APPLICATION FOR PATENT

Title: THROMBIN INHIBITING COMPOSITIONS

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to novel thrombin inhibitor compounds and their use in therapy.

Thrombin is a serine protease present in blood plasma in the form of a precursor, prothrombin. Thrombin plays a central role in the mechanism of blood coagulation by converting the solution plasma protein, fibrinogen, into insoluble fibrin. Blood coagulation occurring inside a blood vessel (thrombosis) may obstruct the flow of blood to tissues thereby causing hypoxia, infarction or even cell death. Thrombosis may lead to

development of diseases such as deep vein thrombosis (DVT), peripheral arterial occlusion, pulmonary embolism, stroke and myocardial infarction.

Present methods for the treatment and prophylaxis of such thrombosis-associated diseases include therapeutic agents which inhibit thrombin activity, such as heparins and cumarins. These agents, however, do not act selectively on thrombin and cause many adverse side effects, such as hemorrhaging or thrombocytopenia.

There is thus a widely recognized need for, and it would be highly advantageous to have, selectively acting and less toxic thrombin inhibitors.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

In the drawings:

FIG. 1 illustrates the HPLC analysis of BL5030-1 Batch No. 036-081.

FIG. 2 illustrates the HPLC analysis of BL5030-2 Batch No. 036-082.

FIG. 3 illustrates the HPLC analysis of BL5030-3 Batch No. 036-083.

FIG. 4 illustrates the HPLC analysis of BL5030-4 Batch No. 036-084.

FIG. 5 illustrates the HPLC analysis of BL5030-5 Batch No. 036-085.

FIG. 6 illustrates the MS analysis of BL5030-1 Batch No. 036-081.

FIG. 7 illustrates the MS analysis of BL5030-2 Batch No. 036-082.

FIG. 8 illustrates the MS analysis of BL5030-3 Batch No. 036-083.

FIG. 9 illustrates the MS analysis of BL5030-4 Batch No. 036-084.

FIG. 10 illustrates the MS analysis of BL5030-5 Batch No. 036-085.

FIG. 11 illustrates the effects of spiking plasma samples with different compounds (BL-5030-1, BL-5030-2, BL-5030-3,BL-5030-4 and BL-5030-5) on the activated partial- prothrombin time (aPPT) and prothrombin time (PT). In the legend the term "comp" refers to "BL-5-30".

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of a novel four amino-acid peptide capable of selectively inhibiting thrombin and of its use in therapy.

The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of the components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and

terminology employed herein is for the purpose of description and should not be regarded as limiting.

U.S. Patent Nos. 7,081,447 and 7,423,021 to the present inventors describe thrombin inhibitor peptides which have been obtained by an accelerated type of computer-assisted drug discovery (CADDIS) approach.

While conceptualizing the present invention the present inventors designed the following novel peptides:

Ac-D-Cha-L-Aze-D-Tyr-L-hArg-NH ₂ (BL-5030-4);

Ac-L-Arg-D-Cha-L-Aze-D-Tyr-L-hArg-NH ₂ (BL-5030-2); and

Ac- Tx-D-Cha-L-Aze-D-Tyr-L-hArg-NH ₂ (BL-5030-5),

wherein Ac stands for acetylated N-Terminus;

Cha stands for cyclohexylalanine;

Aze stands for azetidine;

Tyr stands for tyrosine;

hArg stands for alpha-homo-arginine;

Tx stands for Tranexamic Acid;

Gu stands for guanylated N Terminus; and

NH ₂ stands for amidated C-Terminus.

While reducing the present invention to practice compounds BL-5030-2, BL-5030-4 and BL-5030-5 were synthesized and were found uniquely capable of selectively inhibiting thrombin with low cell toxicity (see details in the Examples hereinbelow)..

Thus, according to one aspect of the present invention, there is provided compound Ac-L-Arg-D-Cha-L-Aze-D-Tyr-L-hArg-NH ₂ (BL-5030-2) having the formula

or a pharmaceutically acceptable salt thereof. In another aspect of the present invention, there is provided compound Ac-D-Cha-L- Aze-D-Tyr-L-hArg-NH ₂ (BL-5030-4) having the formula

or a pharmaceutically acceptable salt thereof.

In another aspect of the present invention, there is provided a compound Ac- Tx-D- Cha-L-Aze-D-Tyr-L-hArg-NH ₂ (BL-5030-5) having the formula

or a pharmaceutically acceptable salt thereof.

In one embodiment of the present invention, the pharmaceutically acceptable salt is an acetate salt. Compounds BL-5030-2, BL-5030-4 and BL-5030-5 can be produced by conventional chemical synthesis technologies of Fmoc SPPS known in the art such as, for example, using the procedures described in Example 1 hereinbelow.

As used herein, any one of compounds BL-5030-2, BL-5030-4 or BL-5030-5 is referred to as "the compound of the present invention".

The compound of the present invention can be used for treating or preventing thrombosis or a thrombosis-associated disease, by administering to a subject of need thereof a therapeutically effective amount of the compound or a pharmaceutically acceptable salt thereof.

