# Patent

## Title
Immunoregulatory compositions

## Abstract

The invention relates to compounds exhibiting immunoregulatory activity as determined by measuring the compound's ability to modulate production of NO by a cell. Preferred compounds include or consist of a sequence

AAL AAQ AAG AAV

wherein AAL is a substituted or unsubstituted non-polar amino acid selected from the group consisting of Ala and Leu; wherein AAQ is a substituted or unsubstituted amino acid selected from the group consisting of Gln, Pro, and Ala; wherein AAG is a substituted or unsubstituted amino acid Gly, and wherein AAV is a substituted or unsubstituted non-polar amino acid selected from the group consisting of Val and Ala. In one embodiment, the compound consists of a tripeptide selected from the group AQG, MTR, VVC, and mixtures thereof.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application is a continuation in part of U.S. Ser. No. 10/028,075, filed Dec. 21, 2001, a continuation of U.S. Ser. No. 10/262,522, filed Sep. 30, 2002, which is a continuation of International Application No. PCT/NL01/00259, designating the United States of America published in English as PCT International Publication No. WO 01/72831 A2 filed Mar. 3, 2001, and a continuation in part of International Application No. PCT/NL02/00639, designating the United States of America and published on Apr. 10, 2002 in English as PCT Internatinal Publication No. WO 03/029292 A2, the contents of all of each of which are incorporated by this reference.


TECHNICAL FIELD
The invention relates generally to biotechnology, and more specifically to compositions having immunoregulatory activity, which compounds include particular oligopeptides derived from human chorionic gonadotropin (hCG).
BACKGROUND
U.S. Pat. No. 5,380,668 to Herron (Jan. 10, 1995), the contents of the entirety of which are incorporated by this reference, discloses, among other things, various compounds having the antigenic binding activity of hCG. The oligopeptides disclosed therein are disclosed generally for use in diagnostice methods.
Various patents and patent applications to Gallo et al. (e.g., U.S. Pat. No. 5,677,275 (corresponding to WO 96/04008 A1), U.S. Pat. No. 5,877,148 (also corresponding to WO 96/04008 A1), WO 97/49721 A1, U.S. Pat. No. 6,319,504 (corresponding to WO 97/49373), U.S. Patent Application 2003/0049273 A1 (also corresponding to WO 97/49373), U.S. Pat. No. 5,968,513 (corresponding to WO 97/49418), U.S. Pat. No. 5,997,871 (corresponding to WO 97/49432), U.S. Pat. No. 6,620,416, U.S. Pat. No. 6,596,688, WO 01/11048 A2, WO 01/10907 A2., and U.S. Pat. No. 6,583,109) relate to various oligopeptides and their use in, among other things, “inhibiting HIV infection”, “treating or preventing HIV infection”, “treating or preventing cancer”, “treating or preventing a condition characterized by loss of body cell mass”, “treating or preventing a condition associated with pathological angiogenesis”, “treating or preventing hematopoietic deficiency”, “ex vivo gene therapy”, “expanding blood cells in vitro”, and/or “providing blood cells to a subject”.
DISCLOSURE OF THE INVENTION
As we described in PCT International Publication No. WO 03/029292 A2 (published Apr. 10, 2003), PCT International Publication No. WO 01/72831 A2 (published Oct. 4, 2001), and U.S. Patent Application Publications 20020064501 A1 (published May 30, 2002), 20030119720 A1 (published Jun. 26, 2003), 20030113733 A1 (published Jun. 19, 2003), and 20030166556 A1 (published Sep. 4, 2003), the contents of all of which are incorporated by this reference, compositions containing purified or isolated oligopeptides described herein have immunoregulatory activity useful in, for example, the treatment of sepsis and other disease states and conditions. They also have gene regulatory activities.
The invention thus includes a composition comprising a purified or isolated peptide consisting of particular four to eight amino acid segments of the sequence MTRVLQGVLPALPQVVC (SEQ ID NO:44 of the incorporated herein SEQUENCE LISTING); and derivatives thereof having one or more conservative substitutions relative to the sequence of SEQ ID NO:44. The particular compositions exhibit immunoregulatory activity as determined by measuring the segment's ability to modulate production of NO by a cell. Preferably, the compositions have the ability to decrease shock in a subject (e.g., a mammal) undergoing sepsis.
In one embodiment, the amino acid segment includes a tetrameric sequence (corresponding to the LQVG (SEQ ID NO: 1) portion of SEQ ID NO:44, i.e.,

    
    
        AAL AAQ AAG AAV

wherein AAL is a substituted or unsubstituted non-polar amino acid selected from the group consisting of Ala and Leu; AAQ is a substituted or unsubstituted amino acid selected from the group consisting of Gln, Pro, and Ala; AAG is a substituted or unsubstituted Gly; and AAV is a substituted or unsubstituted non-polar amino acid selected from the group consisting of Val and Ala. For instance, the peptide could be selected from the group consisting of LQGV (SEQ ID NO:1), the derivative AQGV (SEQ ID NO:2), the derivative LQGA (SEQ ID NO:19), the derivative LAGV (SEQ ID NO:26), and the derivativeLPGC (SEQ ID NO:41).

    
    


In a different embodiment, the segment is the tetramer MTRV (SEQ ID NO:42) or QVVC (SEQ ID NO:43).
In another embodiment, the segment is six or seven amino acids long, and comprises the sequence

    
    
        AAV AAL Pro Arg AAL2 AAP

wherein AAV is substituted or unsubstituted Val or Ala, wherein AAL and AAL2 are independently selected from substituted or unsubstituted Lys or Ala, and wherein AAP is a substituted or unsubstituted Pro or Ala.

    
    


In such an embodiment, the purified or isolated peptide can have a formula selected from the group consisting of VLPALP (SEQ ID NO:3), the derivative ALPALP (SEQ ID NO:21), the derivative VAPALP (SEQ ID NO:22), the derivative ALPALPQ (SEQ ID NO:23), the derivative VLPAAPQ (SEQ ID NO:24), the derivative VLPALAQ (SEQ ID NO:25), the derivative VLPALA (SEQ ID NO:28), VLPALPQ (SEQ ID NO:29), the derivative VLPALPA (SEQ ID NO:31), the derivative GVLPALP (SEQ ID NO:32), and the derivative VLAALP (SEQ ID NO:117).
In another embodiment, the composition has no more than eight amino acids, and includes an amino acid sequence consisting of:

    
    
        AAL AAQ AAG AAV

wherein AAL is a substituted or unsubstituted amino acid selected from the group of amino acids consisting of Ala, Leu, and Met, wherein AAQ is a substituted or unsubstituted amino acid selected from the group of amino acids consisting of Gln, Thr, Ala, and Pro, wherein AAG is substituted or unsubstituted Gly or Arg, and wherein AAV is a substituted or unsubstituted amino acid selected from the group of amino acids consisting of Cys, Ala, and Val. Again such a composition is characterized in having immunoregulatory activity as determined by: measuring its capability of modulating production of NO by a cell.

    
    


