DESCRIPTION BACKGROUND

Field of the Invention

This invention is related to a class of synthetic sulfated beta-04 low molecular weight lignins which are inexpensive to prepare and which have been found to have potent

anticoagulation properties in human plasma and whole blood.

Background of the Invention

Heparin (also known as unfractionated heparin (UFH)) is extensively used in the clinic as anticoagulant.' UFH is obtained from pig intestinal or lung mucosa and is relatively inexpensive. It is then processed to produce low molecular weight heparins (L WHs), which are also powerful anticoagulants. Enoxaparin is one such LMWH and is obtained from UFH by chemical treatment. Other LMWHs available in the clinic are obtained from UFH either through chemical or enzymatic means.

Both UFH and LMWHs are used to treat numerous thrombotic disorders including deep- vein thrombosis (DVT), disseminated intravascular coagulations (DIC), pulmonary embolism (PE), acute myocardial infarction, unstable angina and cerebrovascular thrombosis. ^1"3 These are also used during surgery, organ transplantation and for extracorporeal bypass procedures.

Combined the annual market of heparins is more than $8 billion within the USA alone.

Yet, both heparins have their own disadvantages. Both agents are heterogeneous mixtures of highly sulfated polysaccharide chains, which introduce a number of issues. The biggest drawback is of significant risk of internal bleeding, which might be of either minor bleed or major bleed type. UFH, and sometimes LMWH, is associated with the occurrence of

thrombocytopenia in about 3% of patients. ⁴ Osteoporosis could also arise in some patients upon prolonged heparin usage. In addition to these adverse effects, heparin usage is problematic from quality control and administration perspective. Both UFH and LMWHs possess significant structural variability, which affects their bioavailability and pharmacokinetic parameters. Thus, patient response variability is high. It is also difficult to ascertain the absence of non-heparin-Iike molecules in a preparation of heparin, e.g., chondroitin sulfate in heparin, because of their structural similarity and mode of preparation. Events of 2008, wherein contamination of UFH by oversulfated chondroitin sulfate led to the death of 81 people in the US, demonstrate the difficulty of quality control with these highly heterogeneous preparations. ⁵

It would be advantageous to identify new anticoagulants which possess heparin-like activity without the heterogeneity present in UFH and LMWHs. Further, new anticoagulants, particularly which can be obtained through inexpensive route so as to compete with the cost of UFH therapy, will provide useful alternatives for a number of clinical and other applications.

SUMMARY

Embodiments of the invention relate to sulfated beta-04 low molecular weight lignin (Sb04L), an oligomer which may be prepared using a fully chemical system and possessing highly specific and potent thrombin inhibitory activity. Sb04L has excellent human plasma and blood anticoagulant potential. Sb04L is an oligomeric lignin molecule that contains only one type of inter-monomeric residue linkage i.e. the β-Ο-4 linkage. This reduces heterogeneity dramatically so much so that the agent can be assessed for purity using, for example, standard chromatographic tools. The anticoagulant is easily prepared in few simple steps, and thus the new anticoagulant may be inexpensive. Further, industrial scale preparation of Sb04Ls should be possible, thereby increasing its utility. The new anticoagulants described herein should have significant clinical applications.

According to embodiments of the invention, there is described a generic sulfated beta-04 lignin (Sb04L) scaffold for a family of lignin compounds. These lignin compounds are of low molecular weight (e.g., Molecular Weight ranging from 5000 to 15000 (or 8000 to 12000)). Sb04L has the general chemical structure:

where Y = S0 ₃ M ( = Na ⁺ , Ca ²⁺ , NH ₄ ⁺ and other equivalent positively charged ions) or H; n can be 0 - 50 (e.g., 4-6, 5-10, 5-25, 10-25, 15-25, 10-20, 15-50 as well as other ranges therebetween);

R, - CH ₂ OY;