The phrase "treating or preventing" used herein encompasses the complete range of therapeutically positive effects of administrating the compound of the present invention to the subject, including reversing, alleviating, inhibiting the progress of, or preventing the disease or disorder associated with thrombin inhibition.

A thrombosis-associated disease, according to the present invention, generally refers to all complications characterized by unwanted or excessive thrombin-mediated or thrombin- associated functions and processes. Examples of such diseases or conditions include, but not limited to, deep vein thrombosis (DVT), peripheral arterial occlusion, thrombotic

complications of interventions of atherosclerotic disease, thrombotic complications of surgical or mechanical damage, mechanically-induced thrombin activation, shunt occlusion, thrombosis secondary to vascular damage and inflammation, indications with a diffuse thrombotic/platelet consumption component, venous thrombosis, coronary arterial thrombosis, pathological effects of atherosclerosis and arteriosclerosis, thrombin activity and clot formation in blood and blood products during storage, re -occlusion of an artery or vein following fibrinolytic therapy, thrombotic complications associated with thrombolytic therapy, thrombotic complications associated with coronary and other angioplasty, or thrombotic complications associated with coronary artery bypass procedures. Additional medical or surgical procedures that may cause unwanted thrombin activity include, for example, cardiac interventional procedures, tissue or organ transplantation and angioplastic procedures. These procedures include, without limitation, percutaneous transluminal coronary angioplasty with or without placement of an intracoronary stent, cardiac bypass surgery, hemodialysis, extra-corporeal circulation associated with a surgical procedure, intracranial angioplasty, and angioplasty on peripheral arteries. Additional medical conditions that are amendable to treatment in accordance with the presently described methods an compositions include, without limitation, sickle cell anemia crisis, heparin- induced thrombotic thrombocytopenia (HITT), idiopathic thrombotic thrombocytopenia (ITTP), stroke, atherosclerosis, angiogenesis, pulmonary embolism or thrombophlebitis, disseminated intravascular coagulation or thromboembolic syndromes associated with cancer, sepsis, or obstetrical complications, peripheral arterial occlusive disease, acute coronary syndromes such as unstable angina and myocardial infarction, or diabetes.

The term "subject" used herein refers to a mammal, preferably a human.

The compound of the present invention can also be used for ex-vivo expression, for storage and treatment of blood outside of the body and for coating of invasive devices.

The compound of the present invention can be administered to the subject per se or as part (active ingredient) of a pharmaceutical composition.

As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as

physiologically suitable carriers and excipients. The purpose of a pharmaceutical

composition is to facilitate administration of a compound to an organism.

Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.

Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

A pharmaceutical composition which includes the compound of the present invention may also include one or more anticoagulant or fibrinolytic agents such as, but not limited to, recombinant tissue plasminogen activator (rtPA), streptokinase (SK), urokinase (UK), proUK, heparin, enoxoparin, dalteparin, coumarin anticoagulants, aspirin, dipyrimidamole, aggrennox, ticlopidine, clopidogrel (Plavix), abciximab, RheoPro, integrilin and aggrestat.

Techniques for formulation and administration of drugs may be found in

"Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral, rectal,

transmucosal, especially transnasal, intestinal or parenteral delivery, including

intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, inrtaperitoneal, intranasal, or intraocular injections.

Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a bone tissue region of a patient.

Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. One route of administration which is suited for the pharmaceutical compositions of the present invention is subperiosteal injection, as described in U.S. Pat. No. 6,525,030 to Erikkson. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross- linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. As used herein, the term "oral administration" includes administration of the pharmaceutical compound to any oral surface, including the tongue, gums, palate, or other buccal surfaces. Addition methods of oral administration include provision of the pharmaceutical composition in a mist, spray or suspension compatible with tissues of the oral surface.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g.,

dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions.

Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.

The pharmaceutical composition of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.

Pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount effective to provide a significant therapeutic benefit. The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the disease or disorder, the manner of administration, the judgment of the prescribing physician, etc.

Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and in vivo procedures such as described, for example, in the Examples section which follows.

Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The

Pharmacological Basis of Therapeutics", Ch. 1 p.l).

Dosage amount and interval may be adjusted individually to levels of the active ingredient which are sufficient to, for example, retard tumor progression in the case of blastic metastases (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

Compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by

instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary

administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for use in thrombin inhibition or for treating a thrombosis-associated disease, as further detailed above.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

As used herein, the singular form "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

As used herein the term "about" refers to ± 10 %. Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below.

EXAMPLE 1

Synthesis Protocol of BL-5030

The desired thrombin inhibitors BL5030-1, 2, 3, 4 and 5 were synthesized on an automated peptide synthesizer for SPPS, using general Fmoc chemistry protocol. Several different reactions were carried out, changing different parameters, in order to find the optimal reaction conditions. As a result, the coupling reactions were performed with 3 equivalents of reagents in 60 minutes, using DIPEA as the base and HBTU/HOBt as the coupling reagent.