In such an embodiment, the sequence is preferably selected from the group consisting of Leu Gln Gly Val (SEQ ID NO:1), Ala Gln Gly Val (SEQ ID NO:2), Leu Gln Gly Ala (SEQ ID NO:19), Leu Ala Gly Val (SEQ ID NO:26), Leu Pro Gly Cys (SEQ ID NO:41), and Met Thr Arg Val (SEQ ID NO:42). Preferably, the composition is the sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:19, SEQ ID NO:26, SEQ ID NO:41, SEQ ID NO:42, or a salt of any thereof.
The invention further includes a pharmaceutical composition comprising a purified or isolated peptide, or acid addition salt thereof, the purified or isolated peptide (a) consisting of an amino acid sequence selected from the group consisting of: (i) a four to seven amino acid segment of the sequence of MTRVLQGVLPALPQVVC (SEQ ID NO:44); and (ii) a derivative of the segment of (a) having one or more conservative substitutions relative to the sequence of SEQ ID NO:44; and (b) exhibiting an immunoregulatory activity as determined by measuring its capability of modulating production of NO by a cell.
Such a pharmaceutical composition preferably includes a sequence selected from the group selected from the group consisting of LQGV (SEQ ID NO:1), AQGV (SEQ ID NO:2), VLPALP (SEQ ID NO:3), LQGA (SEQ ID NO:19), ALPALP (SEQ ID NO:21), VAPALP (SEQ ID NO:22), ALPALPQ (SEQ ID NO:23), VLPAAPQ (SEQ ID NO:24), VLPALAQ (SEQ ID NO:25), LAGV (SEQ ID NO:26), VLPALA (SEQ ID NO:28), VLPALPQ (SEQ ID NO:29), VLPALPA (SEQ ID NO:31), and GVLPALP (SEQ ID NO:32), LPGC (SEQ ID NO:41), MTRV (SEQ ID NO:42), QVVC (SEQ ID NO:43), VLAALP (SEQ ID NO:117), and combinations of any thereof, with or without other active or inactive ingredients, presented in a pharmaceutically acceptable form for administration to a human.
In one preferred embodiment, the inventin is a purified or isolated peptide consisting of GVLPALPQ (SEQ ID NO:33), or an acid addition salt thereof. The invention would thus also include a pharmaceutical composition comprising the peptide of SEQ ID NO:33 or an addition salt thereof, together with a pharmaceutically acceptable excipient.
In another embodiment the invention comprises a purified or isolated peptide (or, for example, acid addition salt thereof), selected from the group consisting of AQG, MTR, and VVC.
Finally the invention includes a composition comprising one or more of the following amino acid segments: LQGV (SEQ ID NO:1), AQGV (SEQ ID NO:2), VLPALP (SEQ ID NO:3), LQGA (SEQ ID NO:19), ALPALP (SEQ ID NO:21), VAPALP (SEQ ID NO:22), ALPALPQ (SEQ ID NO:23), VLPAAPQ (SEQ ID NO:24), VLPALAQ (SEQ ID NO:25), LAGV (SEQ ID NO:26), VLPALA (SEQ ID NO:28), VLPALPQ (SEQ ID NO:29), VLPALPA (SEQ ID NO:31), and GVLPALP (SEQ ID NO:32), LPGC (SEQ ID NO:41), MTRV (SEQ ID NO:42), QVVC (SEQ ID NO:43), VLAALP (SEQ ID NO:117), AQG, MTR, or VVC.
In another embodiment, the invention includes a composition comprising a purified or isolated peptide consisting of sequence LQG in an amount sufficient to exhibit an immunoregulatory activity as determined by measuring the sequence LQG's ability to modulate production of NO by a cell.
The invention provides a method for the treatment of bone disease such as osteoporosis comprising administering to a subject believed to be in need of such treatment a composition comprising an oligopeptide, derivative or functional analogue thereof, the particular molecule capable of modulating production of NO and/or TNF-alpha by a cell.
Such a method of treatment is particularly useful in post-menopausal women that no longer experience the benefits of being provided with a natural source of hCG and its breakdown products. Such a treatment can be achieved by systemic administration of a composition of the invention according to the invention, but local administration in joints, bursae or tendon sheaths is provided as well. The molecule can be selected from Table 6 or identified in a method described herein. The treatment comprises administering to the subject a pharmaceutical composition comprising an oligopeptide or functional analogue thereof capable of reducing production of NO by a cell, for example, wherein the composition comprises at least two oligopeptides or functional analogues thereof, each capable of reducing production of NO and/or TNF-alpha by a cell, in particular wherein the at least two oligopeptides are selected from the group LQGV (SEQ ID NO:1), AQGV (SEQ ID NO:2), and VLPALP (SEQ ID NO:3).
Several oligopeptides according to the invention have been tested, both ex vivo and in vivo, and in small animals. A beneficial effect of these oligopeptides on LPS-induced sepsis in mice, namely the inhibition of the effect of the sepsis, was observed. Immunomodulatory effects with these oligopeptides have been observed in vitro and in ex vivo such as in T-cell assays showing the inhibition of pathological Th1 immune responses, suppression of inflammatory cytokines (MIF), increase in production of anti-inflammatory cytokines (IL-10, TGF-beta) and immunomodulatory effects on antigen-presenting cells (APC) like dendritic cells, monocytes and macrophages.
Now knowing the gene modulatory effect of the composition of the inventions such as oligopeptides as provided herein allows for rational design of signal molecule mixtures that better alleviate the symptoms seen with sepsis. One such mixture, a 1:1:1 mixture of LQGV (SEQ ID NO:1), AQGV (SEQ ID NO:2) and VLPALP (SEQ ID NO:3) was administered to primates in a gram-negative induced rhesus monkey sepsis model for prevention of septic shock and found to be effective in this primate model. Accordingly, the invention provides a pharmaceutical composition for the treatment of sepsis in a primate and a method for the treatment of sepsis in a primate comprising subjecting the primate to a composition of the invention according to the invention, preferably to a mixture of such composition of the inventions. Administration of such a composition of the invention or mixture preferably occurs systematically, for example, by intravenous or intraperitoneal administration. In a further embodiment, such treatment also comprises the use of for example an antibiotic, however, only when such use is not contra indicated because of the risk of generating further toxin loads because of lysis of the bacteria subject to the action of those antibiotics in an individual thus treated.
The invention also provides use of a composition according to the invention for the preparation of a pharmaceutical composition or medicament and methods of treating various medical conditions that are other than use in the preparation of a pharmaceutical composition for the treatment of an immune-mediated disorder or a method of treatment of an immune-mediated disorder or treatment of a wasting syndrome.


DETAILED DESCRIPTION OF THE INVENTION
As used herein, a “purified or isolated” peptide is one that has been purified from a natural or biotechnological source, or, more preferably, is synthesized as described herein.
“Composition”, as used herein, refers to chemical compounds which contain or consist of the oligopeptide. The oligopeptide is preferably isolated before inclusion within the composition. The oligopeptide most preferably consists of three (3) to six (6) amino acids.
For instance, the previously described preferred compound could, in one embodiment be:

    
    
        NT AA1 AA2 AA3 AA4 CT

wherein NT at the N-terminus is selected from the group of H—, CH3—, an acyl group, or a general protective group; and CT at the C-terminus is selected from the group of small (e.g. 1 to 5 amino acids) peptides, —OH, —OR1, —NH2, —NHR1, —NR1 R2, or —N(CH2)1-6 NR1 R2, wherein R1 and R2, when present, are independently selected from H, alkyl, aryl, (ar)alkyl, and wherein R1 and R2 can be cyclically bonded to one another.

    
    


