus monkeys to determine their pharmacologic effects, relative efficacy on Factor 11 activity and tolerability in a cynomolgus monkey model. The antisense oligonucleotides were also compared to ISIS 416850 and ISIS 416858 selected from the monkey study described earlier (Example 45). All the ISIS oligonucleotides used in this study target human Factor 11 mRNA and are also fully cross-reactive with the rhesus monkey gene sequence (see Tables 44 and 46). It is expected that the rhesus monkey ISIS oligonucleotides are fully cross-reactive with the cynomolgus monkey gene sequence as well. At the time the study was undertaken, the cynomolgus monkey genomic sequence was not available in the National Center for Biotechnology Information (NCBI) database; therefore, cross-reactivity with the cynomolgus monkey gene sequence could not be confirmed.
Treatment
Groups, each consisting of two male and two female monkeys, were injected subcutaneously with 25 mg/kg of ISIS 416850, ISIS 449709, ISIS 445522, ISIS 449710, ISIS 449707, ISIS 449711, ISIS 449708, 416858, and ISIS 445531. Antisense oligonucleotide was administered to the monkeys at 25 mg/kg three times per week for week 1 and 25 mg/kg twice per week for weeks 2 to 8. A control group, consisting of two male and two female monkeys was injected subcutaneously with PBS according to the same dosing regimen. Body weights were taken 14 days and 7 days before the start of treatment and were then measured weekly throughout the treatment period. Blood samples were collected 14 days and 5 days before the start of treatment and subsequently several times during the dosing regimen for PT and aPTT measurements, and measurement of various hematologic factors. On day 55, the monkeys were euthanized by exsanguination while under deep anesthesia, and organs harvested for further analysis.
RNA Analysis
On day 55, RNA was extracted from liver tissue for real-time PCR analysis of Factor 11 using primer probe set LTS00301. Results are presented as percent inhibition of Factor 11, relative to PBS control. As shown in Table 146, treatment with ISIS 416850, ISIS 449709, ISIS 445522, ISIS 449710, ISIS 449707, ISIS 449708, ISIS 416858, and ISIS 445531 resulted in significant reduction of Factor 11 mRNA in comparison to the PBS control.







TABLE 146







Inhibition of Factor 11 mRNA in the cynomolgus


monkey liver relative to the PBS control










Oligo ID
% inhibition














416850
68



449709
69



445522
89



449710
52



449707
47



449711
0



449708
46



416858
89



445531
66










Protein Analysis

Plasma samples from all monkey groups taken on different days were analyzed by a sandwich-style ELISA assay (Affinity Biologicals Inc.) using an affinity-purified polyclonal anti-Factor 11 antibody as the capture antibody and a peroxidase-conjugated polyclonal anti-Factor 11 antibody as the detecting antibody. Monkey plasma was diluted 1:50 for the assay. Peroxidase activity was expressed by incubation with the substrate o-phenylenediamine. The color produced was quantified using a microplate reader at 490 nm and was considered to be proportional to the concentration of Factor 11 in the samples.
The results are presented in Table 147, expressed as percentage reduction relative to that of the PBS control. Treatment with ISIS 416850, ISIS 449709, ISIS 445522, and ISIS 416858 resulted in a time-dependent decrease in protein levels.







TABLE 147







Inhibition of Factor 11 protein in the cynomolgus monkey liver relative to the PBS control

















ISIS No.
Day −14
Day −5
Day 10
Day 17
Day 24
Day 31
Day 38
Day 45
Day 52
Day 55




















416850
0
0
20
31
38
52
51
53
53
58


449709
1
0
27
35
44
45
46
48
47
50


445522
2
0
36
50
61
70
73
77
80
82


449710
1
0
10
14
17
25
20
23
4
24


449707
0
0
16
19
21
29
28
35
29
32


449711
0
1
5
3
6
9
2
4
3
5


449708
1
0
7
15
3
14
9
2
6
6


416858
4
0
36
49
62
68
74
79
81
81


445531
0
1
9
22
23
27
29
32
32
37









PT and aPTT Assay

PT and aPTT were measured using platelet poor plasma (PPP) from mice treated with ISIS oligonucleotides. PT and aPTT values are provided in Tables 148 and 149 and are reported as International Normalized Ratio (INR) values. INR values for PT and aPTT were determined by dividing the PT or aPTT value for each experimental group by the PT or aPTT for the PBS treated group. This ratio was then raised to the power of the International Sensitivity Index (ISI) of the tissue factor used. As shown in Table 148, PT was not significantly prolonged in mice treated with ISIS oligonucleotides. However, aPTT was significantly prolonged in groups treated with ISIS 416850, ISIS 445522, and ISIS 416858, as presented in Table 149. These data suggest that antisense reduction of Factor 11 affects the contact activation pathway, but not the extrinsic pathway of blood coagulation. Therefore, antisense reduction of Factor 11 with these ISIS oligonucleotides is useful for inhibiting the formation of a thrombus or clot in response to an abnormal vessel wall, but not in response to tissue injury.







