# Patent

## Title
Compounds and methods for treatment of sickle cell or complications associated therewith

## Abstract

Compounds, compositions and methods are provided for treatment of sickle cell disease or a complication associated therewith, or graft versus host disease, in an individual. More specifically, the use of particular glycomimetics for the treatment is described.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser. No. 12/218,213 filed Jul. 11, 2008, now allowed; which application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 60/959,984 filed Jul. 18, 2007; whereby these applications are incorporated herein by reference in their entirety.


BACKGROUND
1. Technical Field
The present invention relates generally to compounds, compositions and methods for treating sickle cell disease or complications associated therewith, and more specifically to the use of particular glycomimetics for the treatment. The glycomimetics may also be used to treat graft versus host disease.
2. Description of the Related Art
Sickle cell disease is an inheritable hematological disorder based on a mutation in the β-globin gene of hemoglobin. Upon deoxygenation, this mutated hemoglobin polymerizes and causes a shape change (sickling) of the red blood cell. This change in red blood cells leads to obstruction of blood vessels causing a wide variety of complications such as stroke, pulmonary hypertension, end-organ disease and death.
In addition to the fatal or potentially fatal complications, there are serious non-fatal complications of sickle cell disease such as pain. The severity of the pain may vary, but normally requires some form of medical attention. Hospitalization may be necessary.
In the U.S. alone, approximately 70,000-80,000 people suffer from sickle cell disease. Sickle cell disease is estimated to affect one of every 1,300 infants in the general population, and one of every 400 of African descent. Currently, there is no cure for sickle cell disease. The disease is chronic and lifelong. Life expectancy is typically shortened.
Accordingly, there is a need in the art for the treatment of sickle cell disease or the complications associated therewith. The present invention fulfills these needs and further provides other related advantages.
BRIEF SUMMARY
Briefly stated, compounds, compositions and methods for treating sickle cell disease or the complications associated therewith, or graft versus host disease, are provided. In the present invention, the compounds used for treatment comprise, or consist of, a particular glycomimetic. Such a compound may be combined with a pharmaceutically acceptable carrier or diluent to form a pharmaceutical composition.
In one embodiment, the present invention provides a method for the treatment of sickle cell disease or a complication associated therewith in an individual who is in need thereof, comprising administering to the individual a compound in an amount effective for treatment, the compound with the formula:




wherein

L=linker group; and
n=0-1.
In one embodiment, the present invention provides a method for the treatment of graft versus host disease in an individual who is in need thereof, comprising administering to the individual a compound in an amount effective for treatment, the compound with the formula:




wherein

L=linker group; and
n=0-1.
In other embodiments, the above compounds or compositions thereof may be used in the manufacture of a medicament, for any of the uses recited herein.
These and other aspects of the present invention will become apparent upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.



BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
FIG. 1 is a diagram illustrating the synthesis of a component of Compound #1.
FIG. 2 is a diagram illustrating the synthesis of a component of Compound #1.
FIG. 3 is a diagram illustrating the modification of the component of FIG. 1.
FIG. 4 is a diagram illustrating the reaction of the components of FIGS. 2 and 3 to form Compound #1. Compound XIX of FIG. 2 is reacted with ethylenediamine (EDA) to form EDA-XIX.
FIG. 5 is a diagram illustrating the synthesis of Compound #2. Compound XIX of FIG. 2 is reacted with ethylenediamine (EDA) to form EDA-XIX.
FIG. 6 is a schematic of the protocol for inducing a vaso-occlusive crisis (VOC) in sickle cell-affected mice and recording blood flow.
FIG. 7 shows the effects of a compound or anti-E&P monoclonal antibodies on blood flow in the sickle cell mouse.
FIG. 8 shows the effects of a compound or anti-E&P monoclonal antibodies on the adhesion of sickle red blood cells to leukocytes in sickle cell mice in which a vaso-occlusive crisis was induced.
FIG. 9 is a schematic of a sickle cell mouse model for use with both prevention and treatment protocols. The black arrows underneath Comp. #2 are for the prevention protocol. The gray arrow underneath Comp. #2 is for the treatment protocol. Recording is started at the 90 minute mark for the prevention protocol. Recording is started at the 120 minute mark for the treatment protocol.
FIG. 10 shows the effects of a compound on delayed treatment of VOC in sickle cell mice as measured by blood flow rate. Compound #2 normalizes the rate of blood flow. The control is phosphate buffered saline (PBS) without compound.
FIG. 11 shows the effects of a compound on delayed treatment of VOC in sickle cell mice as measured by adherent white blood cells (WBCs). Compound #2 causes a significant reduction in the number of WBCs adherent to the vascular endothelium. The control is PBS without compound.
FIG. 12 shows the effects of a compound on delayed treatment of VOC in sickle cell mice as measured by interactions of sickle red blood cells (RBCS) with leukocytes (white blood cells). Compound #2 dramatically reduces interactions of sickle RBCs with leukocytes. The control is PBS without compound.
FIG. 13 depicts Kaplan-Meier survival curves. The curve with black diamond symbols is for Compound #2. The curve with gray squares is for the control of PBS without compound.