“Alkyl” as used herein, is preferably a saturated branched or unbranched hydrocarbon having one to six carbon atoms, e.g. methyl, ethyl, and isopentyl.
“Aryl” as used herein, is an aromatic hydrocarbon group, preferably having 6 to 10 carbon atoms, such as phenyl or naphthyl.
“(Ar)alkyl”, as used herein, is an arene group (having both aliphatic and aromatic portions), preferably having 7 to 13 carbon atoms such as benzyl, ethylbenzyl, n-propylbenzyl, and isobutylbenzyl.
“Oligopeptide”, as used herein are peptides having from 3 to 8 amino acids joined together by peptide bonds. Equivalent to oligopeptides are compounds having the same or equivalent sidechains as the particular amino acids used in an oligopeptide, and arranged sequentially in the same order as the peptides, but joined together by non-peptide bonds, e.g., by isosteric linkages such as the keto isostere, hydroxy isostere, diketo isostere, or the keto-difluoromethylene isostere.
“Composition” also includes, for example, an acceptable salt of the oligopeptide or a labeled oligopeptide. As used herein, “acceptable salt” refers to salts that retain the desired activity of the oligopeptide or equivalent compound, but preferably do not detrimentally affect the activity of the oligopeptide or other component of a system in which uses the oligopeptide. Examples of such salts are acid addition salts formed with inorganic acids, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like. Salts may also be formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, and the like. Salts may be formed with polyvalent metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel and the like or with an organic cation formed from N,N′-dibenzylethylenediamine or ethylenediamine, or combinations thereof (e.g., a zinc tannate salt).
The thus developed chemical entity can be administered and introduced in-vivo systemically, topically, or locally. The peptide, or is modification or derivative, can be administered as the entity as such or as a pharmaceutically acceptable acid- or baseaddition salt, formed by reaction with an inorganic acid (such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid); or with an organic acid (such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxahc acid, malonic acid, succinic acid, maleic acid, and fumaric acid); or by reaction with an inorganic base (such as sodium hydroxide, ammonium hydroxide, potassium hydroxide); or with an organic base (such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines). A selected peptide and any of the derived entities may also be conjugated to sugars, lipids, other polypeptides, nucleic acids and PNA; and function in-situ as a conjugate or be released locally after reaching a targeted tissue or organ.
A “substitution” with regard to the various amino acids generally relate to substituting a group such as alkoxy, halogen, hydroxy, nitro, or lower alkyl onto an aromatic ring for a hydrogen that would usually be present. Substitutions can also be made on the alkyl chain connecting the aromatic portion to the peptide backbone, with, for instance lower alkyl groups substituting for a hydrogen. Still further substitutions can be made at the alpha position of an amino acid, also using an alkyl group.
Substitutions with regard to the amino acid phenylalanine include compounds such as L/D-homophenylalanyl, N methyl phenylalanyl, .alpha.-methylphenylalanyl, and alpha.-methyl-tyrosyl.
Preferred substitutions involve the use of fluorine or chlorine as a halogen, and methoxy as an alkoxy group. With regard to alkyl and lower alkyl, generally alkyl groups having fewer (1 to 3) carbon atoms are preferred.
The compounds according to the general formula may be prepared in a manner conventional for such compounds. To that end, suitably N alpha. protected (and side-chain protected if reactive side-chains are present) amino acid derivatives or peptides are activated and coupled to suitably carboxyl protected amino acid or peptide derivatives either in solution or on a solid support. Protection of the .alpha.-amino functions generally takes place by urethane functions such as the acid-labile tertiary-butyloxycarbonyl group (“Boc”), benzyloxycarbonyl (“Z”) group and substituted analogs or the base-labile 9-fluoremyl-methyloxycarbonyl (“Fmoc”) group. The Z group can also be removed by catalytic hydrogenation, Other suitable protecting groups include the Nps, Bmv, Bpoc, Aloc, MSC, etc. A good overview of amino protecting groups is given in The peptides, Analysis, Synthesis, Biology, Vol. 3 E. Gross and J. Meienhofer, eds., (Academic Press, New York, 1981). Protection of carboxyl groups can take place by ester formation, for example, base-labile esters like methyl or ethyl, acid labile esters like tert. butyl or, substituted, benzyl esters or hydrogenolytically. Protection of side-chain functions like those of lysine and glutamic or aspartic acid can take place using the aforementioned groups. Protection of thiol, and although not always required, of guanidino, alcohol and imidazole groups can take place using a variety of reagents such as those described in The Peptides, Analysis, Synthesis, Biology, id. or in Pure and Applied Chemistry, 59(3), 331-344 (1987). Activation of the carboxyl group of the suitably protected amino acids or peptides can take place by the azide, mixed anhydride, active ester, or carbodiimide method especially with the addition of catalytic and racemization-suppressing compounds like 1-N—N-hydroxybenzotriazole, N-hydroxysuccin-imide, 3-hydroxy-4-oxo-3,4-dihydro-1,2,3,-benzotriazine, N-hydroxy-5 norbornene-2,3-dicarboxyimide. Also the anhydrides of phosphorus based acids can be used. See, e.g., The Peptides, Analysis, Synthesis, Biology, supra and Pure and Applied Chemistry, 59(3), 331-344 (1987).
It is also possible to prepare the compounds by the solid phase method of Merrifield. Different solid supports and different strategies are known see, e.g. Barany and Merrifield in The Peptides, Analysis, Synthesis, Biology, Vol. 2, E. Gross and J. Meienhofer, eds., (Acad. Press, New York, 1980), Kneib-Cordonier and Mullen Int. J. Peptide Protein Res., 30, 705-739 (1987) and Fields and Noble Int. J. Peptide Protein Res., 35, 161-214 (1990). The synthesis of compounds in which a peptide bond is replaced by an isostere, can, in general, be performed using the previously described protecting groups and activation procedures. Procedures to synthesize the modified isosteres are described in the literature e.g. for the—CH2—NH—isostere and for the—CO—CH2—isostere.
Removal of the protecting groups, and, in the case of solid phase peptide synthesis, the cleavage from the solid support, can take place in different ways, depending on the nature of those protecting groups and the type of linker to the solid support. Usually deprotection takes place under acidic conditions and in the presence of scavengers. See, e.g. volumes 3, 5 and 9 of the series on The Peptides Analysis, Synthesis, Biology, supra.
Another possibility is the application of enzymes in synthesis of such compounds; for reviews see, e.g., H. D. Jakubke in The Peptides, Analysis, Synthesis, Biology, Vol. 9, S. Udenfriend and J. Meienhofer, eds., (Acad. Press, New York, 1987).
Although possibly not desirable from an economic point of view, oligopeptides according to the invention could also be made according to recombinant DNA methods. Such methods involve the preparation of the desired oligopeptide thereof by means of expressing recombinant polynucleotide sequence which codes for one or more of the oligopeptides in question in a suitable microorganism as host. Generally the process involves introducing into a cloning vehicle (e.g., a plasmid, phage DNA, or other DNA sequence able to replicate in a host cell) a DNA sequence coding for the particular oligopeptide or oligopeptides, introducing the cloning vehicle into a suitable eucaryotic or procaryotic host cell, and culturing the host cell thus transformed. When a eucaryotic host cell is used, the compound may include a glycoprotein portion.
As used herein, a “functional analogue” or “derivative” of a peptide includes an amino acid sequence, or other sequence monomers, which has been altered such that the functional properties of the sequence are essentially the same in kind, not necessarily in amount. An analogue or derivative can be provided in many ways, for instance, through “conservative amino acid substitution”. Also peptidomimetic compounds can be designed that functionally or structurally resemble the original peptide taken as the starting point but that are for example composed of non-naturally occurring amino acids or polyamides. With “conservative amino acid substitution”, one amino acid residue is substituted with another residue with generally similar properties (size, hydrophobicity), such that the overall functioning is likely not to be seriously affected. However, it is often much more desirable to improve a specific function. A derivative can also be provided by systematically improving at least one desired property of an amino acid sequence. This can, for instance, be done by an Ala-scan and/or replacement net mapping method. With these methods, many different peptides are generated, based on an original amino acid sequence but each containing a substitution of at least one amino acid residue. The amino acid residue may either be replaced by alanine (Ala-scan) or by any other amino acid residue (replacement net mapping). This way, many positional variants of the original amino acid sequence are synthesized. Every positional variant is screened for a specific activity. The generated data are used to design improved peptide derivatives of a certain amino acid sequence.
A derivative or analogue can also be, for instance, generated by substitution of an L-amino acid residue with a D-amino acid residue. This substitution, leading to a peptide which does not naturally occur in nature, can improve a property of an amino acid sequence. It is, for example, useful to provide a peptide sequence of known activity of all D-amino acids in retro inversion format, thereby allowing for retained activity and increased half-life values. By generating many positional variants of an original amino acid sequence and screening for a specific activity, improved peptide derivatives comprising such D-amino acids can be designed with further improved characteristics.
A person skilled in the art is well able to generate analogous compounds of an amino acid sequence. This can, for instance, be done through screening of a peptide library. Such an analogue has essentially the same functional properties of the sequence in kind, not necessarily in amount. Also, peptides or analogues can be circularized, for example, by providing them with (terminal) cysteines, dimerized or multimerized, for example, by linkage to lysine or cysteine or other compounds with side-chains that allow linkage or multimerization, brought in tandem- or repeat-configuration, conjugated or otherwise linked to carriers known in the art, if only by a labile link that allows dissociation.