TABLE 148







Effect of antisense oligonucleotide treatment on PT ratio in cynomolgus monkeys


















Day −14
Day −5
Day 10
Day 17
Day 24
Day 31
Day 38
Day 45
Day 52
Day 55





















ISIS 416850
1.02
1.00
0.99
1.00
0.97
1.00
1.01
1.00
1.02
1.07


ISIS 449709
1.00
0.96
0.95
0.95
0.95
0.95
0.97
0.97
0.99
1.03


ISIS 445522
1.00
0.94
0.95
0.96
0.94
0.96
0.97
0.96
0.98
1.01


ISIS 449710
1.03
0.96
0.98
1.00
0.97
0.98
0.99
0.97
0.98
1.06


ISIS 449707
1.01
0.94
0.95
0.97
0.95
0.96
1.00
0.96
0.96
1.00


ISIS 449711
1.00
0.95
0.94
0.95
0.94
0.98
1.02
1.01
1.00
1.07


ISIS 449708
1.03
0.95
0.98
1.00
0.95
1.06
0.99
0.99
0.99
1.04


ISIS 416858
1.01
0.96
0.96
0.98
0.95
1.00
0.97
1.00
0.99
1.01


ISIS 445531
1.06
1.00
1.00
1.06
1.02
1.04
1.03
1.01
1.04
1.06
















TABLE 149







Effect of antisense oligonucleotide treatment on aPTT ratio in cynomolgus monkeys


















Day −14
Day −5
Day 10
Day 17
Day 24
Day 31
Day 38
Day 45
Day 52
Day 55





















ISIS 416850
0.99
0.90
0.98
1.01
1.05
1.22
1.25
1.34
1.32
1.45


ISIS 449709
0.99
0.91
0.99
1.03
1.05
1.08
1.08
1.15
1.09
1.17


ISIS 445522
0.96
0.91
1.06
1.10
1.14
1.25
1.32
1.39
1.39
1.42


ISIS 449710
1.07
0.98
1.00
0.97
1.00
1.04
1.02
1.06
1.03
1.07


ISIS 449707
0.90
0.87
0.92
0.94
0.93
0.95
0.99
1.00
0.99
1.04


ISIS 449711
0.94
0.96
0.92
0.90
0.92
0.89
0.93
0.94
0.92
0.96


ISIS 449708
1.07
1.01
1.06
1.05
1.01
1.09
1.06
1.06
1.08
1.11


ISIS 416858
1.03
0.96
1.07
1.13
1.21
1.32
1.41
1.49
1.53
1.61


ISIS 445531
1.00
0.89
0.95
1.05
1.00
1.07
1.06
1.13
1.15
1.19









Body and Organ Weights

Body weights of each group are given in Table 150 expressed in grams. The results indicate that treatment with the antisense oligonucleotides did not cause any adverse changes in the health of the animals, which may have resulted in a significant alteration in weight compared to the PBS control. Organ weights were taken after the animals were euthanized on day 55, and livers, kidneys and spleens were harvested. The results are presented in Table 150 expressed as a percentage of the body weight and also show no significant alteration in weights compared to the PBS control, with the exception of ISIS 449711, which caused increase in spleen weight.