Product BL5030-1, 036-081 (638 mg) was obtained as the acetate salt in >99% purity. Product BL5030-2, 036-082 (340 mg) was obtained as the acetate salt in >99% purity. Product BL5030-3, 036-083 (620 mg) was obtained as the acetate salt in >99% purity. Product BL5030-4, 036-084 (630 mg) was obtained as the acetate salt in >99% purity. Product BL5030-5, 036-085 (442 mg) was obtained as the acetate salt in >99% purity.

Introduction The project involves the synthesis of five peptides synthesized from un-natural amino acids, as potential thrombin inhibitors. Since all amino acids in the BL5030 sequence are unnatural, these molecules are expected to be relatively stable for oral administration. The BL5030 peptides have the same core sequence of homo-arginine (L-hArg), D-tyrosine (D- Tyr), azetidine (L-Aze) and D-cyclohexyl alanine (D-Cha), which are all important for thrombin recognition. Peptides 1 , 3 and 5 have an additional amino acid, tranexamic acid (TX) at the N-terminal; however in peptide 1 the amine is left uncapped, in peptide 3 it is coupled with guanidine and in peptide 5 it is acetylated. Peptide 2 has arginine as its fifth amino acid and is also acetylated. Peptide 4 is the shortest of all five peptides as it does not have a fifth amino acid but it has an acetyl group on the N-terminal.

BL5030 structure

BL5030-1

TX-D-Cha-L-Aze-D-Tyr-L-hArg-NHz

Mw: Free base: 725.92

Acetate salt: 846.03 • BL -2

Ac-L-Arg-D-Cha-L-Aze-D-Tyr-L-hArg-NHz

Mw: Free base: 784.95

Acetate salt: 905.05

Acetate salt: 888.06

• -4

Ac-D-Cha-L-Aze-D-Tyr-L-hAr

Mw: Free base: 628.76

Acetate salt: 688.81

Ac- TX-D-C h a-L- Aze-D-Ty r-L-h Ar -NH ₂

Mw: Free base: 767.96

Acetate salt: 828.01 Materials

• Fmoc Rink Amide methylbenzhydrylamme (MBHA) resin, 100-200 mesh - Novabiochem Cat No. 01640037 (0.7 mmol/g resin)

• Fmoc-HomoArg(pbf)OH - Megapharm, Cat No. 60004-5

• Fmoc-D-Tyr(tBu)-OH - Mercury, Cat No. 4131037

• Fmoc-(S)-azetidine-2-carboxylic acid - Megapharm, Cat No. 20339

• N-a-Fmoc- -cyclohexyl-D-alanine - Mercury, Cat No. 4131058

• Trans-4-(Fmoc-aminomethyl)cyclohexane carboxylic acid - Sigma- Aldrich, Cat No.

58446

• Fmoc-Arg(Pbf)-OH - Mercury, Cat No. 4121 145

• Acetic anhydride - Bio Lab, Cat No. 480881

• N,N'-Di-Boc-lH-pyrazole-l-carboxamidine - Sigma-Aldrich, Cat No. 434167

• DIPEA (N,N-Diisopropylethylamine) - Sigma-Aldrich, Cat No. 49621 -9

• HOBt anhydrous (N-Hydroxybenzotriazole) - Megapharm., Cat No. 81002

• HBTU(O-(Benzotriazol- 1 -yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate) - Megapharm, Cat No. 200241

• Piperidine - Bio Lab, Cat No. 560621

• TIS (Triisopropylsilane) - Sigma-Aldrich, Cat No. 23378-1

• TFA (Trifluoroacetic acid) - Bio Lab, Cat No. 578981

• NMP (N-Methyl-2-pyrrolidone) - Bio Lab, Cat No. 57871 1

• DMF (N,N-Dimethylformamide) - Bio Lab, Cat No. 641681

• Acetonitrile - J. T. Baker, Cat No. 9017-03

• Dichloromethane - Bio Lab, Cat No. 573621

• Acetic acid - J. T. Baker, Cat No. 9508-01 Synthesis of BL5030

Optimization of reaction conditions

Initially, compounds BL5030-1 and BL5030-4 were selected as being most likely to give the best results and therefore these compounds were synthesized first and they were the ones upon which the optimization for the best reaction conditions were carried out.

3.1.1 BL5030-1

A number of small scale reactions were carried out on BL5030-1 using different activators, bases and other reaction variables in order to find the best conditions for synthesis of this peptide. The different activators compared were HATU, PyBOP and HBTU/HOBt. The different bases used were NMM and DIPEA. Comparisons were also made between double coupling of the first amino acid, homo-arginine, versus single coupling.

Based on the purity of the crude product obtained from each reaction it was found that the optimum conditions were those using HBTU/HOBt, DIPEA and single coupling of homo- arginine.

3.1.2 BL5030-4

A number of different experimental conditions were carried out on BL5030-4 to see if the purity of the reaction could be improved in any way. The reaction was carried out using HBTU/HOBt as the activator and both NMM and DIPEA as the base. The results of these two reactions confirmed that DIPEA is the preferred base.

As it was hypothesized that the most difficult amino acid to couple would be azetidine, due to the ring strain within, a further reaction was carried out using 5 equivalents of azetidine relative to the resin, instead of the 3 equivalents used for all previous reactions. In addition the coupling time was increased from 60 minutes to 90 minutes for the azetidine only. However these changes in reaction condition showed no effect in purity. Another reaction was carried out using HATU as the activator for the azetidine, also with no major effect on the purity.