Synthetic versions of these oligopeptides as described above, and functional analogues or derivatives of these breakdown products, are herein provided to modulate gene expression in a cell and be used in methods to rectify errors in gene expression or the treatment of disease.
The term “pharmaceutical composition” as used herein is intended to cover both the active composition of the invention alone or a composition containing the composition of the invention together with a pharmaceutically acceptable carrier, diluent or excipient. Acceptable diluents of an oligopeptide as described herein in the detailed description are for example physiological salt solutions or phosphate buffered salt solutions. In one embodiment, a signal molecule is administered in an effective concentration to an animal or human systemically, e.g., by intravenous, intramuscular or intraperitoneal administration. Another way of administration comprises perfusion of organs or tissue, be it in vivo or ex vivo, with a perfusion fluid comprising a signal molecule according to the invention. Topical administration, e.g., in ointments or sprays, may also apply, e.g., in inflammations of the skin or mucosal surfaces of for example mouth, nose and/or genitals. Local administration can occur in joints, bursae, tendon sheaths, in or around the spinal cord at locations where nerve bundles branch off, at the location of hernias, in or around infarcted areas in brain or heart, etc. The administration may be done as a single dose, as a discontinuous sequence of various doses, or continuously for a period of time sufficient to permit substantial modulation of gene expression. In the case of a continuous administration, the duration of the administration may vary depending upon a number of factors which would readily be appreciated by those skilled in the art.
The administration dose of the active molecule may be varied over a fairly broad range. The concentrations of an active molecule which can be administered would be limited by efficacy at the lower end and the solubility of the compound at the upper end. The optimal dose or doses for a particular patient should and can be determined by the physician or medical specialist involved, taking into consideration well-known relevant factors such as the condition, weight and age of the patient, etc.
The active molecule may be administered directly in a suitable vehicle, such as, for example, phosphate-buffered saline (PBS) or solutions in alcohol or DMSO. Pursuant to preferred embodiments of the present invention, however, the active molecule is administered through a single dose delivery using a drug-delivery system, such as a sustained-release delivery system, which enables the maintenance of the required concentrations of the active molecule for a period of time sufficient for adequate modulation of gene expression. A suitable drug-delivery system would be pharmacologically inactive or at least tolerable. It should preferably not be immunogenic nor cause inflammatory reactions, and should permit release of the active molecule so as to maintain effective levels thereof over the desired time period. Alternatives are known in the art as suitable for purposes of sustained release and are contemplated as within the scope of the present invention. Suitable delivery vehicles include, but are not limited to, the following: microcapsules or microspheres; liposomes and other lipid-based release systems; viscous instillates; absorbable and/or biodegradable mechanical barriers and implants; and polymeric delivery materials, such as polyethylene oxide/polypropylene oxide block copolymers, polyesters, cross-linked polyvinylalcohols, polyanhydrides, polymethacrylate and polymethacrylamide hydrogels, anionic carbohydrate polymers, etc. Useful delivery systems are well known in the art.
A highly suitable formulation to achieve the active molecule release comprises injectable microcapsules or microspheres made from a biodegradable polymer, such as poly(dl-lactide), poly(dl-lactide-co-glycolide), polycaprolactone, polyglycolide, polylactic acid-co-glycolide, poly(hydroxybutyric acid), polyesters or polyacetals. Injectable systems comprising microcapsules or microspheres having a diameter of about 50 to about 500 micrometers offer advantages over other delivery systems. For example, they generally use less active molecules and may be administered by paramedical personnel. Moreover, such systems are inherently flexible in the design of the duration and rate of separate drug release by selection of microcapsule or microsphere size, drug loading and dosage administered. Further, they can be successfully sterilized by gamma irradiation.
The design, preparation, and use of microcapsules and microspheres are well within the reach of persons skilled in the art and detailed information concerning these points is available in the literature. Biodegradable polymers (such as lactide, glycolide and caprolactone polymers) may also be used in formulations other than microcapsules and microspheres; e.g., premade films and spray-on films of these polymers containing the active molecule would be suitable for use in accordance with the present invention. Fibers or filaments comprising the active molecule are also contemplated as within the scope of the present invention.
Another highly suitable formulation for a single-dose delivery of the active molecule in accordance with the present invention involves liposomes. The encapsulation of an active molecule in liposomes or multilamellar vesicles is a well-known technique for targeted drug delivery and prolonged drug residence. The preparation and use of drug-loaded liposomes is well within the reach of persons skilled in the art and well documented in the literature.
Yet another suitable approach for single-dose delivery of an active molecule in accordance with the present invention involves the use of viscous installates. In this technique, high molecular weight carriers are used in admixture with the active molecule, giving rise to a structure which produces a solution with high viscosity. Suitable high molecular weight carriers include, but are not limited to, the following: dextrans and cyclodextrans; hydrogels; (cross-linked) viscous materials, including (cross-linked) viscoelastics; carboxymethylcellulose; hyaluronic acid; and chondroitin sulfate. The preparation and use of drug-loaded viscous instillates is well known to persons skilled in the art.
Pursuant to yet another approach, the active molecule may be administered in combination with absorbable mechanical barriers such as oxidized regenerated cellulose. The active molecule may be covalently or non-covalently (e.g., ionically) bound to such a barrier, or it may simply be dispersed therein.
The invention is further explained with the aid of the following illustrative examples.
EXAMPLES
Example I
Material and Methods
PEPTIDE SYNTHESIS: The peptides as mentioned herein such as LQG, AQG, LQGV (SEQ ID NO:1), AQGV (SEQ ID NO:2), LQGA (SEQ ID NO:19), VLPALP (SEQ ID NO:13), ALPALP (SEQ ID NO:21), VAPALP (SEQ ID NO:22), ALPALPQ (SEQ ID NO:23), VLPAAPQ (SEQ ID NO:24), VLPALAQ (SEQ ID NO:25), LAGV (SEQ ID NO:26), VLAALP (SEQ ID NO:27), VLPALA (SEQ ID NO:28), VLPALPQ (SEQ ID NO:29), VLAALPQ (SEQ ID NO:30), VLPALPA (SEQ ID NO:31), GVLPALP (SEQ ID NO:32), VVCNYRDVRFESIRLPGCPRGVNPVVSYAVALSCQCAL (SEQ ID NO:35), RPRCRPINATLAVEKEGCPVCITVNTTICAGYCPT (SEQ ID NO:45), SKAPPPSLPSPSRLPGPS (SEQ ID NO:38), LQGVLPALPQVVC (SEQ ID NO:34), SIRLPGCPRGVNPVVS (SEQ ID NO:39), LPGCPRGVNPVVS (SEQ ID NO:40), LPGC (SEQ ID NO:41), MTRV (SEQ ID NO:42), MTR, and VVC were prepared by solid-phase synthesis (R. B. Merrifield, J. Am. Chem. Soc., 85:2149-2165 (1963)) using the fluorenylmethoxycarbonyl (Fmoc)/tert-butyl-based methodology (Atherton, 1985) with 2-chlorotrityl chloride resin (Barlos et al., Int. J. Peptide Protein res., 37:513-520 (1991)) as the solid support.
The side-chain of glutamine was protected with a trityl function. The peptides were synthesized manually. Each coupling consisted of the following steps: (i) removal of the alpha-amino Fmoc-protection by piperidine in dimethylformamide (DMF), (ii) coupling of the Fmoc amino acid (3 eq) with diisopropylcarbodiimide (DIC)/1-hydroxybenzotriazole (HOBt) in DMF/N-methylformamide (NMP) and (iii) capping of the remaining amino functions with acetic anhydride/diisopropylethylamine (DIEA) in DMF/NMP. Upon completion of the synthesis, the peptide resin was treated with a mixture of trifluoroacetic acid (TFA)/H2O/triisopropylsilane (TIS) 95:2.5:2.5. After 30 minutes, TIS was added until decolorization. The solution was evaporated in vacuo and the peptide precipitated with diethylether.
The crude peptides were dissolved in water (50-100 mg/ml) and purified by reverse-phase high-performance liquid chromatography (RP-HPLC). HPLC conditions were: column: Vydac TP21810C18 (10×250 mm); elution system: gradient system of 0.1% TFA in water v/v (A) and 0.1% TFA in acetonitrile (ACN) v/v (B); flow rate 6 ml/min; absorbance was detected from 190-370 nm. There were different gradient systems used. For example, for peptides LQG and LQGV (SEQ ID NO:1): 10 minutes 100% A followed by linear gradient 0-10% B in 50 minutes. For example for peptides VLPALP (SEQ ID NO:3) and VLPALPQ (SEQ ID NO:29): 5 minutes 5% B followed by linear gradient 1% B/minute. The collected fractions were concentrated to about 5 ml by rotation film evaporation under reduced pressure at 40° C. The remaining TFA was exchanged against acetate by eluting two times over a column with anion exchange resin (Merck II) in acetate form. The eluate was concentrated and lyphilized in 28 hours. Peptides later were prepared for use by dissolving them in PBS.
Example II
Endotxin Shock Model (Sepsis)
Sepsis. For the endotoxin model, BALB/c mice were injected i.p. with 8-9 mg/kg LPS (E. coli 026:B6; Difco Lab., Detroit, Mich., USA). Control groups (PBS) were treated with PBS i.p. only. To test the effect of NMPF from different sources (synthetic, commercial hCG preparation [c-hCG]), we treated BALB/c mice with a dose of 300-700 IU of different hCG preparations (PG23; PREGNYLTM batch no. 235863, PG25; PREGNYLTM batch no. 255957 from NV Organon of Oss, NL) and with synthetic peptides (5 mg/kg) after two hours of LPS injection. In other experiments, BALB/c mice were injected i.p. either with 10 mg/kg or with 11 mg/kg LPS (E. coli 026:B6; Difco Lab., Detroit, Mich., USA). Subsequently, mice were treated after 2 hours and 24 hours of LPS treatment with NMPF peptides.
Semi-quantitative sickness measurements. Mice were scored for sickness severity using the following measurement scheme:

    
    
        1 Percolated fur, but no detectable behaviour differences compared to normal mice.
        2 Percolated fur, huddle reflex, responds to stimuli (such as tap on cage), just as active during handling as healthy mouse.
        3 Slower response to tap on cage, passive or docile when handled, but still curious when alone in a new setting.
        4 Lack of curiosity, little or no response to stimuli, quite immobile.
        5 Labored breathing, inability or slow to self-right after being rolled onto back (moribund)
        6 Sacrificed

Results

Endotoxin Shock Model (Sepsis)

    
    


Sepsis experiments. To determine the effect of synthetic peptides (NMPF) in high-dose LPS shock model, BALB/c mice were injected intraperitoneally with different doses of LPS and survival was assessed daily for 5 days. In this experiment (for the LPS endotoxin model), BALB/c mice were injected i.p. with 8-9 mg/kg LPS (E. coli 026:B6; Difco Lab., Detroit, MI, USA). Control groups (PBS) were treated with PBS i.p. only. We treated BALB/c mice with a dose of 300-700 IU of different hCG preparations (PG23; PREGNYL batch no. 235863, PG25; PREGNYL batch no. 255957) or with peptides (5 mg/kg) after two hours of LPS injection.
These experiments showed (Table 1) that NMPF peptides 4, 6, 66 and PG23 inhibited shock completely (all mice had in first 24 hours sickness scores not higher than 2; shortly thereafter they recovered completely and had sickness scores of 0), while peptides 2, 3 and 7 accelerated shock (all mice had in first 24 hours sickness scores of 5 and most of them died, while the control mice treated with LPS+PBS had sickness scores of 3-4 in first 24 hours and most of them died after 48 hours with sickness scores of 5; 17% survival rate at 72 hours). In addition, peptides 1, 5, 8, 9, 11, 12, 13, 14 and 64 showed in a number of different experiments variability in effectiveness as well as in the kind (inhibitory vs accelerating) of activity. This variability is likely attributable to the rate of breakdown of the various peptides and the different effects the various peptides and their breakdown products have in vivo. In addition, these experiments also showed the variability in anti-shock activity in c-hCG preparations that is likely attributable to the variation in the presence of anti-shock and shock-accelerating NMPF. Visible signs of sickness were apparent in all of the experimental animals, but the kinetics and obviously the severity of this sickness were significantly different. These data are representative of at least 10 separate experiments.
In Table 2, we see the effect of ALA-replacement (PEPSCAN) in peptide LQG, LQGV (SEQ ID NO:1), VLPALP (SEQ ID NO:3), VLPALPQ (SEQ ID NO:29) in septic shock experiments. We conclude that the change in even one amino acid by a neutral amino acid can lead to different activity. So, genomic differences as well as polymorphism in these peptides can regulate the immune response very precisely. Derivatives of these peptides, for example (but not limited to) by addition of classical and non-classical amino acids or derivatives that are differentially modified during or after synthesis, for example benzylation, amidation, glycosylation, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. could also lead to a better effectiveness of the activity.
To determine whether treatment of BALB/c mice with NMPF inhibits septic shock at different stages of disease, synthetic peptides (NMPF) were injected i.p. at 2 and 24 hours after the induction of septic shock with high dose LPS (10 mg/kg).
As shown in Tables 3 and 4, control mice treated with PBS after the shock induction reached a sickness score of 5 at 14 and 24 hours, and remained so after the second injection with PBS. The survival rate in control group mice was 0% at 48 hours. In contrast to control mice, mice treated with NMPF 9, 11, 12, 43, 46, 50 and 60 reached a maximum sickness score of 2-3 at 24 hours after the induction of septic shock and further reached a maximum sickness score of 1-2 at 48 hours after the second injection of NMPF. In addition, mice treated with NMPF 5, 7, 8, 45, 53 and 58 reached a sickness score of 5 and after the second injection with NMPF all mice returned to a sickness score of 1-2 and survival rates in NMPF groups were 100%. Mice treated with NMPF 3 reached sickness scores of 3-4 and the second NMPF injection did save these mice. These experiments show that NMPF peptides have anti-shock activity at different stages of the disease and NMPF have anti-shock activity even at the disease stage when otherwise irreversible damage had been done. This indicates that NMPF have effects on different cellular levels and also have repairing and regenerating capacity.
Example III
NOD Experiment
Mice. Female NOD mice at the age of 13-14 weeks were treated i.p. with PBS (n=6) or NMPF peptides (VLPALPQVVC (SEQ ID NO:20), LQGV (SEQ ID NO:1), GVLPALPQ (SEQ ID NO:33), VLPALP (SEQ ID NO:3), VLPALPQ (SEQ ID NO:29), MTRV (SEQ ID NO:42), LPGCPRGVNPVVS (SEQ ID NO:40), CPRGVNPVVS (SEQ ID NO:50), LPGC (SEQ ID NO:41), MTRVLQGVLPALPQVVC (SEQ ID NO:44), VVCNYRDVRFESIRLPGCPRGVNPVVSYAVALSCQCAL (SEQ ID NO:35)) (5 mg/kg, n=6) three times a week for 2 weeks. Every four days urine was checked for the presence of glucose (Gluketur Test; Boehringer Mannheim, Mannheim, Del.). All mice used in these studies were maintained in a pathogen-free facility. They were given free access to food and water. The experiments were approved by the Animal Experiments Committee of the Erasmus University Rotterdam. Diabetes was assessed by measurement of the venous blood glucose level using an Abbott Medisense Precision glucometer. Mice were considered diabetic after two consecutive glucose measurements≧11 mmol/l (200 mg/dl). Onset of diabetes was dated from the first consecutive reading.
Glucose tolerance test (GTT) was performed at 28 weeks of age in fasted mice (n=5) by injecting 1 g/kg D-glucose intraperitoneally (i.p.). At 0 (fasting), 5, 30 and 60 minutes, blood samples were collected from the tail and tested for glucose content.
Example IV
NO Experiment
Cell culture. The RAW 264.7 murine macrophage cell line, obtained from American Type Culture Collection (Manassas, Va., USA), were cultured at 37° C. in 5% CO2 using DMEM containing 10% fetal calf serum (FCS), 50 U/ml penicillin, 50 μg/ml streptomycin, 0.2 M Na-pyruvate, 2 mM glutamine and 50 μM 2-mercaptoethanol (Bio Whittaker, Europe). The medium was changed every 2 days.
Nitrite measurements. Nitrite production was measured in the RAW 264.7 macrophage supernatants. The cells (7.5×105/ml) were cultured in 48-well plates in 500 μl of culture medium. The cells were stimulated with LPS (10 microg/ml) and/or NMPF (1 pg/ml, 1 ng/ml, 1 μg/ml) for 24 hours, then the culture media were collected. Nitrite was measured by adding 100 microl of Griess reagent (Sigma) to 100 microl samples of culture medium. The OD540 was measured using a microplate reader, and the nitrite concentration was calculated by comparison with the OD540 produced using standard solutions of sodium nitrite in the culture medium.
Results
NOD Experiment
In order to determine whether NMPF has effect on the disease development in NOD mice, we tested NMPF on pre-diabetic female NOD mice at the age of 13-14 weeks. After only two weeks of treatment (injection of NMPF (5 mg/kg) every other day), glucosuria data of all NOD mice was analyzed at the of 17 weeks. Profound anti-diabetic effect (mice negative for glucosuria) was observed in different NMPF groups as compared to the PBS group, especially in NMPF groups treated with peptide VLPALPQVVC (SEQ ID NO:20), VLPALP (SEQ ID NO:3), MTRV (SEQ ID NO:42), LPGCPRGVNPVVS (SEQ ID NO:40) and LPGC. In addition, impairment of the glucose tolerance test was positively correlated to insulitis, but negatively correlated to the number of functional beta cells; also this test showed that NOD mice successfully treated with NMPF were tolerant for glucose as compared to the PBS group. Our results show that PBS treated NOD mice were all diabetic at the age of 23 weeks. Whereas, NOD mice treated three times a week for two weeks with NMPF showed profound inhibition of diabetes development. The strongest anti-diabetic effects were seen with NMPF-1, -4, -5, -6, -7, -65, -66 and commercial hCG preparation (PREGNYL, batch no. 235863). These mice had a low fasting blood glucose level and were tolerant for glucose (data partially shown). However, NMPF-71 showed no effect on the incidence of diabetes, while NMPF-64 and NMPF-11 had a moderate anti-diabetic effect.
NO Experiment
NO production is a central mediator of the vascular and inflammatory response. Our results show that macrophages (RAW 264.7) stimulated with LPS produce large amounts of NO. However, these cells co-stimulated with most of the NMPF peptides (NMPF peptides 1 to 14, 43 to 66 and 69) even in a very low dose (1 pg/ml) inhibited the production of NO.
Results
apoE Experiment
The invention provides a method and a composition of the invention for the treatment of conditions that are associated with dysfunctional LDL receptors such as apoE and other members of the apolipoprotein family. In particular, use of a composition of the invention comprising GVLPALPQ (SEQ ID NO:33) (NMPF-5) and/or VLPALP (SEQ ID NO:3) (NMPF-6) or a functional analogue or derivative thereof is preferred. Groups of apoE deficient mice (n=6 per group) were fed a high cholesterol food and given PBS or NMPF every other day intraperitoneally. After 2.5 weeks, body weight was determined as shown in the Table below.

















Average Weight (g)
SD (g)
p-value









ApoE-/-PBS
31.667
1.007




ApoE-/-NMPF-4
31.256
1.496
 0.536



ApoE-/-NMPF-5
29.743
1.160
 0.019



Background/PBS
26.760
1.582
10−6



ApoE-/-NMPF-6
29.614
1.064
 0.004


















TABLE 1







Results of shock experiments in mice











% SURVIVAL




IN TIME (HRS)












  0
 16
 40
 72

















TEST








SUBSTANCE


PBS

100
100
 67
 17


 


PG23

100
100
100
100


 


PG25

100
 83
 83
 83


PEPTIDE


NMPF
SEQUENCE


 1
VLPALPQVVC (SEQ ID NO:20)
100
100
 50
 17


 


 2
LQGVLPALPQ (SEQ ID NO:49)
100
 67
  0
  0


 


 3
LQG
100
 83
 20
 17


 


 4
LQGV (SEQ ID NO:1)
100
100
100
100


 


 5
GVLPALPQ (SEQ ID NO:33)
100
100
 80
 17


 


 6
VLPALP (SEQ ID NO:3)
100
100
100
100


 


 7
VLPALPQ (SEQ ID NO:168)
100
 83
  0
  0


 


 8
GVLPALP (SEQ ID NO:32)
100
100
 83
 67


 


 9
VVC
100
100
 50
 50


 


11
MTRV (SEQ ID NO:42)
100
100
 67
 50


 


12
MTR
100
100
 67
 50


 


13
LQGVLPALPQVVC
100
100
100
100


 



(SEQ ID NO:34)


 


14
(CYCLIC) LQGVLPALPQVVC
100
 83
 83
 83


 



(SEQ ID NO:34)


 


64
LPGCPRGVNPVVS
100
100
100
100


 



(SEQ ID NO:40)