R ₂ , R ₃ , R ₅ , and R ₆ can be hydrogen (-H), CI -10 linear alkyl (e.g., -CH ₃ , -CH ₂ CH ₃ , etc.), C 1-10 branched alkyl (e.g., -CH(CH ₃ ) ₂ , -CH ₂ CH(CH ₃ ) ₂ , etc.), C 1-10 and 01 -10 oxyalkyl (e.g., -OCH ₃ , -OCH ₂ OCH ₃ , etc.), electron-donating (e.g., -OH, -OR (R = CI -10 alkyl, aryl, allyl, and benzyl), -NH ₂ , -NHR (R = C 1-10 alkyl, aryl, allyl, and benzyl), electron- withdrawing (e.g., -N0 ₂ , -S0 ₃ H, -S0 ₃ M (M - Na ⁺ , Ca ²⁺ , NH ₄ ⁺ and other equivalent positively charged ions) or halogens (-F, -Br, -CI and -I), and can be the same or different; and chiral centers denoted by (*) are either R or S, and can be the same or different.

Examples of Sb04L ligands include tetrameric-hexameric molecules and decameric- pentadecameric molecules represented by the following generalized structures:

In each of the two exemplary Sb04L families of compounds above, Y is preferably H or S0 ₃ Na. in particular embodiments, mixtures are provided wherein the mixtures contain two or more different sulfated lignin scaffolds, each of which has the generic Sb04L scaffold. For example, the two or more different sulfated lignin scaffolds could have different sizes or have different constituents (i.e., R ₂ , R ₃ , R ₅ or R ₆ differ between successive aromatic units), etc. (e.g., different lignins in a mixture could have varying numbers of repeats (n) or one or more different substitutions (e.g., different charged ions, different moieties, such as ary!, alkyl, allyl, oxyalkyl, etc.).

In other embodiments, compositions are provided which including one or more carriers or matrices mixed with one or more sulfated lignins, wherein at least one of the sulfated lignins has the generic Sb04L scaffold. In some compositions, mixtures of two or more different sulfated lignins, where each sulfated lignin has the generic Sb04L scaffold. In some compositions, at least one carrier which is a pharmaceutically acceptable liquid (e.g., saline, oil, water (deionized, distilled, etc.), etc.) is included in the composition. In some compositions, at least one matrix which is a pharmaceutically acceptable sold (e.g. cornstarch, etc.) is included in the composition.

In still other embodiments, methods of producing sulfated lignins with the generic Sb04L scaffold are disclosed. In particular embodiments, a simple three step process of polymerization, reduction, and sulfation is used.

In further embodiments, methods of using sulfated lignins with the generic Sb04L scaffold are disclosed. Exemplary embodiments include using the sulfated lignins, or mixtures, or compositions thereof as an anticoagulant (e.g., through direct, a!losteric inhibition of clotting enzymes) and in any application where UFH and LMW may be employed.

DESCRIPTION OF THE DRAWINGS

Figure 1. Structures of unfractionated heparin (UFH) (A) and sulfated p-04 ligtitn (Sb04L) (B), Clinically used UFH (and LMW heparins) are a polydisperse mixture of large number of sulfated polysaccharide chains with variations in Y and R groups (shown in A) UFH is prepared from pig mucosa, while LMW heparins are prepared from UFH. Sb04L (B) was synthesized in high yields in three simple steps - alkaline polymerization using ₂ C0 (step i), sodium borohydride reduction of carbonyl groups (step ii) and sulfation of available hydroxyl groups (step iii) - from ethyl 2-bromo-3-(4-hydroxy-3-methoxyphenyl)-3-oxopropanoate monomer.