DETAILED DESCRIPTION
As noted above, the present invention provides compounds, compositions and methods for the treatment of sickle cell disease or a complication associated therewith, or graft versus host disease, in an individual.
Compounds useful in the compositions and methods of the present invention include embodiments with the formula:




In the above formula, “L” represents a linker. There may be no linkers present (i.e., “n” is 0) or a linker may be present (i.e., “n” is 1). Where no linker is present, the compound is with the formula:




Where n is 1, a linker is present. A linker may include a spacer group, such as —(CH2)p— or —O(CH2)p— where p is generally about 1-20 (including any whole integer range therein). Other examples of spacer groups include a carbonyl or carbonyl containing group such as an amide.
Embodiments of linkers include the following:




Other linkers, e.g., polyethylene glycols (PEG) or —C(═O)—NH—(CH2)p—C(═O)—NH2 where p is as defined above, will be familiar to those in the art or in possession of the present disclosure.

In another embodiment, the linker is




which produces:





In another embodiment, the linker is




which produces:





All compounds of the present invention or useful thereto (e.g., for pharmaceutical compositions or methods of treating), include physiologically acceptable salts thereof. Examples of such salts are Na, K, Li, Mg, Ca and Cl.
Compounds as described herein may be present within a pharmaceutical composition. A pharmaceutical composition comprises one or more compounds in combination with (i.e., not covalently bonded to) one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide) and/or preservatives. Within yet other embodiments, compositions of the present invention may be formulated as a lyophilizate. Compositions of the present invention may be formulated for any appropriate manner of administration, including for example, topical, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous, or intramuscular administration.
The compositions described herein may be administered as part of a sustained release formulation (La, a formulation such as a capsule or sponge that effects a slow release of compound following administration). Such formulations may generally be prepared using well known technology and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target site. Carriers for use within such formulations are biocompatible, and may also be biodegradable; preferably the formulation provides a relatively constant level of compound release. The amount of compound contained within a sustained release formulation depends upon the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.
The above described compounds including equivalents thereof are useful in methods of the present invention as it relates to sickle cell disease and as it relates to graft versus host disease. In an embodiment, an individual who is in need of treatment for sickle cell disease or a complication associated therewith is administered at least one (i.e., one or more) of the above described compounds in an amount effective for the treatment. As used herein, the term “treatment” (including variations such as “treating”) includes prevention. For example, a complication associated with sickle cell disease may not have presented itself in an individual with the disease, and a compound may be administered to prevent presentation of the complication in the individual. Sickle cell disease and complications associated therewith include, for example, anemia, red blood cells becoming stuck in blood vessels, ischemia, infarction, stroke, acute chest crisis, splenic sequestration crisis, shortened life expectancy, organ damage and periodic or chronic pain.
In another embodiment, an individual who is in need of treatment for graft versus host disease (GVHD) is administered at least one (i.e., one or more) of the above-described compounds in an amount effective for the treatment. GVHD commonly arises in patients post stem cell transplantation. A preferred route of administration is via an orally available formulation.
The term “treatment,” as set forth above, refers to any of a variety of positive effects from the treatment including, for example, eradicating a complication associated with the disease, relieving to some extent a complication, slowing or stopping progression of the disease, and prolonging the survival time of the recipient. The treatment may be used in conjunction with one or more other therapies for sickle cell disease or complications associated therewith, or therapies for graft versus host disease.