TABLE 150







Weekly measurements of body weights (g) of cynomolgus monkeys



















ISIS
ISIS
ISIS
ISIS
ISIS
ISIS
ISIS
ISIS
ISIS


Days
PBS
416850
449709
445522
449710
449707
449711
449708
416858
445531




















−14
2069
2061
2044
2050
2097
2072
2049
2096
2073
2079


−7
2107
2074
2093
2042
2114
2083
2105
2163
2092
2092


1
2131
2083
2112
2047
2131
2107
2123
2130
2115
2125


8
2186
2072
2075
2094
2120
2088
2123
2148
2149
2119


15
2201
2147
2085
2092
2145
2120
2103
2125
2162
2109


22
2206
2139
2117
2114
2177
2142
2171
2110
2188
2143


29
2204
2159
2068
2125
2149
2155
2203
2095
2196
2148


36
2246
2136
2064
2121
2180
2158
2227
2100
2210
2191


43
2304
2186
2106
2142
2227
2197
2251
2125
2238
2233


50
2274
2143
2147
2127
2201
2185
2227
2076
2225
2197
















TABLE 151







Organ weights (g) of cynomolgus monkeys


after antisense oligonucleotide treatment











Liver
Spleen
Kidney
















PBS
2.3
0.16
0.48



ISIS 416850
2.5
0.17
0.51



ISIS 449709
2.6
0.21
0.57



ISIS 445522
2.6
0.23
0.55



ISIS 449710
2.6
0.24
0.58



ISIS 449707
2.5
0.24
0.53



ISIS 449711
2.6
0.32
0.54



ISIS 449708
2.6
0.19
0.60



ISIS 416858
2.6
0.24
0.47



ISIS 445531
2.8
0.24
0.49










Liver Function

To evaluate the impact of ISIS oligonucleotides on hepatic function, plasma concentrations of ALT and AST were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma concentrations of alanine transaminase (ALT) and aspartate transaminase (AST) were measured and the results are presented in Tables 152 and 153 expressed in IU/L. Plasma levels of bilirubin were also measured and results are presented in Table 154 expressed in mg/dL. As observed in Tables 152-154, there were no significant increases in any of the liver metabolic markers after antisense oligonucleotide treatment.







TABLE 152







Effect of antisense oligonucleotide treatment on


ALT (IU/L) in the liver of cynomolgus monkeys












Day −14
Day −5
Day 31
Day 55















PBS
57
55
53
57


ISIS 416850
48
42
45
55


ISIS 449709
73
77
65
102


ISIS 445522
43
45
40
60


ISIS 449710
37
42
37
45


ISIS 449707
54
56
52
63


ISIS 449711
49
137
48
54


ISIS 449708
48
54
44
46


ISIS 416858
43
66
46
58


ISIS 445531
84
73
57
73
















TABLE 153







Effect of antisense oligonucleotide treatment on


AST (IU/L) in the liver of cynomolgus monkeys












Day −14
Day −5
Day 31
Day 55















PBS
65
45
44
47


ISIS 416850
62
45
46
57


ISIS 449709
62
51
45
71


ISIS 445522
62
47
46
79


ISIS 449710
52
38
37
64


ISIS 449707
64
53
50
52


ISIS 449711
58
78
47
47


ISIS 449708
74
53
56
50


ISIS 416858
64
100
60
69


ISIS 445531
78
46
47
49
















TABLE 154







Effect of antisense oligonucleotide treatment on bilirubin


(mg/dL) in the liver of cynomolgus monkeys












Day −14
Day −5
Day 31
Day 55















PBS
0.25
0.20
0.20
0.17


ISIS 416850
0.26
0.22
0.26
0.17


ISIS 449709
0.24
0.19
0.15
0.18


ISIS 445522
0.24
0.20
0.14
0.18


ISIS 449710
0.24
0.19
0.15
0.22


ISIS 449707
0.27
0.19
0.13
0.16


ISIS 449711
0.23
0.16
0.13
0.13


ISIS 449708
0.27
0.21
0.14
0.14


ISIS 416858
0.25
0.23
0.16
0.16


ISIS 445531
0.22
0.18
0.13
0.11









Kidney Function

To evaluate the impact of ISIS oligonucleotides on kidney function, urine samples were collected on different days. BUN levels were measured at various time points using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.) and the results are presented in Table 155. The ratio of urine protein to creatinine in urine samples after antisense oligonucleotide treatment was also calculated for day 49 and results are presented in Table 156. As observed in Tables 155 and 156, there were no significant increases in any of the kidney metabolic markers after antisense oligonucleotide treatment.







TABLE 155







Effect of antisense oligonucleotide treatment


on BUN levels (mg/dL) in cynomolgus monkeys












Day −14
Day −5
Day 31
Day 55















PBS
22
21
22
22


ISIS 416850
24
23
21
26


ISIS 449709
22
21
20
28


ISIS 445522
23
22
22
22


ISIS 449710
19
19
19
23


ISIS 449707
25
21
21
20


ISIS 449711
26
22
20
23


ISIS 449708
25
23
23
23


ISIS 416858
25
24
23
24


ISIS 445531
22
18
20
22
















TABLE 156







Effect of antisense oligonucleotide treatment on urine


protein to creatinine ratio in cynomolgus monkeys









Urine protein/



creatinine ratio














PBS
0.02



ISIS 416850
0.08



ISIS 449709
0.05



ISIS 445522
0.01



ISIS 449710
0.00



ISIS 449707
0.03



ISIS 449711
0.01



ISIS 449708
0.00



ISIS 416858
0.05



ISIS 445531
0.08










Hematology Assays

Blood obtained from all the monkey groups on different days were sent to Korea Institute of Toxicology (KIT) for HCT, MCV, MCH, and MCHC measurements, as well as measurements of the various blood cells, such as WBC (neutrophils and monocytes), RBC and platelets, as well as total hemoglobin content. The results are presented in Tables 157-166.