 


66
LPGC (SEQ ID NO:41)
100
100
100
100

















TABLE 2







Additional results of shock experiments








NMPF SEQUENCE ID:












ANTI-SHOCK EFFECT


LQGV (SEQ ID NO:1)
+++


 


AQGV (SEQ ID NO:2)
+++


 


LQGA (SEQ ID NO:19)
+++


 


VLPALP (SEQ ID NO:3)
+++


 


ALPALP (SEQ ID NO:21)
++


 


VAPALP (SEQ ID NO:22)
++


 


ALPALPQ (SEQ ID NO:23)
++


 


VLPAAPQ (SEQ ID NO:24)
++


 


VLPALAQ (SEQ ID NO:25)
+++



SHOCK ACCELERATING EFFECT


LAGV (SEQ ID NO:26)
+++


 


LQAV (SEQ ID NO:52)
+++


 


VLAALP (SEQ ID NO:27)
+++


 


VLPAAP (SEQ ID NO:117)
+++


 


VLPALA (SEQ ID NO:28)
+++


 


VLPALPQ (SEQ ID NO:29)
+++


 


VLAALPQ (SEQ ID NO:30)
+++


 


VLPALPA (SEQ ID NO:31)
+++
















TABLE 3







Further additional results of shock experiments










% SURVIVAL IN TIME (HRS)















Tx

Tx




NMPF PEPTIDES
0
14
24
48







PBS
100
100
100
 0



NMPF-3
100
100
100
 0



NMPF-5
100
100
100
100



NMPF-7
100
100
100
 67



NMPF-8
100
100
100
100



NMPF-9
100
100
100
100



NMPF-11
100
100
100
100



NMPF-12
100
100
100
100



NMPF-43
100
100
100
100



NMPF-45
100
100
100
100



NMPF-46
100
100
100
100



NMPF-50
100
100
100
100



NMPF-53
100
100
100
100



NMPF-58
100
100
100
100



NMPF-60
100
100
100
100

















TABLE 4







Further additional results









SICKNESS SCORES












Tx

Tx



NMPF PEPTIDES
0
14
24
48





PBS
0, 0, 0, 0, 0, 0
5, 5, 5, 5, 4, 4
5, 5, 5, 5, 5, 5
††††††


NMPF-3
0, 0, 0, 0, 0, 0
3, 3, 3, 3, 3, 4
4, 4, 4, 4, 4, 4
††††††


NMPF-5
0, 0, 0, 0, 0, 0
5, 5, 5, 5, 5, 5
5, 5, 5, 5, 5, 5
2, 2, 2, 2, 2, 2


NMPF-7
0, 0, 0, 0, 0, 0
1, 1, 4, 4, 4, 4
5, 5, 5, 5, 5, 5
2, 2, 2, 2, ††


NMPF-8
0, 0, 0, 0, 0, 0
3, 3, 5, 5, 5, 5
5, 5, 5, 5, 5, 5
2, 2, 4, 4, 4, 5


NMPF-9
0, 0, 0, 0, 0, 0
3, 3, 4, 4, 5, 5
2, 2, 2, 2, 2, 2
1, 1, 2, 2, 2, 2


NMPF-11
0, 0, 0, 0, 0, 0
1, 1, 3, 3, 4, 4, 
2, 2, 2, 2, 4, 4
1, 1, 1, 1, 1, 1


NMPF-12
0, 0, 0, 0, 0, 0
1, 1, 1, 1, 3, 3
1, 1, 1, 1, 1, 1
1, 1, 1, 1, 1, 1


NMPF-43
0, 0, 0, 0, 0, 0
1, 1, 4, 4, 4, 4
1, 1, 1, 1, 3, 3
2, 2, 2, 2, 2, 2


NMPF-45
0, 0, 0, 0, 0, 0
5, 5, 5, 5, 4, 4
3, 3, 4, 4, 5, 5
2, 2, 4, 4, 5, 5


NMPF-46
0, 0, 0, 0, 0, 0
1, 1, 2, 2, 3, 3
1, 1, 2, 2, 2, 2
1, 1, 1, 1, 1, 1


NMPF-50
0, 0, 0, 0, 0, 0
1, 1, 1, 1, 3, 3
2, 2, 2, 2, 3, 3
1, 1, 1, 1, 1, 1


NMPF-53
0, 0, 0, 0, 0, 0
5, 5, 5, 5, 5, 5
5, 5, 5, 5, 5, 5
1, 1, 2, 2, 2, 2


NMPF-58
0, 0, 0, 0, 0, 0
5, 5, 5, 5, 3, 3
5, 5, 5, 5, 3, 3
1, 1, 1, 1, 1, 1


NMPF-60
0, 0, 0, 0, 0, 0
1, 1, 4, 4, 2, 2
2, 2, 2, 2, 4, 4
1, 1, 1, 1, 1, 1

















TABLE 5







Summary of results of the various peptides in the various experiments.














ID
SEQUENCE
SEPSIS
ANGIOGENSIS
CAO
DC
NOD

















NMPF-1
VLPALPQVVC (SEQ ID NO:20)
−+

+
+




 


NMPF-2
LQGVLPALPQ (SEQ ID NO:49)
−+


+


 


NMPF-3
LQG
−+
+
+
+


 


NMPF-4
LQGV (SEQ ID NO:1)
+
+
+
+


 


NMPF-5
GVLPALPQ (SEQ ID NO:33)
−+


+


 


NMPF-6
VLPALP (SEQ ID NO:3)
+
+
+
+


 


NMPF-7
VLPALPQ (SEQ ID NO:29)
+
+

+


 


NMPF-8
GVLPALP (SEQ ID NO:32)
−+


+


 


NMPF-9
VVC
+
+

+


 


NMPF-10
QVVC (SEQ ID NO:43)


 


NMPF-11
MTRV (SEQ ID NO:42)
+
+

+
+


 


NMPF-12
MTR
−+
+

+


 


NMPF-13
LQGVLPALPQVVC (SEQ ID NO:34)
+


+


 


NMPF-14
cyclic—LQGVLPALPQVVC (SEQ ID NO:34)
+


 


NMPF-43
AQG
+
+

+


 


NMPF-44
LAG

+


 


NMPF-45
LQA
+
+


 


NMPF-46
AQGV (SEQ ID NO:2)
+
+

+


 


NMPF-47
LAGV (SEQ ID NO:26)
−+

+
+


 


NMPF-48
LQAV (SEQ ID NO:52)


 


NMPF-49
LQGA (SEQ ID NO:19)
+


 


NMPF-50
ALPALP (SEQ ID NO:21)
+


+


 


NMPF-51
VAPALP (SEQ ID NO:22)
+
+


 


NMPF-52
VLAALP (SEQ ID NO:27)


 


NMPF-53
VLPAAP (SEQ ID NO:117)
+


+


 


NMPF-54
VLPALA (SEQ ID NO:28)


 


NMPF-55
ALPALPQ (SEQ ID NO:23)
+


 


NMPF-56
VAPALPQ (SEQ ID NO:173)

+


 


NMPF-57
VLAALPQ (SEQ ID NO:30)


 


NMPF-58
VLPAAPQ (SEQ ID NO:24)
+


+


 


NMPF-59
VLPALAQ (SEQ ID NO:25)
+
+


 


NMPF-60
VLPALPA (SEQ ID NO:31)
+


+


 


NMPF-61
VVCNYRDVRFESIRLPGCPRGVNPVVSYAVALSCQCAL
−+

+


 



(SEQ ID NO:35)


 


NMPF-62
VVCNYRDVRFESIRLPGCPRGVNPVVSYAVALSCQ


 



(SEQ ID NO:169)


 


NMPF-63
SIRLPGCPRGVNPVVS (SEQ ID NO:39)
−+


 


NMPF-64
LPGCPRGVNPVVS (SEQ ID NO:40)


+


 


NMPF-65
CPRGVNPVVS (SEQ ID NO:50)


 


NMPF-66
LPGC (SEQ ID NO:41)
+
+
+


 


NMPF-67
CPRGVNP (SEQ ID NO:170)


 


NMPF-68
PGCP (SEQ ID NO:10)
−+


 


NMPF-69
RPRCRPINATLAVEKEGCPVCITVNTTICAGYCPT


 



(SEQ ID NO:45)


 


NMPF-70
MTRVLQGVLPALPQ (SEQ ID NO:171)
−+


 


NMPF-71
MTRVLPGVLPALPQVVC (SEQ ID NO:174)
−+


 


NMPF-74
CALCRRSTTDCGGPKDHPLTC (SEQ ID NO:46)


 


NMPF-75
SKAPPPSLPSPSRLPGPC (SEQ ID NO:172)


 


NMPF-76
TCDDPRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ


 



(SEQ ID NO:48)





+ = effects;


−+ = variable effect;


no entry is no effect or not yet tested when table was assembled














TABLE 6







MODULATION OF NO AND/OR TNF-α












ID
SEQUENCE
TNF-A
NO
TNF-A and NO















NMPF-1
VLPALPQVVC
++
++++
++++



 



(SEQ ID NO:20)