A further reaction was carried out using 6 equivalents of all the amino acids. This showed a small increase in purity.

As a result of all these reactions, it was concluded that the best reaction conditions obtained, and thus those to be used for all subsequent syntheses of BL5030 require using 0.5M

HBTU/HOBt as the activator, DIPEA as the base, single coupling of all the amino acids and 3 equivalents of all amino acids. Though the reaction using 6 equivalents did show an increase in purity of crude product, the increase was not enough to justify the significant extra expense involved in doubling the quantity of amino acid in each reaction. Synthesis of BL5030-1, BL5030-2 and BL5030-4 using the CEM Liberty microwave peptide synthesizer

Synthesis of 2mmol of BL5030 peptides 1, 2 and 4 was performed on an automated peptide synthesizer in 2 batches of lmmol, using general Fmoc chemistry protocol: Rink amide methylbenzhydrylamine (MBHA) resin, 100-200, loading: 0.7mmol/g (714mg, lmmol) was added to a centrifuge tube and the tube attached to the synthesiser.

The coupling cycle

· Swelling: Initial swelling of the resin in 12 mL NMP, occurred for 120 minutes, prior to initialization of the reaction program on the synthesizer. Following transfer of the resin to the reaction vessel, under nitrogen pressure, swelling was continued for a further 15 minutes. Each coupling cycle includes the following steps: Fmoc deprotection, resin wash, coupling of amino acid and resin wash:

• Fmoc Deprotection: Deprotection of the Fmoc group was achieved using a solution of 20% piperidine in NMP. The resin was mixed with 20 mL of this solution for 20 minutes and then with an additional 20 mL of this solution for a further 20 minutes.

• Resin Wash: The resin was washed 4 times with a 20 mL portion of NMP for 2 minutes.

• Coupling: The first 4 amino acids are common to all 3 peptides:

Amino acid according to the sequence (3 equiv.; 1.988 g of Fmoc-HomoArg(pbf)OH for the first coupling cycle 1.379 g of Fmoc-D-TyrOH for the second coupling cycle, 970 mg of Fmoc-Aze-OH for the third coupling cycle or 1.181 g of Fmoc-Cha-OH for the fourth coupling cycle), in a 10 mL solution of 1 : 1 NMP:DMF, was transferred to the reaction vessel under nitrogen pressure. 0.5M HBTU/HOBt in DMF (3 eq., 6 ml) and 2M

DIPEA in NMP (6 eq., 6 ml) were added to the resin. Nitrogen was bubbled through the reaction mixture for 60 minutes.

For compound 1, a fifth amino acid was added on using the same conditions: 1.138 g of Fmoc-tranexamic acid in a 10 mL solution of 1 : 1 NMP:DMF.

For compound 2, a fifth amino acid was added on using the same conditions: 1.446 g of Fmoc-L-Arg(pbf)OH) in a 10 mL solution of 1 : 1 NMP:DMF

• Acetylation: An acetate cap was attached to compounds 2 and 4 only:

Following the final deprotection, N-termini acetylation was carried out using 15 mL of

10% acetic anhydride in NMP, and 1.5 mL of 2M DIPEA. The resin was mixed with this solution for 60 minutes.

Cleavage from the resin

On completion of the peptide synthesis, the resin was transferred to a reaction vessel with a sintered glass bottom. The resin was washed with 20mL DCM and dried under vacuum. Afterwards, the peptide was cleaved from the resin by treatment with a solution of 95:2.5:2.5 TFA:TIS:H ₂ 0 (30 mL) for 2 hours. The solution was concentrated to

approximately one third and the peptide was precipitated into cold diethyl ether. The ether solution was removed by decantation following centrifugation. The crude product was solubilised with H ₂ 0:CH ₃ CN 80:20 solution and lyophilized.

Synthesis of BL5030-3 and BL5030-5 using the CS Bio automated peptide synthesizer Synthesis of 2mmol of BL5030 peptides 3 and 5 was performed on an automated peptide synthesizer using general Fmoc chemistry protocol: Rink amide methylbenzhydrylamine (MB HA) resin, 100-200, loading: 0.7mmol/g (714 mg, 1 mmol) was added to the reaction vessel of the synthesiser. The coupling cycle

• Swelling: Initial swelling of the resin in 25 mL of NMP, occurred for 120 minutes, as the first step of the reaction program on the synthesizer. Each coupling cycle includes the following steps: Fmoc deprotection, resin wash, coupling of amino acid and resin wash:

• Fmoc Deprotection: Deprotection of the Fmoc group was achieved using a solution of 20% piperidine in NMP. The resin was shaken with 25 mL of this solution for 5 minutes and then with an additional 20 mL of this solution for a further 20 minutes.

• Resin Wash: The resin was washed 6 times with a 25 mL portion of NMP for 2 minutes.