TABLE 157







Effect of antisense oligonucleotide treatment


on HCT (%) in cynomolgus monkeys














Day −14
Day −5
Day 17
Day 31
Day 45
Day 55

















PBS
40
42
43
43
41
40


ISIS 416850
41
44
42
42
42
40


ISIS 449709
41
42
43
42
41
40


ISIS 445522
42
42
41
43
41
39


ISIS 449710
41
44
43
44
43
41


ISIS 449707
40
43
42
43
43
42


ISIS 449711
41
41
42
39
39
38


ISIS 449708
41
44
44
43
44
42


ISIS 416858
41
44
43
43
41
39


ISIS 445531
41
42
43
41
41
41
















TABLE 158







Effect of antisense oligonucleotide treatment on platelet


count (×100/μL) in cynomolgus monkeys














Day −14
Day −5
Day 17
Day 31
Day 45
Day 55

















PBS
361
441
352
329
356
408


ISIS 416850
462
517
467
507
453
396


ISIS 449709
456
481
449
471
418
441


ISIS 445522
433
512
521
425
403
333


ISIS 449710
411
463
382
422
313
360


ISIS 449707
383
464
408
408
424
399


ISIS 449711
410
431
325
309
257
259


ISIS 449708
387
517
444
378
381
348


ISIS 416858
369
433
358
289
287
257


ISIS 445531
379
416
380
376
345
319
















TABLE 159







Effect of antisense oligonucleotide treatment


on neutrophils (%) in cynomolgus monkeys














Day −14
Day −5
Day 17
Day 31
Day 45
Day 55

















PBS
81
84
75
75
91
118


ISIS 416850
88
109
95
100
85
108


ISIS 449709
73
101
89
81
77
115


ISIS 445522
61
84
81
66
69
125


ISIS 449710
93
86
80
94
97
132


ISIS 449707
85
106
80
89
89
98


ISIS 449711
64
71
52
58
45
70


ISIS 449708
73
84
61
57
61
75


ISIS 416858
65
84
54
54
61
73


ISIS 445531
60
80
85
116
93
91
















TABLE 160







Effect of antisense oligonucleotide treatment


on monocytes (%) in cynomolgus monkeys














Day −14
Day −5
Day 17
Day 31
Day 45
Day 55

















PBS
1.9
2.8
3.1
2.8
3.9
2.2


ISIS 416850
1.9
2.9
3.2
3.7
3.8
3.4


ISIS 449709
4.0
2.0
3.0
2.8
3.6
3.4


ISIS 445522
2.1
2.3
3.6
3.9
4.4
3.0


ISIS 449710
1.3
2.0
2.5
2.4
3.4
1.6


ISIS 449707
1.3
2.3
3.2
4.2
4.0
4.8


ISIS 449711
1.2
2.3
5.9
6.9
7.6
7.8


ISIS 449708
1.7
2.6
5.4
5.8
7.0
6.2


ISIS 416858
2.0
2.7
4.0
4.7
4.6
4.6


ISIS 445531
1.3
2.2
3.4
4.1
4.4
4.1
















TABLE 161







Effect of antisense oligonucleotide treatment on


hemoglobin content (g/dL) in cynomolgus monkeys














Day −14
Day −5
Day 17
Day 31
Day 45
Day 55

















PBS
12.3
12.5
12.9
12.7
12.4
12.1


ISIS 416850
13.0
13.5
13.3
13.1
13.1
12.7


ISIS 449709
12.8
12.8
13.2
13.1
12.6
12.5


ISIS 445522
13.3
12.7
12.7
12.9
12.6
12.0


ISIS 449710
13.0
13.2
13.4
13.1
13.0
12.7


ISIS 449707
12.7
12.8
12.7
12.7
12.9
12.6


ISIS 449711
12.7
12.7
12.5
11.8
11.5
11.3


ISIS 449708
13.0
13.2
13.5
13.0
13.3
13.0


ISIS 416858
12.8
13.0
13.0
12.8
12.3
12.0


ISIS 445531
12.6
12.6
12.7
12.3
12.0
12.1
















TABLE 162







Effect of antisense oligonucleotide treatment on WBC


count (×103/μL) in cynomolgus monkeys














Day −14
Day −5
Day 17
Day 31
Day 45
Day 55

















PBS
10
10
11
12
11
12


ISIS 416850
12
13
11
12
12
10


ISIS 449709
11
10
11
11
11
10


ISIS 445522
10