The above described compounds may be administered in a manner appropriate to the disease to be treated. Appropriate dosages and a suitable duration and frequency of administration may be determined by such factors as the condition of the patient, the type and severity of the patient's disease and the method of administration. In general, an appropriate dosage and treatment regimen provides the compound(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit. Within particularly preferred embodiments of the invention, a compound may be administered at a dosage ranging from 0.001 to 1000 mg/kg body weight (more typically 0.01 to 1000 mg/kg), on a regimen of single or multiple daily doses. Appropriate dosages may generally be determined using experimental models and/or clinical trials. In general, the use of the minimum dosage that is sufficient to provide effective therapy is preferred. Patients may generally be monitored for therapeutic effectiveness using assays suitable for the condition being treated, which will be familiar to those of ordinary skill in the art.
The following Examples are offered by way of illustration and not by way of limitation.
EXAMPLES
Example 1
Synthesis of BASA (FIG. 1)
Synthesis of compound 4: 3-nitro-benzyl iodide (1) (48.3 g) is added to an aqueous solution (pH 11) of commercially available (Aldrich Chemical Co., Milwaukee, Wis.), 8-aminonaphthalene-1,3,5-trisulfonic acid (2) (29.5 g) with stirring at room temperature. pH of the solution is adjusted to 1 and after evaporation of the solvent, the product 3 (6.4 g) is precipitated out from ethanol.
Platinum catalyzed hydrogenation of compound 3 affords compound 4 (the BASA of FIG. 1) in 96% yield.
Example 2
Synthesis of Glycomimetic (FIG. 2)
Synthesis of intermediate II: (−)-Shikimic acid (20 g) in MeOH (200 ml) and sulfuric acid (2 ml, 98%) are stirred at room temperature (rt) for 50 h. The reaction mixture is neutralized with 2N aqueous NaOH in the cold. After evaporation to dryness, the residue is purified by silica gel chromatography to afford II (19.2 g).
Synthesis of intermediate III: Methyl shikimate (II, 10 g), 2,2 dimethoxypropane (10 ml) and p-TsOH (0.8 g) are dissolved in acetonitrile (125 ml) and stirred at rt for 1 h. The reaction mixture is then neutralized with triethylamine (2 ml) and evaporated to dryness. The residue is chromatographed on silica gel to yield III (11 g).
Synthesis of intermediate IV: The shikimic acid derivative III (10 g) and PtO2/C (10%, 250 mg) in MeOH (40 ml) are hydrogenated at rt under vigorous stirring. After 16 h the reaction mixture is filtered over celite and evaporated to dryness. The residue is chromatographed on silica gel to yield IV.
Synthesis of intermediate V: To a solution of IV (8 g) in DCM (100 ml) at 0° C. are added pyridine (12 ml), acetic anhydride (7 ml) and a DMAP (25 mg). The reaction mixture is stirred at rt for 1 h, and diluted with EtOAc (250 ml). After washing with 0.5 M aqueous HCl (3×50 ml), saturated solution of KHCO3 (3×50 ml) and brine (3×50 ml), the combined organic layers are dried (Na2SO4) and evaporated to dryness. The residue is purified by chromatography on silica gel to yield V (6.8 g).
Synthesis of intermediate VI: A solution of V (6.0 g) in acetic acid (30 ml, 80%) is stirred at 80° C. for 1 h. Solvent is evaporated off and the residue is purified by chromatography on silica gel (DCM/MeOH 14:1) to yield VI (3.6 g).
Synthesis of intermediate VII: A solution of VI (3 g) and p-TsCl (3.5 g) in pyridine (30 ml) is stirred at rt for 6 h. MeOH (5 ml) is added and the solvent is evaporated at reduced pressure, the residue dissolved in EtOAc (3×150 ml) and the organic layers are washed with 0.5 M aqueous HCl (0° C.), water (cold) and brine (cold). The combined organic layers are dried (Na2SO4), filtered on Celite and evaporated to dryness. The residue is purified by chromatography on silica gel (toluene/EtOAc 4:1) to yield VII (3.7 g).