• Coupling: Both peptides have the same sequence of 5 amino acids:

Amino acid according to the sequence (3 equiv.; 3.977 g of Fmoc-HomoArg(pbf)OH for the first coupling cycle, 2.758 g of Fmoc-D-TyrOH for the second coupling cycle, 1.940 g of Fmoc-Aze-OH for the third coupling cycle, 2.361 g of Fmoc-Cha-OH for the fourth coupling cycle and 2.277 g of Fmoc-tranexamic acid for the fifth coupling cycle) was dissolved in 0.5M HBTU/HOBt in DMF (3 eq., 12 ml) and the solution made up to 25 mL with NMP. This was transferred to the reaction vessel under nitrogen pressure. 1M

DIPEA in NMP (6 eq., 12 ml) was added to the resin. The resin was mixed through repeated and continuous inversion of the reaction vessel for 60 minutes.

• N-Termini guanidine group: A guanidine group was attached to compound 3:

Following the final deprotection, 25 mL of 0.24M N,N'-Di-Boc-lH-pyrazole-l- carboxamidine in NMP (3 eq), and 12 mL of 1M DIPEA were added. The resin was mixed with this solution for 4 hours.

Acetylation: An acetate cap was attached to compound 5:

Following the final deprotection, N-termini acetylation was carried out using 25 mL of

10% acetic anhydride in NMP, and 12 mL of 1M DIPEA. The resin was mixed with this solution for 60 minutes.

Cleavage from the resin

On completion of the peptide synthesis, the resin was transferred to a reaction vessel with a sintered glass bottom. The resin was washed with 20 mL DCM and dried under vacuum. Afterwards, the peptide was cleaved from the resin by treatment with a solution of 95:2.5:2.5 TFA:TIS:H ₂ 0 (30mL) for 2 hours. The solution was concentrated to

approximately one third and the peptide was precipitated into cold diethyl ether. The ether solution was removed by decantation following centrifugation. The crude product was solubilised with H ₂ 0:CH ₃ CN 80:20 solution and lyophilized.

Purification methods

Purification method for BL5030-1 The crude product was obtained in 1.568 g yield (after merging the two reactions). Purity of crude product was detected by HPLC as 84%.

The crude product was purified by Gilson preparative HPLC under the following conditions: Column: Xbridge C18 5μιη 30X150mm column

Mobile phase: 0.1% TFA in water and 0.1 % TFA in ACN

Diluent: Water/ ACN 80/20

Detector: UV - 215 nm for collection and 254 nm for monitoring

Flow: 30 mL/min

Injection volume: 3 mL Method:

From all collected fractions only the fractions of >99% purity were combined to obtain the final product 036-075 (769 mg) as a white powder in >99 % purity.

Purification method for BL5030-2

The crude product was obtained in 1.983 g yield. Purity of crude product was detected by HPLC as 72%.

The crude product was purified by Gilson preparative HPLC under the following conditions: Column: Xbridge C18 5μιη 30X150mm column

Mobile phase: 0.1% TFA in water and 0.1 % TFA in ACN

Diluent: Water/ ACN 80/20

Detector: UV - 215 nm for collection and 254 nm for monitoring

Flow: 35 mL/min

Injection volume: 3 mL Time (min) 0.1% TFA in Water 0.1% TFA in Acetonitrile

0 90 10

6 90 10

18 77 23

21 77 23

22 25 75

24 25 75

25 90 10

30 90 10

From all collected fractions only the fractions of >99% purity were combined to obtain the final product 036-077 (419mg) as a white powder in >99% purity.

Purification method for BL5030-3

The crude product was obtained in 1.983 g yield. Purity of crude product was detected by HPLC as 78%.

The crude product was purified by Gilson preparative HPLC under the following conditions: Column: Xbridge C18 5μιη 30X150mm column

Mobile phase: 0.1% TFA in water and 0.1% TFA in ACN

Diluent: Water/ ACN 80/20

Detector: UV - 215 nm for collection and 254 nm for monitoring

Flow: 30 mL/min

Injection volume: 3 mL

Method:

From all collected fractions only the fractions of >99% purity were combined to obtain the final product 036-026 (753 mg) as a white powder in >99 % purity. Purification method for BL5030-4

The crude product was obtained in 1.769 g yield. Purity of crude product was detected by HPLC as 82%.

The crude product was purified by Gilson preparative HPLC under the following conditions: Column: Xbridge C18 5μιη 30X150mm column

Mobile phase: 0.1% TFA in water and 0.1 % TFA in ACN

Diluent: Water/ ACN 80/20

Detector: UV - 215 nm for collection and 254 nm for monitoring

Flow: 35 mL/min

Injection volume: 3 mL Method:

From all collected fractions only the fractions of >99% purity were combined to obtain the final product 036-079 (776 mg) as a white powder in >99 % purity.

Purification method for BL5030-5

The crude product was obtained in 1.935 g yield. Purity of crude product was detected by HPLC as 83%.