9
11
13
10
11


ISIS 449710
11
11
12
12
11
15


ISIS 449707
13
11
12
11
12
8


ISIS 449711
13
12
10
9
9
7


ISIS 449708
14
10
11
11
10
10


ISIS 416858
10
11
10
9
8
9


ISIS 445531
20
15
17
17
20
15
















TABLE 163







Effect of antisense oligonucleotide treatment on RBC


count (×106/μL) in cynomolgus monkeys














Day −14
Day −5
Day 17
Day 31
Day 45
Day 55

















PBS
5.6
5.6
5.8
5.8
5.6
5.5


ISIS 416850
5.5
5.7
5.6
5.6
5.7
5.6


ISIS 449709
5.8
5.8
5.9
5.9
5.7
5.7


ISIS 445522
5.9
5.6
5.6
5.8
5.7
5.4


ISIS 449710
5.6
5.8
5.8
5.8
5.7
5.6


ISIS 449707
5.7
5.8
5.7
5.7
5.9
5.8


ISIS 449711
5.6
5.7
5.6
5.4
5.4
5.3


ISIS 449708
5.7
5.9
5.9
5.8
6.0
5.8


ISIS 416858
5.5
5.5
5.6
5.6
5.5
5.3


ISIS 445531
5.7
5.7
5.8
5.6
5.5
5.6
















TABLE 164







Effect of antisense oligonucleotide treatment


on MCV (fL) in cynomolgus monkeys














Day −14
Day −5
Day 17
Day 31
Day 45
Day 55

















PBS
72
74
75
73
73
73


ISIS 416850
74
77
76
75
75
73


ISIS 449709
72
74
73
73
71
71


ISIS 445522
72
74
74
75
73
72


ISIS 449710
75
77
75
75
75
73


ISIS 449707
71
75
74
74
73
73


ISIS 449711
73
74
75
73
73
73


ISIS 449708
73
75
75
75
74
74


ISIS 416858
75
79
78
76
75
75


ISIS 445531
72
74
75
75
75
74
















TABLE 165







Effect of antisense oligonucleotide treatment


on MCH (pg) in cynomolgus monkeys














Day −14
Day −5
Day 17
Day 31
Day 45
Day 55

















PBS
22.1
22.4
22.3
22.1
22.0
22.0


ISIS 416850
23.7
23.7
23.7
23.3
22.7
22.9


ISIS 449709
22.4
22.3
22.5
22.2
21.0
22.0


ISIS 445522
22.6
22.5
22.8
22.4
22.4
22.2


ISIS 449710
23.0
22.8
23.1
22.6
21.8
22.7


ISIS 449707
22.2
22.2
22.1
22.1
22.6
21.9


ISIS 449711
22.6
22.7
22.2
22.1
21.7
21.3


ISIS 449708
22.9
22.7
22.9
22.7
22.2
22.5


ISIS 416858
23.2
23.5
23.1
23.0
22.2
22.8


ISIS 445531
22.2
22.2
22.1
22.0
21.6
21.7
















TABLE 166







Effect of antisense oligonucleotide treatment


on MCHC (g/dL) in cynomolgus monkeys














Day −14
Day −5
Day 17
Day 31
Day 45
Day 55

















PBS
30.8
30.0
30.1
29.9
30.3
30.2


ISIS 416850
32.0
30.7
31.3
31.0
31.0
30.9


ISIS 449709
31.4
30.3
30.7
30.7
31.1
31.2


ISIS 445522
31.4
30.4
30.9
30.0
30.7
31.0


ISIS 449710
31.2
29.7
30.7
30.1
30.4
31.1


ISIS 449707
31.4
29.8
30.0
29.8
29.8
30.0


ISIS 449711
31.0
30.7
29.9
29.8
29.6
29.5


ISIS 449708
31.4
30.2
30.7
29.9
30.6
31.8


ISIS 416858
31.1
29.8
29.9
31.0
30.3
30.4


ISIS 445531
30.9
30.0
29.5
29.7
29.0
29.6









Cytokine and Chemokine Assays

Blood samples obtained from all monkey groups were sent to Pierce Biotechnology (Woburn, Mass.) for measurements of chemokine and cytokine levels. Levels of IL-1β, IL-6, IFN-γ, and TNF-α were measured using the respective primate antibodies and levels of IL-8, MIP-1α, MCP-1, MIP-1β and RANTES were measured using the respective cross-reacting human antibodies. Measurements were taken 14 days before the start of treatment and on day 55, when the monkeys were euthanized. The results are presented in Tables 167 and 168.