Synthesis of compound VIII: A solution of VII (3 g) and NaN3 (2.5 g) in DMF (20 ml) is stirred at 80° C. The reaction mixture is cooled to rt and diluted with EtOAc (200 ml) and water (50 ml). The organic layer is additionally washed twice with water (2×50 ml) and once with brine (50 ml). All aqueous layers are extracted twice with EtOAc (2×50 ml). The combined organic layers are dried with Na2SO4, filtered and the solvent is evaporated off. The residue is purified by chromatography on silica gel (petroleum ether/EtOAc 5:2) to give VIII (2.2 g).
Synthesis of compound X: To a solution of ethyl 2,3,4-tri-O-benzyl-α-L-fucothiopyanoside IX (1.5 g) in DCM (3 ml), bromine (150 μl) is added at 0° C. under argon. After 5 min the cooling bath is removed and the reaction mixture is stirred for additional 25 min at rt. Cyclohexene (200 μl) is added and the reaction mixture is added to a solution of VIII (400 mg), (Et)4NBr (750 mg) and powdered 4 Å molecular sieves in DCM (10 ml) and DMF (5 ml). After 16 h, triethylamine (1.5 ml) is added and stirred for an additional for 10 min, diluted with EtOAc (50 ml) and washed with sat. aqueous NaHCO3, water and brine. The aqueous layers are extracted twice with EtOAc (2×50 ml). The combined organic layers are dried (Na2SO4), filtered and evaporated to dryness. The residue is purified by chromatography on silica gel (toluene/EtOAc 9:1) to yield X (700 mg).
Synthesis of compound XI: To a solution of X (1.5 g) in MeOH (20 ml) is added freshly prepared NaOMe (80 mg) and the reaction mixture is stirred in a pressure tube at 80° C. for 20 h. The reaction mixture is cooled to rt and neutralized with acetic acid. Solvent is evaporated to dryness and the residue is dissolved in ether. Freshly prepared diazomethane is added and the excess diazomethane is neutralized with acetic acid. Solvent is evaporated off to give XI (1.25 g).
Synthesis of building block XV: This synthesis is done exactly in same way as described previously (Helvetica Chemica Acta 83:2893-2907 (2000)).
Synthesis of compound XVI: A mixture of XI (1.6 g), XV(3 g) and activated powdered molecular sieves 4 Å (1 g) in DCM (17 ml) is stirred at rt under argon for 2 h. Then DMTST (2 g) is added in 4 equal portions over a period of 1.5 h. After 24 h the reaction mixture is filtered over Celite and the filtrate is diluted with DCM (100 ml). The organic layer is washed with sat. aqueous NaHCO3 and brine and the aqueous layers are extracted twice with DCM. The combined organic layers are dried (Na2SO4), filtered and evaporated to dryness. The residue is purified by chromatography on silica gel (toluene/EtOAc 8:1) to yield XVI (1.5 g).
Synthesis of compound XVII: To a solution of XVI (500 mg) and orotic acid chloride (500 mg) in dichloromethane (10 ml) is added a solution of triphenylphosphine (500 mg in 5 ml dichloromethane) dropwise during 10 min. The reaction mixture is stirred at rt for 25 h and the solvent is evaporated off. The residue is purified (chromatography on silica gel DCM/MeOH 19:1) to give XVII (250 mg).
Synthesis of compound XVIII: To a solution of XVII (200 mg) in dioxane-water (5:1, 12 ml) is added 10% Pd—C (100 mg) and the reaction mixture is stirred vigorously under hydrogen (55 psi) for 24 h. Catalyst is filtered through a bed of celite and the solvent is evaporated off. Residue is purified by silica gel chromatography to give compound XVIII (150 mg).
Synthesis of XIX: To a solution of compound XVIII (145 mg) in MeOH (5 ml) is added a solution of NaOMe in MeOH (25%, 0.025 ml) and the reaction mixture is stirred at rt for 4 h, neutralized with acetic acid and the solvent is evaporated off. Residue is dissolved in water and passed through a bed of Dowex 50wX-8 (Na-form) resin. Water wash is evaporated off to afford compound XIX (100 mg).
Synthesis of EDA-XIX: XIX (80 mg) is heated at 70° C. with ethylenediamine (EDA) (1 ml) with stirring for 5 h. Solvent is evaporated off and the purified by sephadex G-25 column to give EDA-XIX (82 mg).
Example 3
Synthesis of PEGylated BASA (FIG. 3)
To a solution of 3,6-dioxaoctanedioic acid (PEG, 200 mg, Aldrich Chemical Co., Milwaukee, Wis.) in DMF (1 ml) is added Hunig base (0.4 ml), and then HATU (0.35 g) is added after 5 min. The solution is stirred at RT for 10 min. and then a solution of the BASA of Example 2 (50 mg) in DMF (0.1 ml) is added. The reaction mixture is stirred for 4 h at rt and the solvent is evaporated off. The residue is purified by hplc (reverse-phase C18 column) to give XX (40 mg).
Example 4
Synthesis of Glycomimetic-BASA Compound #1 (FIG. 4)