 


NMPF-2
LQGVLPALPQ
−+
++++
++++


 



(SEQ ID NO:49)


 


NMPF-3
LQG
+
++++
++++


 


NMPF-4
LQGV
++++
++++
+++++++


 



(SEQ ID NO:1)


 


NMPF-5
GVLPALPQ
++++
++++
+++++++


 



(SEQ ID NO:33)


 


NMPF-6
VLPALP
++++
++++
+++++++


 



(SEQ ID NO:3)


 


NMPF-7
VLPALPQ
++++
++++
+++++++


 



(SEQ ID NO:29)


 


NMPF-8
GVLPALP
++++
++++
+++++++


 



(SEQ ID NO:32)


 


NMPF-9
VVC
++++
++++
+++++++


 


NMPF-10
QVVC
++++
+++
++++


 



(SEQ ID NO:43)


 


NMPF-11
MTRV
++++
++++
++++


 



(SEQ ID NO:42)


 


NMPF-12
MTR
++++
++++
++++


 


NMPF-13
LQGVLPALPQVVC
++
++++
++++


 



(SEQ ID NO:34)


 


NMPF-14
cyclic-
++
++++
++++


 



LQGVLPALPQVVC


 



(SEQ ID NO:34)


 


NMPF-43
AQG
++++
++++
+++++++


 


NMPF-44
LAG
−+
++++
++++


 


NMPF-45
LQA
++++
++++
+++++++


 


NMPF-46
AQGV
++++
++++
+++++++


 



(SEQ ID NO:2)


 


NMPF-47
LAGV
++
++++
++++


 



(SEQ ID NO:26)


 


NMPF-48
LQAV
++
++++
++++


 



(SEQ ID NO:52)


 


NMPF-49
LQGA
++
++++
++++


 



(SEQ ID NO:19)


 


NMPF-50
ALPALP
++++
++++
+++++++


 



(SEQ ID NO:21)


 


NMPF-51
VAPALP
+
+++
++++


 



(SEQ ID NO:22)


 


NMPF-52
VLAALP
++
++++
++++


 



(SEQ ID NO:27)


 


NMPF-53
VLPAAP
++++
++++
+++++++


 



(SEQ ID NO:117)


 


NMPF-54
VLPALA
+
++++
++++


 



(SEQ ID NO:28)


 


NMPF-55
ALPALPQ
+
++++
++++


 



(SEQ ID NO:23)


 


NMPF-56
VAPALPQ
−+
++++
++++


 



(SEQ ID NO:173)


 


NMPF-57
VLAALPQ
+
++++
++++


 



(SEQ ID NO:30)


 


NMPF-58
VLPAAPQ
++++
++++
+++++++


 



(SEQ ID NO:24)


 


NMPF-59
VLPALAQ
++
++++
++++


 



(SEQ ID NO:25)


 


NMPF-60
VLPALPA
++++
++++
+++++++


 



(SEQ ID NO:31)


 


NMPF-61
VVCNYRDVRFESIRLPGCP
−+
++++
++++


 



RGVNPVVSYAVALSCQCAL


 



(SEQ ID NO:35)


 


NMPF-62
VVCNYRDVRFESIRLPGCP
−+
+++
++++


 



RGVNPVVSYAVALSCQ


 



(SEQ ID NO:169)


 


NMPF-63
SIRLPGCPRGVNPVVS
−+
++
++


 



(SEQ ID NO:39)


 


NMPF-64
LPGCPRGVNPVVS
++
++++
++++


 



(SEQ ID NO:40)


 


NMPF-65
CPRGVNPVVS
++
+++
+++


 



(SEQ ID NO:50)


 


NMPF-66
LPGC
+++
++
+++


 



(SEQ ID NO:41)


 


NMPF-67
CPRGVNP
−+
+
+


 



(SEQ ID NO:170)


 


NMPF-68
PGCP
+
+
+++


 



(SEQ ID NO:10)


 


NMPF-69
RPRCRPINATLAVEKEGCP
−+
++
++


 



VCITVNTTICAGYCPT


 



(SEQ ID NO:45)


 


NMPF-70
MTRVLQGVLPALPQ
−+
+
+


 



(SEQ ID NO:171)


 


NMPF-71
MTRVLPGVLPALPQVVC
−+
−+
−+


 



(SEQ ID NO:174)


 


NMPF-74
CALCRRSTTDCGGPKDHPL
−+
++
+


 



TC


 



(SEQ ID NO:46)


 


NMPF-75
SKAPPPSLPSPSRLPGPS
+
++
++


 



(SEQ ID NO:172)


 


NMPF-76
TCDDPRFQDSSSSKAPPPS
+
+
+


 



LPSPSRLPGPSDTPILPQ


 



(SEQ ID NO:48)


 


NMPF-78
CRRSTTDCGGPKDHPLTC
+
+
+


 



(SEQ ID NO:47)





from −+ to +++++++ indicates from barely active to very active in modulating






Example V
Monkey Experiment
Efficacy of NMPF, here a mixture 1:1:1 of LQGV (SEQ ID NO:1), AQGV (SEQ ID NO:2) and VLPALP (SEQ ID NO:3), administered in a gram-negative induced rhesus monkey sepsis model for prevention of septic shock.
Overwhelming inflammatory and immune responses are essential features of septic shock and play a central part in the pathogenesis of tissue damage, multiple organ failure, and death induced by sepsis. Cytokines, especially tumor necrosis factor (TNF)-α interleukin (IL)-1β, and macrophage migration inhibitory factor (MIF), have been shown to be critical mediators of septic shock. Yet, traditional anti-TNF and anti-IL-1 therapies have not demonstrated much benefit for patients with severe sepsis. We have designed peptides that block completely LPS induced septic shock in mice, even when treatment with these peptides is started up to 24 hours after LPS injection. These peptides are also able to inhibit the production of MIF. This finding provides the possibility of therapeutic use of these peptides for the treatment of patients suffering from septic shock. Since primates are evolutionary more closer to humans, we tested these peptides for their safety and effectiveness in a primate system.
Experimental Design














EXPERIMENTAL





TREATMENT



(independent variable,



e.g., placebo treated


GROUP
control group)
BIOTECHNIQUES
NUMBER







animal I
i.v. infusion of a lethal
Live E. coli infusion
N = 1



dose of live
Blood sampling



Escherichia. coli (10E10
No recovery (section)



CFU/kg) + antibiotics +



placebo treated


animal II
i.v. infusion of a lethal
Live E. coli infusion
N = 1



dose of live
Blood sampling



Escherichia. coli (10E10
No recovery (section)



CFU/kg) + antibiotics +



oligopeptide (5 mg/kg



of each of 3 peptides)









Only naive monkeys were used in this preclinical study to exclude any interaction with previous treatments. The animals were sedated with ketamine hydrochloride. Animals were intubated orally and allowed to breathe freely. The animals were kept anesthetized with O2/N2O/isoflurane. The animals received atropin as pre-medication for O2/N20/isoflurane anesthesia. A level of surgical anesthesia was maintained during the 2 h infusion of E. coli and for 6 h following E. coli challenge, after which the endothracheal tubes were removed and the animals were euthanized. Before bacteria were induced, a 1 hour pre-infusion monitoring of heart-rate and blood pressure was performed.
Two rhesus monkeys were infused with a 10 10CFU per kg of the Gram negative bacterium E. coli to induce a fatal septic shock. One monkey received placebo-treatment and was sacrificed within 7 hours after infusion of the bacteria without recovery from the anesthesia. The second monkey received treatment with test compound and was sacrificed at the same time point.
In a limited dose-titration experiment performed with the same bacterium strain in 1991, the used dose proved to induce fatal shock within 8 hours. In recent experiments, a 3-fold lower dose was used inducing clear clinical and pathomorphological signs of septic shock without fatal outcome.
The monkeys were kept anesthetized throughout the observation period and sacrificed 7 hours after the start of the bacterium infusion for pathological examination. The animals underwent a gross necropsy in which the abdominal and thorax cavities were opened and internal organs examined in situ.
Full Description of the Experiment with Three Rhesus Monkeys
The study was conducted in rhesus monkeys (Maccaca mulatta). Only experimentally naive monkeys were used in the study to exclude any interaction with previous treatments. Prior to the experiment, the state of health of the animals was assessed physically by a veterinarian. All animals had been declared to be in good health and were free of pathogenic ecto- and endoparasites and common bacteriological infections: Yersinia pestis, Yersinia enterocolitica, Yersinia pseudotuberculosis, Shigella, Aeromonas hydrophilia, pathogenic Campylobacter species and Salmonella. 
Reagents. The Escherichia coli strain was purchased from ATCC (E. coli; 086a: K61 serotype, ATCC 33985). In a control experiment, the strain proved equally susceptible to bactericidal factors in human and rhesus monkey serum. Prior to the experiment, a fresh culture was set-up; the E. coli strain was cultured for one day, harvested and washed thoroughly to remove free endotoxine. Prior to infusion into the animal, the number and viability of the bacteria were assessed. Serial dilutions of the E. coli stock were plated on BHI agar and cultured overnight at 37° C. The colonies on each plate were counted and the number of colony-forming units per ml was calculated. The body weight measurement of the day of the experiment was used to calculate the E. coli dose and E. coli stock was suspended in isotonic saline (N.P.B.I., Emmer-Compascuum, NL) at the concentration needed for infusion (total dose volume for infusion approximately 10 ml/kg. The E. coli suspension was kept on ice until infusion.
Antibiotic was used to synchronize the shock induction in the monkeys. Baytril (Baytril 2.5%, Bayer, Del.) was used instead of gentamycin, as the strain proved only marginally susceptible to the latter antibiotic. Individual animals were identified by a number or letter combination tattooed on the chest.
Experimental design.