TABLE 167







Effect of antisense oligonucleotide treatment on cytokine/chemokine


levels (pg/mL) in cynomolgus monkeys on day −14

















IL-1β
IL-6
IFN-γ
TNF-α
IL-8
MIP-1α
MCP-1
MIP-1β
RANTES




















PBS
350
3
314
32
82
27
277
8
297


ISIS 416850
215
1
115
4
45
14
434
31
4560


ISIS 449409
137
1
37
9
34
13
290
14
2471


ISIS 445522
188
5
172
16
32
22
297
27
3477


ISIS 449710
271
7
1115
72
29
20
409
18
1215


ISIS 449707
115
1
34
6
106
16
294
13
3014


ISIS 449711
79
2
29
6
156
20
264
24
3687


ISIS 449708
35
1
27
12
184
11
361
19
11666


ISIS 416858
103
0
32
4
224
11
328
37
6521


ISIS 445531
101
2
68
9
83
25
317
22
7825
















TABLE 168







Effect of antisense oligonucleotide treatment on cytokine/chemokine


levels (pg/mL) in cynomolgus monkeys on day 55

















IL-1β
IL-6
IFN-γ
TNF-α
IL-8
MIP-1α
MCP-1
MIP-1β
RANTES




















PBS
453
3
232
191
68
21
237
34
775


ISIS 416850
106
1
19
16
620
17
887
50
27503


ISIS 449409
181
0
25
8
254
17
507
47
8958


ISIS 445522
341
2
83
18
100
22
592
63
16154


ISIS 449710
286
2
176
26
348
27
474
53
22656


ISIS 449707
97
1
24
16
48
12
264
49
1193


ISIS 449711
146
7
22
31
110
17
469
91
3029


ISIS 449708
131
0
18
17
85
23
409
128
4561


ISIS 416858
28
1
9
15
167
11
512
47
5925


ISIS 445531
155
1
15
16
293
12
339
84
5935









Example 47
Measurement of Viscosity of ISIS Antisense Oligonucleotides Targeting Human Factor 11
The viscosity of antisense oligonucleotides targeting human Factor 11 was measured with the aim of screening out antisense oligonucleotides which have a viscosity more than 40 cP at a concentration of 165-185 mg/mL.
ISIS oligonucleotides (32-35 mg) were weighed into a glass vial, 120 μL of water was added and the antisense oligonucleotide was dissolved into solution by heating the vial at 50° C. Part of (75 μL) the pre-heated sample was pipetted to a micro-viscometer (Cambridge). The temperature of the micro-viscometter was set to 25° C. and the viscosity of the sample was measured. Another part (20 μL) of the pre-heated sample was pipetted into 10 mL of water for UV reading at 260 nM at 85° C. (Cary UV instrument). The results are presented in Table 169.







TABLE 169







Viscosity and concentration of ISIS antisense


oligonucleotides targeting human Factor 11










Viscosity
Concentration


ISIS No.
(cP)
(mg/mL)












412223
8
163


412224
98
186


412225
>100
162


413481
23
144


413482
16.
172


416848
6
158


416850
67
152


416851
26
187


416852
29
169


416856
18
175


416858
10
166


416859
10
161


416860
>100
154


416861
14
110


416863
9
165


416866
>100
166


416867
8
168


445498
21
157


445504
20
139


445505
9
155


445509
>100
167


445513
34
167


445522
63
173


445522
58
174


445530
25
177


445531
15
155


445531
20
179


449707
7
166


449708
9
188


449709
65
171


449710
7
186


449711
6
209


451541
10
168

## Claims

1. A compound, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 12 contiguous nucleobases of SEQ ID NO: 217 or 223, wherein the nucleobase sequence of the modified oligonucleotide is at least 90% complementary to SEQ ID NO: 1 as measured over the entirety of the modified oligonucleotide.

2. The compound of claim 1, consisting of a single-stranded modified oligonucleotide.

3. The compound of claim 2, wherein at least one internucleoside linkage is a modified internucleoside linkage.

4. The compound of claim 3, wherein each internucleoside linkage is a phosphorothioate internucleoside linkage.

5. The compound of claim 2, wherein at least one nucleoside comprises a modified sugar.

6. The compound of claim 5, wherein at least one modified sugar is a bicyclic sugar.

7. The compound of claim 6, wherein each of the at least one bicyclic sugar comprises a 4′-(CH2)n—O-2′ bridge, wherein n is 1 or 2.