The crude product was purified by Gilson preparative HPLC under the following conditions: Column: Xbridge C18 5μιη 30X150mm column

Mobile phase: 0.1% TFA in water and 0.1 % TFA in ACN

Diluent: Water/ ACN 80/20

Detector: UV - 215 nm for collection and 254 nm for monitoring

Flow: 30 mL/min

Injection volume: 3 mL Method:

From all collected fractions only the fractions of >99% purity were combined to obtain the final product 036-021 (505 mg) as a white powder in >99 % purity.

5 Formation of acetate salts

The formation of the acetate salt of BL5030 was carried out using an ion exchange resin:

The resin was preconditioned with ImM acetic acid. The product was dissolved in 1.5 mL acetic acid and the solution made up to 6 mL with water. This solution was filtered through the preconditioned resin and then washed a further two times with ImM acetic acid. The combined washes were lyophilized. The final product was dissolved in 80/20 water/ ACN then filtered through a sterilized vacuum driven 0.22μιη Millipore filtration system and lyophilized a few times to remove traces of acetic acid. The product was obtained as white powder. Results

Analytical Results

HPLC results

HPLC results are shown in Figs. 1-10. EXAMPLE 2

Thrombin Inhibition in vitro

Materials and Methods

Chemicals

The test compound BL-5030-4 was manufacture at as described in Example 1 hereinabove and was stored at -20 ± 2 °C and protected from light until use.

The reagents Actin FSL, Actin FS, Citrol (QC) and Owren's Buffer where purchased from Dade Behring (Deerfield, IL USA) and were stored at 4-8 °C until use.

Activated Partial Prothrombin Time (APPT) assay

The ATTP assay was performed essentially as described by Sterling et al (Eds.)

Laboratory Hemostasis: A Practical Guide for Pathologists, 2007, Springer. Briefly, fresh human blood was mixed with sodium citrate solution (3.2 % w/v). The mixture was centrifuged at 3,000 rpm for 10 minutes. The plasma was then removed and stored at -20 °C until use (up to 5 days). Plasma samples were supplemented with compound BL-5030-4 to final concentrations of 1, 3 and 10 ug/ml then timed for coagulation (clot formation) using Sysmex CA-1500 and Sysmex CA-1500 blood coagulation analyzers. Untreated plasma was used as a negative control. Heparin-supplemented plasma (0.5 unit/ml) was used as a positive control.

Results

As shown in Table 2 below, the untreated plasma (negative control) coagulated after 29.4 seconds under Sysmex CA-7000 blood coagulation analyzer. The addition of BL-5030- 4 to final concentrations of 1, 3 and 10 ug/ml delayed the plasma coagulation by 63.9 seconds (117%), 78.4 seconds (166.7%) and 96.6 seconds (228.6%), respectively.

Under Sysmex CA-1500 blood coagulation analyzer, the untreated plasma (negative control) coagulated after 30.1 seconds. The addition of BL-5030-4 to final concentrations of 1, 3 and 10 ug/ml delayed the plasma coagulation by after 64.7 seconds (15.0 %), 81.5 seconds (70.8 %) and 103.2 seconds (142.9 %), respectively.

Hence, the results clearly indicate that BL-5030-4 is capable of inhibiting blood coagulation in vitro in a dose response fashion.

Table 2

The effect of BL-5030-4 on blood coagulation time using Sysmex CA-7000 Analyzer

EXAMPLE 3

Thrombin Inhibition in vivo

Materials and Methods

Test compound

The test compound BL-5030-4 was manufacture at as described in Example 1 hereinabove and was stored at -20 ± 2 °C and protected from light until use.

Animals

Male Sprague-Dawley rats, 6-7 wk old weighing 180-200 g at the time of treatment initiation were used.

Rat tail bleeding time assay

The assay was performed essentially as described by Bernata et al. (Chronobiology International 10: 360 - 363, 1993). Briefly, different doses of test compound (1 , 5 and 15 mg/kg), or saline vehicle, were administered to rats by oral gavage. Fifteen or 120 min following administration the rats were anaesthetized then had their tails cut about 5 mm from the distal end of the tail. Each cut tail was immediately positioned vertically within saline (10 ml saline contained in a 15 ml collecting tube kept at 37 °C) and observed for bleeding. The time measured between the tail's cut until the tail bleeding stopped (when no bleeding was observed for at least 30 seconds) was recorded as the "tail bleeding time" (TBT).

Results

As shown in Table 3 below, oral administration of 1 mg/kg BL-5030-4 to rats, followed by tail cutting 15 seconds later, resulted in an increased tail bleeding time of 77.8% (P = 0.047) and an increased the volume of bleeding red blood cells of 120% (P = 0.013), as compared with the saline control.

Hence, the results clearly indicate that orally administered BL-5030-4 is capable of substantially inhibiting blood coagulation in rats. Table 3

The effect of orally administered BL-5030-4 on time and volume of bleeding from cut rat tail.

means ± SD of 5 replications (animals)

1 vs. saline control using unpaired t test.

1 accumulated RBC in the collecting tube.