To a solution of XX from Example 3 (0.015 g) in DMF (0.1 ml) is added Hunig base (0.015 ml) and then HATU (0.007 g). The reaction mixture is stirred for 10 min at RT. A solution of EDA-XIX from Example 2 (0.010 g in DMF ml) is added and the reaction mixture is stirred at RT for 8 h. Solvent is evaporated off and the residue is purified by sephadex G-25 chromatography to give Glycomimetic-BASA #1 of FIG. 4 (0.008 g).
Example 5
Synthesis of Glycomimetic-BASA Compound #2 (FIG. 5)
Synthesis of compound XXI: To a solution of 3,6-dioxaoctanedioic acid (PEG, 200 mg, available commercially) in DMF (1 ml) is added Hunig base (0.4 ml) and then HATU (0.35 g) is added after 5 min. The solution is stirred at RT for 10 min and then solution of 8-aminonaphthalene-1,3,6-trisulfonic acid (50 mg, available commercially) in DMF is added. The reaction mixture is stirred for 4 h at RT and the solvent is evaporated off. The residue is purified by hplc (reverse-phase C18 column) to give XXI (25 mg).
Synthesis of compound XXII: This synthesis is performed in the same way as described in example 4 except using EDA-XIX from example 2 and XXI to give compound XXII (4 mg).
Example 6
Effects of Compound #2 on Microvascular Flow in Sickle Cell Mice as Determined by Intravital Microscopy
Sickle cell disease is a genetic condition caused by a point mutation (βS) in the β-chain of hemoglobin. This single mutation leads to abnormal microvascular flow, endothelial activation and episodic vaso-occlusion. Impairment of blood flow is responsible for the severe pain, end organ damage and eventual death of these patients.
An animal model of sickle cell disease exists in fully chimeric mice constructed by bone marrow transplantation and expressing >97% human globin containing the βS mutation. A vaso-occlusive crisis is induced by stimulation with TNFα and is monitored by observing and recording blood flow by intravital microscopy of the venules in the cremaster muscle. A schematic diagram of the protocol is shown in FIG. 6.
Each mouse is prepared by cannulating the right carotid artery and by undergoing a tracheotomy to facilitate ventilation under anesthesia. Twenty (20) minutes prior to stimulation with TNFα, the cremaster muscle is gently exteriorized and the venules are set on a microscope stage for observation and recording of blood flow. Just prior to administration of TNFα and again prior to the recording of the venules (90 min later), Compound #2 is administered through the cannulated carotid artery and data is obtained by monitoring blood flow and recording time of death.
The effects of Compound #2 on blood flow in the sickle cell mouse are shown in FIG. 7. In the control mice, in which only vehicle is injected, blood flow is very slow and indicative of the sickle disease state after stimulation with TNFα. Either Compound #2 or a mixture of antibodies against E- and P-selectin have dramatic effects by restoring blood flow to a velocity observed in normal mice.
During a vaso-occlusive crisis which is simulated in the sickle cell mouse model, blood cells adhere to each other, form aggregates, and decrease blood flow to vital organs, resulting in organ failure, and in severe cases, death. The effects of Compound #2 on the adhesion of sickle red blood cells (RBC) to leukocytes was determined in sickle cell mice in which TNFα was used to induce a vaso-occlusive crisis. As shown in FIG. 8, both Compound #2 and a mixture of antibodies against E- and P-selectins provide significant inhibition of adhesion among these cell types.
All of the above U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet, are incorporated herein by reference, in their entirety.
From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention.

## Claims

1. A method for the treatment of sickle cell disease or a complication associated therewith in an individual who is in need thereof, comprising administering to the individual a compound in an amount effective for treatment, the compound with the formula:



wherein L=linker group; n=0-1; and the compound is the sodium salt thereof.

2. The method according to claim 1, wherein in the compound n=0.

3. The method according to claim 1, wherein the compound is the sodium salt of:

4. The method according to claim 1, wherein the compound
is the sodium salt of:

5. The method of any one of claims 1-4, wherein the compound is in combination with a pharmaceutically acceptable carrier or diluent.

6. The method of claim 5, wherein the compound is in a buffer solution.