8. The compound of claim 6, wherein each of the at least one bicyclic sugar comprises a 4′-CH(CH3)—O-2′ bridge.

9. The compound of claim 5, wherein at least one modified sugar comprises a 2′-O-methoxyethyl group.

10. The compound of claim 2, comprising at least one tetrahydropyran modified nucleoside wherein a tetrahydropyran ring replaces the furanose ring.

11. The compound of claim 10, wherein each of the at least one tetrahydropyran modified nucleoside has the structure:



wherein Bx is an optionally protected heterocyclic base moiety.

12. The compound of claim 2, wherein at least one nucleoside comprises a modified nucleobase.

13. The compound of claim 12, wherein the modified nucleobase is a 5-methylcytosine.

14. The compound of claim 2, wherein the modified oligonucleotide comprises:
a gap segment consisting of linked deoxynucleosides;
a 5′ wing segment consisting of linked nucleosides;
a 3′ wing segment consisting of linked nucleosides;
wherein the gap segment is positioned immediately adjacent to and between the 5′ wing segment and the 3′ wing segment, and wherein each nucleoside of each wing segment comprises a modified sugar.

15. The compound of claim 14, wherein the modified oligonucleotide consists of 20 linked nucleosides.

16. The compound of claim 15, wherein the modified oligonucleotide comprises:
a gap segment consisting of ten linked deoxynucleosides;
a 5′ wing segment consisting of five linked nucleosides;
a 3′ wing segment consisting of five linked nucleosides;
wherein the gap segment is positioned immediately adjacent and between the 5′ wing segment and the 3′ wing segment, wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, wherein each internucleoside linkage is a phosphorothioate linkage, and wherein each cytosine is a 5-methylcytosine.

17. The compound of claim 14, wherein the nucleobase sequence of the modified oligonucleotide comprises the sequence of SEQ ID NO: 217 or 223.

18. The compound of claim 16, wherein the nucleobase sequence of the modified oligonucleotide consists of the sequence of SEQ ID NO: 217 or 223.

19. The compound of claim 2, wherein the nucleobase sequence of the modified oligonucleotide comprises the sequence of SEQ ID NO: 217 or 223.

20. The compound of claim 2, wherein the nucleobase sequence of the modified oligonucleotide consists of the sequence of SEQ ID NO: 217 or 223.

21. A composition, comprising:
a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 12 contiguous nucleobases of SEQ ID NO: 217 or 223, or a salt thereof, wherein the nucleobase sequence of the modified oligonucleotide is at least 90% complementary to SEQ ID NO: 1 as measured over the entirety of the modified oligonucleotide; and
a pharmaceutically acceptable carrier or diluent.

22. The composition of claim 21, further comprising any of the group consisting of aspirin, clopidogrel, dipyridamole, heparin, lepirudin, ticlopidine, warfarin, apixaban, rivaroxaban, LOVENOX, and Factor Xa inhibitor.

23. The composition of claim 22, wherein said any of the group consisting of aspirin, clopidogrel, dipyridamole, heparin, lepirudin, ticlopidine, warfarin, apixaban, rivaroxaban, LOVENOX, and Factor Xa inhibitor is in single pharmaceutical composition with said modified oligonucleotide and pharmaceutically acceptable carrier or diluent.

24. A compound, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 12 contiguous nucleobases of any of the group consisting of SEQ ID NOs: 51-53, 114-115, 213-232, wherein the nucleobase sequence of the modified oligonucleotide is at least 90% complementary to SEQ ID NO: 1 as measured over the entirety of the modified oligonucleotide.

25. The compound of claim 16, wherein the nucleobase sequence of the modified oligonucleotide consists of the sequence of SEQ ID NO: 217.

26. The compound of claim 16, wherein the nucleobase sequence of the modified oligonucleotide consists of the sequence of SEQ ID NO: 223.

27. The compound of claim 24, wherein the nucleobase sequence comprises any of the group consisting of SEQ ID NOs: 51-53, 114-115, 213-232.

28. The compound of claim 24, wherein the nucleobase sequence consists of any of the group consisting of SEQ ID NOs: 51-53, 114-115, 213-232.

29. The compound of claim 24, consisting of a single-stranded modified oligonucleotide.

30. The compound of claim 29, wherein at least one internucleoside linkage is a modified internucleoside linkage.

31. The compound of claim 30, wherein each internucleoside linkage is a phosphorothioate internucleoside linkage.

32. The compound of claim 29, wherein at least one nucleoside comprises a modified sugar.

33. The compound of claim 32, wherein at least one modified sugar is a bicyclic sugar.

34. The compound of claim 33, wherein each of the at least one bicyclic sugar comprises a 4′-(CH2)n—O-2′ bridge, wherein n is 1 or 2.