EXAMPLE 4

Activated Partial Prothrombin Time (aPPT) & Prothrombin Time (PT) assays

Materials and Methods

Chemicals

The test compounds: Ac- L-Arg-D-Cha-L-Aze-D-Tyr-L-hArg-NH ₂ (BL-5030-2), Ac- D-Cha-L-Aze-D-Tyr-L-hArg-NH ₂ (BL-5030-4) and Ac-TX-D-Cha-L-Aze-D-Tyr-L-hArg- NH ₂ (BL-5030-5) were manufactured as described in Example 1 hereinabove and were stored at -20 ± 2 °C and protected from light until use.

Reference compounds TX-D-Cha-L-Aze-D-Tyr-L-hArg-NH ₂ (BL-5030-1) and Gu- TX-D-Cha-L-Aze-D-Tyr-L-hArg-NH2 (BL-5030-3) were synthesized essentially as described in U.S. Patent No. 7,423,021 and were stored at -20 ± 2 °C and protected from light until use.

The reagents Actin FSL, Actin FS, Citrol (QC) and Owren's Buffer where purchased from Dade Behring (Deerfield, IL USA) and were stored at 4-8 °C until use.

Human donors

A total of three male human donors were used in this study. Animals

A total of three male experimentally naive Cynomolgus monkeys, three female experimentally naive Gottingen minipigs®, three male experimentally naive beagle dogs, three male experimentally naive New Zealand White Hra:(NZW)SPF rabbits and three male experimentally naive CD® [Crl:CD®(SD)] rats were used in this study.

Clinical Pathology

The primate blood samples were collected from three male animals from the femoral vein and were divided into six tubes (approximately 1.8 mL/tube). Two additional blood samples were collected from two primates.

The swine blood samples were collected from three female animals from the anterior vena cava and divided into six tubes (approximately 1.8 mL/tube). On occasion, blood samples were redrawn.

The canine and rabbit blood samples were collected from three male animals via the jugular vein and divided into six tubes each (approximately 1.8 mL/tube). On occasion, rabbit blood samples were redrawn.

The rat blood samples were collected from three male animals via vena cava after carbon dioxide inhalation and divided into seven tubes (approximately 2 mL/tube).

The human donor samples were collected from three humans and were divided into seven tubes (approximately 2.7 mL/tube). The donors were fasted for at least 8 hours, alcohol-free during the fasting period, and drug naive for 7 days.

All samples were collected into tubes containing sodium citrate for evaluation of coagulation parameters. The samples were centrifuged after collection. The plasma was harvested, aliquoted, and evaluated at room temperature within 4 hours of collection.

Sample Treatment

The plasma was pooled together into one tube for each individual species subject, with the exception of the two additional primate blood samples (animal numbers 323 and 324) which were not pooled with the previously collected samples, and 3 of each analogue concentration was added. Three separate analyses (heparin spiked, analogue spiked, and non-spiked) were conducted per subject for the human plasma. For each individual subject, the human plasma was spiked with heparin for a final concentration of 0.25, 0.5, and 1.0 U/mL, each of the five BL-5030 analogues at concentrations of 100, 300, and 1000 μg/mL, and one control plasma sample (non-spiked) were prepared. A total of 57 plasma samples were analyzed for aPTT and PT.

For each individual animal subject, the plasma was spiked with each of the five BL-5030 analogues at concentrations of 100, 300, and 1000 μg/mL, and one control sample (non-spiked) was prepared. A total of 48 samples for each species were analyzed for aPTT and PT.

The volume of each test article spike for each species was 1% of the volume of the samples or 3 spike per 300 μΐ, for final concentrations of 1, 3, or 10 μg/mL, as presented in Table 4 hereinbelow.

Results

As shown in Figure 11 and Table 4 below, all compounds (BL-5030-1, BL-5030-2, BL-5030-3, BL-503-4 and BL-5030-5) substantially increased the activated partial thromboplastin time (aPTT) and prothrombin time (PT) with all plasma types, when administered at a high dose (10 μg/mL). Compounds BL-5030-3 and BL-5030-5 increased the aPTT and PT values more effectively than the other compounds, when administered at a low dose (1 μg/mL).

Table 4

The effects of different compounds (BL-5030-1, BL-5030-2, BL-5030-3, BL-5030-4 and BL-5030-5) spiked to different plasma types on the activated partial thromboplastin time

(aPTT) and prothrombin time (PT)

EXAMPLE 5

Hemolytic Assays Materials and Methods

Chemicals

The test compounds: TX-D-Cha-L-Aze-D-Tyr-L-hArg-NH ₂ (BL-5030-1), Ac- L- Arg-D-Cha-L-Aze-D-Tyr-L-hArg-NH ₂ (BL-5030-2), Gu-TX-D-Cha-L-Aze-D-Tyr-L-hArg- NH ₂ (BL-5030-3), Ac-D-Cha-L-Aze-D-Tyr-L-hArg-NH ₂ (BL-5030-4), Ac-TX-D-Cha-L- Aze-D-Tyr-L-hArg-NH ₂ (BL-5030-5) were manufactured as described in Example 1 hereinabove and were stored at -20 ± 2 °C and protected from light until use.

The reagents Actin FSL, Actin FS, Citrol (QC) and Owren's Buffer where purchased from Dade Behring (Deerfield, IL USA) and were stored at 4-8 °C until use.