EXPERIMENTAL





GROUP
TREATMENT


(number/
(independent variable,


letter or other
e.g., placebo treated


identification
control group)

NUMBER
SEX







Animal I
i.v. infusion of a lethal
Live E. coli infusion
N = 1
F



dose of live
Blood sampling



Escherichia. coli (10E10
No recovery



CFU/kg) + antibiotic +



placebo treated


Animal II
i.v. infusion of a lethal
Live E. coli infusion
N = 1
F



dose of live
Blood sampling



Escherichia. coli (10E10
No recovery (section)



CFU/kg) + antibiotic +



NMPF-4, -6, -46; each



5 mg/kg


Animal III
i.v. infusion of a lethal
Live E. coli infusion
N = 1
F



dose of live
Blood sampling



Escherichia. coli (10E10
Recovery and survival



CFU/kg) + antibiotic +



NMPF-4, -6, -46; each



5 mg/kg









Anesthesia. All animals were fasted overnight prior to the experiment. On the morning of the experiment, the animals were sedated with ketamine hydrochloride (Tesink, NL) and transported to the surgery. The animal was placed on its side on a temperature-controlled heating pad to support body temperature. Rectal temperature was monitored using a Vet-OX 5700. The animals were intubated orally and were allowed to breathe freely. The animals were kept anesthesized using O2/N2O/isoflurane inhalation anesthesia during the E. coli infusion and the 7 hour observation period following E. coli challenge, after which the endothracheal tubes were removed and the animals were euthanized or allowed to recover from anesthesia. The femoral or the cephalic vein was cannulated and used for infusing isotonic saline, live E. coli and antibiotic administration. Insensible fluid loss was compensated for by infusing isotonic saline containing 2.5% glucose (Fresenius, 's Hertogenbosch, NL) at a rate of 3.3 ml/kg/hr.
Preparative actions. During anesthesia the animals were instrumented for measurement of blood pressure (with an automatic cuff), heart rate and body temperature. Isotonic saline was infused at 3.3 ml/kg/hr to compensate for fluid loss. Femoral vessels were cannulated for infusion of E. coli and antibiotics. Temperature-controlled heating pads were used to support body temperature. The monkeys were continuously monitored during the E. coli challenge and for the 6 hr period following E. coli administration. After 7 hrs, 2 animals (the control animal and one treated with NMPF) were sacrificed to compare the direct effect of the compound at the level of histology. The 3rd animal, treated with NMPF, was allowed to recover from anesthesia and was intensively observed during the first 12 hours after recovery followed by frequent daily observation. The decision to allow the 3rd animal to recover was made after consulting with the veterinarian.
Induction of septic shock. Before the infusion of E. coli, a 1 hr pre-infusion monitoring of heart-rate and blood pressure was performed. All three animals received an i.v. injection of E. coli 086 (k61 serotype; ATCC 33985) at a lethal dose of 10×109 CFU/kg body weight. In a dose titration study with this batch performed in 1991, this bacterial dose induced lethal shock within 8 hrs after the start of the infusion. The infusion period was 2 hrs.
Antibiotics. Baytril was administered intravenously immediately after completion of the 2 h.E. coli infusion (i.v.; dose 9 mg/kg).
Treatment with NMPF. 30 minutes post-onset of E. coli infusion, the animals were administered a single intravenous bolus injection of a mixer of NMPF oligopeptides. The oligopeptide mixer contained the following NMPF peptides: LQGV (SEQ ID NO: 1) (5 mg/kg), AQGV (SEQ ID NO:2) (5 mg/kg) and VLPALP (SEQ ID NO:3) (5 mg/kg). These NMPF peptides were dissolved in 0.9% sodium chloride for injection (N.P.B.I., Emmer Compascuum, NL).
Results
Preliminary Monkey Results
An anti-shock effect of the test compound on sepsis in the monkey treated with the oligopeptide mixture, namely the inhibition of the effect of the sepsis in this early 7-hour trajectory of this primate model, was observed. Immunomodulatory effects with these peptides have been observed in vitro/ex vivo such as in T-cell assays, the inhibition of pathological Th1 immune responses, suppression of inflammatory cytokines (MIF), increase in production of anti-inflammatory cytokines (IL-10, TGF-beta) and immunomodulatory effects on antigen-presenting cells (APC) like dendritic cells and macrophages.
The following organs were weighed and a bacterial count was performed: kidneys, liver, lungs, lymph nodes, and gross lesions.
Tissues of all organs were preserved in neutral aqueous phosphate buffered 4% solution of formaldehyde. Lymphoid organs were cryopreserved. All tissues will be processed for histopathological examination.
Further Results Obtained in the Three-monkey Experiment
Monkey 429(control). Female monkey (5.66 kg) received an i.v. injection of E. coli 086 (10E10 CFU/kg). In a dose titration study with this batch performed in 1991, this bacterial dose induced lethal shock within 8 hrs after the start of the infusion. The infusion period was 2 hrs. Baytril was administered intravenously immediately after completion of the 2 h. E. coli infusion (i.v.; dose 9 mg/kg). After the E. coli injection, the monkey was observed by the authorized veterinarian without knowing which of the monkeys received NMPF treatment. The clinical observations were as follows: vomiting, undetectable pulse, heart arythmia, abnormalities in ECG: signs of ventricle dilatation/heart decompensation (prolonged QRS complex, extra systoles), decreased blood clotting and forced respiration. In addition, there was big fluctuation in heart rate (30-150 beats per minute), collapse of both systolic and diastolic blood pressure (35/20 mmHg) and decrease in blood oxygen concentration (80-70%). Seven hours after the start of the E. coli infusion, monkey began to vomit blood and feces, and have convulsions. After final examination, the veterinarian did not give permission to let this monkey awake. At this time point, the control monkey was euthanized. Hereafter, post-mortem examination was conducted and internal organs were examined in situ. A number of internal bleedings were found by the pathologist.
Monkey 459(NMPF). Female monkey (5.44 kg) received an i.v. injection of E. coli 086 (10E10 CFU/kg). In a dose titration study with this batch performed in 1991, this bacterial dose induced lethal shock within 8 hrs after the start of the infusion. The infusion period was 2 hrs. Thirty minutes after the initiation of E. coli infusion, NMPF was i.v. injected in a single bolus injection. Baytril was administered intravenously immediately after completion of the 2 h. E. coli infusion (i.v.; dose 9 mg/kg). After the E. coli injection, this monkey was also observed by the authorized veterinarian without knowing which of the monkeys received NMPF treatment. The clinical observations were as follows: normal pulse, heart sounds normal, normal ECG, higher heart-rate but otherwise stable (180 beats per minute), no hypotension (75/30 mmHg), normal blood oxygen concentration (95-85%), lungs sound normal, normal turgor. Seven hours after the start of the E. coli infusion, the clinical condition of the monkey was stable. After final examination, the veterinarian did give permission to let this monkey awake due to her stable condition. In order to compare the hematological and immunological parameters between the control and NMPF-treated monkey, at this time point the NMPF-treated monkey 459 was euthanized. Hereafter, post-mortem examination was conducted and internal organs were examined in situ. No macroscopic internal bleedings were found by the pathologist.
Monkey 427(NMPF). Female monkey (4.84 kg) received an i.v. injection of E. coli 086 (10E10 CFU/kg). In a dose titration study with this batch performed in 1991, this bacterial dose induced lethal shock within 8 hrs after the start of the infusion. The infusion period was 2 hrs. Thirty minutes after the initiation of E. coli infusion, NMPF was i.v. injected. Baytril was administered intravenously immediately after completion of the 2 h. E. coli infusion (i.v.; dose 9 mg/kg). After the E. coli injection, this monkey was also observed by the authorized veterinarian doctor without knowing which of the monkeys received NMPF treatment. The clinical observations were as follows: normal pulse, heart sounds normal, normal ECG, moderately higher heart-rate but otherwise stable (160 beats per minute), no hypotension (70/30 mmHg), normal blood oxygen concentration (95-90%), lungs sound normal, normal turgor. Seven hours after the start of the E. coli infusion, the clinical condition of the monkey was stable. After final examination, the veterinarian did give permission to let this monkey wake up due to her stable condition. The monkey woke up quickly, she was alert and there was a slow disappearance of oedema.

## Claims

1. A composition comprising one or more purified or isolated peptides of the sequence AQGV (SEQ ID NO:2).