35. The compound of claim 33, wherein each of the at least one bicyclic sugar comprises a 4′-CH(CH3)—O-2′ bridge.

36. The compound of claim 32, wherein at least one modified sugar comprises a 2′-O-methoxyethyl group.

37. The compound of claim 29, comprising at least one tetrahydropyran modified nucleoside wherein a tetrahydropyran ring replaces the furanose ring.

38. The compound of claim 37, wherein each of the at least one tetrahydropyran modified nucleoside has the structure:



wherein Bx is an optionally protected heterocyclic base moiety.

39. The compound of claim 29, wherein at least one nucleoside comprises a modified nucleobase.

40. The compound of claim 39, wherein the modified nucleobase is a 5-methylcytosine.

41. The compound of claim 29, wherein the modified oligonucleotide comprises:
a gap segment consisting of linked deoxynucleosides;
a 5′ wing segment consisting of linked nucleosides;
a 3′ wing segment consisting of linked nucleosides;
wherein the gap segment is positioned immediately adjacent to and between the 5′ wing segment and the 3′ wing segment, and wherein each nucleoside of each wing segment comprises a modified sugar.

42. The compound of claim 41, wherein the modified oligonucleotide consists of 20 linked nucleosides.

43. The compound of claim 42, wherein the modified oligonucleotide comprises:
a gap segment consisting of ten linked deoxynucleosides;
a 5′ wing segment consisting of five linked nucleosides;
a 3′ wing segment consisting of five linked nucleosides;
wherein the gap segment is positioned immediately adjacent and between the 5′ wing segment and the 3′ wing segment, wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, wherein each internucleoside linkage is a phosphorothioate linkage, and wherein each cytosine is a 5-methylcytosine.

44. The compound of claim 41, wherein the nucleobase sequence of the modified oligonucleotide comprises the sequence of any of the group consisting of SEQ ID NOs: 51-53, 114-115, 213-232.

45. The compound of claim 43, wherein the nucleobase sequence of the modified oligonucleotide consists of the sequence of any of the group consisting of SEQ ID NOs: 51-53, 114-115, 213-232.

46. The compound of claim 1, wherein the nucleobase sequence of the modified oligonucleotide is at least 95% complementary to SEQ ID NO: 1 as measured over the entirety of the modified oligonucleotide.

47. The compound of claim 1, wherein the nucleobase sequence of the modified oligonucleotide is 100% complementary to SEQ ID NO: 1 as measured over the entirety of the modified oligonucleotide.

48. The compound of claim 14, wherein the nucleobase sequence of the modified oligonucleotide is at least 95% complementary to SEQ ID NO: 1 as measured over the entirety of the modified oligonucleotide.

49. The compound of claim 14, wherein the nucleobase sequence of the modified oligonucleotide is 100% complementary to SEQ ID NO: 1 as measured over the entirety of the modified oligonucleotide.

50. The composition of claim 21, wherein the nucleobase sequence of the modified oligonucleotide is at least 95% complementary to SEQ ID NO: 1 as measured over the entirety of the modified oligonucleotide.

51. The composition of claim 21, wherein the nucleobase sequence of the modified oligonucleotide is 100% complementary to SEQ ID NO: 1 as measured over the entirety of the modified oligonucleotide.

52. The compound of claim 24, wherein the nucleobase sequence of the modified oligonucleotide is at least 95% complementary to SEQ ID NO: 1 as measured over the entirety of the modified oligonucleotide.

53. The compound of claim 24, wherein the nucleobase sequence of the modified oligonucleotide is 100% complementary to SEQ ID NO: 1 as measured over the entirety of the modified oligonucleotide.

54. The compound of claim 41, wherein the nucleobase sequence of the modified oligonucleotide is at least 95% complementary to SEQ ID NO: 1 as measured over the entirety of the modified oligonucleotide.

55. The compound of claim 41, wherein the nucleobase sequence of the modified oligonucleotide is 100% complementary to SEQ ID NO: 1 as measured over the entirety of the modified oligonucleotide.

56. The compound of claim 43, wherein the nucleobase sequence of the modified oligonucleotide is at least 95% complementary to SEQ ID NO: 1 as measured over the entirety of the modified oligonucleotide.

57. The compound of claim 43, wherein the nucleobase sequence of the modified oligonucleotide is 100% complementary to SEQ ID NO: 1 as measured over the entirety of the modified oligonucleotide.