Whole Blood and Plasma Preparation

A total of 36 whole blood samples/sex (12 samples containing 4 mL each) collected into tubes with sodium heparin anticoagulant were received from three male (donor numbers 1 to 3) and three female (donor numbers 4 to 6) donors. The donors had free access to drinking water, but were fasted overnight for at least 8 hours. The donors were non- smokers, drug naive for at least 7 days, had no previous use of recreational drugs, and had abstained from alcohol for at least 48 hours. Samples were received within 1 to 2 hours of sample collection at room temperature.

12 samples (4 mL each) received from each donor were pooled together into six sets of 8 mL samples. Using an 8 mL sample, 0.25 mL aliquots of well-mixed blood was placed into tubes labeled "Blood A" (one aliquot), "Blood B" (five aliquots), and "Blood C" (one aliquot). The remaining blood (approximately 6 mL) was centrifuged at approximately

3000 rpm for 10 minutes. After centrifugation, 0.25 mL aliquots of plasma was placed into tubes labeled "Plasma A" (one aliquot) and "Plasma B" (five aliquots). A negative control was prepared from any additional tubes of plasma. This procedure was repeated for all five BL-5030 analogues at each of the five concentrations for all donor samples.

The processed whole blood samples were stored frozen at -10 to -30°C. The processed plasma samples were discarded following experimentation. Experimental Procedures

Assessment of Test System Quality (Blood A)

A volume of 0.25 mL of negative control, 0.9% Sodium Chloride for Injection, USP, was added to each tube labeled "Blood A". The tube was gently mixed, incubated for 45 minutes at approximately 34 to 39°C, and centrifuged at approximately 3000 rpm for 10 minutes.

Compatibility with Whole Blood (Blood B)

A volume of 0.25 mL of each test article concentration (0.6, 2, 6, 20, and 200 μg/mL) for each of the five BL-5030 analogues was added to the tubes labeled "Blood B". The tubes were gently mixed, incubated for 45 minutes at approximately 34 to 39°C, and centrifuged at approximately 3000 rpm for 10 minutes. The final nominal concentrations of analogue in whole blood were 0.3, 1, 3, 10, and 100 μg/mL.

Compatibility with Whole Blood (Blood C)

A volume of 0.25 mL of positive control, distilled, deionized water, was added to each tube labeled "Blood C". The tube was gently mixed, allowed to stand for approximately 45 minutes, and centrifuged at approximately 3000 rpm for 10 minutes.

Compatibility with Plasma (Plasma A and B)

A volume of 0.25 mL of negative control, 0.9% Sodium Chloride for Injection, USP, was added to each tube labeled "Plasma A". A volume of 0.25 mL of each test article concentration (0.6, 2, 6, 20, and 200 μg/mL) for each of the five BL-5030 analogues was added to the tubes labeled "Plasma B". The tubes were gently mixed, allowed to stand for 45 minutes at 34 to 39°C, and visually inspected for precipitation and/or turbidity. The final nominal concentrations of analogue in whole blood were 0.3, 1, 3, 10, and 100 μg/mL.

Assessment Methods

Visual Evaluation of Plasma

The plasma samples were visually inspected for the presence or absence of precipitation and/or turbidity as compared to the negative control. Hemoglobin Testing

Plasma free hemoglobin levels were measured with an Olympus chemistry analyzer and plasma free hemoglobin reagent kit provided by Catachem Inc., Bridgeport, Connecticut.

For assessing hemolysis in the test article analogue concentrations, free hemoglobin concentration was measured using the chemistry analyzer as indicated above. If the hemoglobin concentration was equivalent to that in the negative control (saline), the treatment was not considered to have hemolytic potential in human blood. If the hemoglobin concentration was equivalent to that in the positive control (water), the treatment was considered to have hemolytic potential in human blood. Hemoglobin concentrations in between these extremes were assessed on an individual basis in consideration of values obtained in the negative control. The positive control was analyzed to confirm the susceptibility for cell lysis.

Results

Measurement of Plasma Free Hemoglobin Levels

Plasma free hemoglobin values are summarized in Table 5.

This in vitro evaluation of hemolytic potential (Plasma Free Hemoglobin) compared five dose levels of five compounds in human blood samples. At the dose levels evaluated, there was no evidence of any meaningful hemolysis with any of the compounds. Although no hemolytic effects were observed in this in vitro study, the in vivo effects could vary.

Plasma

Incubation of plasma with each of the five BL-5030 analogues (BL-5030-1 through BL-5030-5) at concentrations of 0.3, 1, 3, 10, and 100 μg/mL did not produce precipitation or turbidity. The negative control (saline) also did not produce precipitation or turbidity.

Conclusion

None of the tested compounds, BL-5030-1, BL-5030-2, BL-5030-3, BL-5030-4 and BL-5030-5, provided to human blood at different concentrations (0.3, 1, 3, 10, and

100 μg/mL), caused hemolysis under the experimental conditions. Table 5

The hemolytic potential of different compounds (BL-5030-1, BL-5030-2, BL-5030-3, BL-5030-4 and BL- 5030-5) in human whole blood

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents, patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent, patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.