Figure 2 Direct inhibition of serine proteases by Sb04L. A) The inhibition of factors Ha (thrombin), Xa, XIa, IXa and Vila, Vila-tissue factor (TF) complex, and plasmin by Sb04L was studied using chromogenic substrate hydrolysis assay. Solid lines represent sigmoidal dose- response fits (Eq. 1 ) to the data to obtain /C ₅₍ ). B) Sb04L inhibition of other heparin-binding serine proteases including cathepsin G, human leukocyte elastase (HLE), porcine pancreatic elastase (PPE), activated protein C (APC), plasmin, chymotrypsin and trypsin at a fixed 373 pg l (~1800-fold excess over the / 50 of thrombin inhibition). Error bars were derived from nonlinear regression and represent ± 1 S.E. Figure 3 Sb04L Inhibition of thrombin-thrombomodulin activation of protein C.

Formation of APC (o) in the presence of Sb04L was followed using S-2366 hydrolysis by thrombin - TM complex under standard assay conditions after neutralization of thrombin activity using argatroban (see Experimental for details). For comparison, Sb04L inhibition of thrombin's proteolytic activity is also shown (€). Solid lines represent sigmoidal dose-response fits (Eq. 1 ) to the data to obtain /C ₅₀ .

Figure 4 Michaelis-Menten kinetics of Spectrozyme TH hydrolysis by thrombin in the presence of Sb04L. The initial rate of hydrolysis at various substrate concentrations was measured in a pH 7.4 buffer (50 mM Tris-HCl buffer, pH 7.4, containing 100 mM NaCl, and 1 mM CaCl ₂ at 37°C). The concentrations of Sb04L were 0 (♦), 0.012 (€), 0.1 15 (·), 0.230 (Δ) and 2.3 n /ml (*). Solid lines represent non- linear fits to the data using the standard Michaelis- Menten equation.

Figure 5 Competitive effect of a hirudin peptide HirP (A) and UFH (B) on the direct inhibition of thrombin by Sb04L. Thrombin inhibition by Sb04L in the presence of a constant concentration of HirP (A) or UFH (B) was measured through the Spectrozyme TH hydrolysis assay at pH 7.4. Solid lines represent fits by the dose-response equation to obtain the apparent 7C ₅ o (50 mM Tris-HCl buffer, pH 7.4, containing 100 mM NaCl, and 1 mM CaCl ₂ at 37°C). The concentrations of HirP chosen for study include 0 (♦), 8.6 (a), and 86 iiM (O), while that of UFH were 0 (O), 0.2 (■), 2 (Δ), 20 (·) and 100 μΜ (O). Figure 6. Effect of Sb04L in human plasma. A) Prolongation of clotting time as a function of Sb04L concentration in either the prothrombin time (PT) or the activated partial thromboplastin time (APTT) assay. The solid lines are trend lines, and not exponential fits. Errors in clotting time measurement were in the range of symbol size and have been omitted. B) Effect of serum albumin on the anticoagulant potential of Sb04L. Solid line to the data represents a rectangular hyperbolic fit to the data to derive the maximal thrombin activity at limiting concentrations of BSA. The dotted line represents the maximal thrombin activity.

Figure 7. Comparison of the effect of Sb04L on platelet function in whole blood using hemostasis analysis system (HAS™)- A) and B) show the change in platelet contractile force (PCF) and clot elastic modulus (CEM), respectively, with time at various fixed concentrations of Sb04L (0-74 g/ml).

Figure 8. Protamine-mcdiated reversal of Sb04L inhibition of thrombin. Recovery of thrombin activity at varying levels of protamine following 50% inhibition by Sb04L was measured through the Spectrozyme TH hydrolysis assay at pH 7.4. Solid line represents nonlinear fit of the data by a logistic function similar to equation 1 to obtain the RC _50i the concentration of protamine necessary to recover 50% thrombin activity.

Figure 9. Tabular data showing human whole blood clotting parameters of Sb04L by hemostasis analysis and thromboelastography. " Analysis was performed using

Hemostasis Analysis System (HAS) on human whole blood. Parameters deduced from this analysis included TOT (thrombin onset time), PCF (platelet contractile force) and CEM (clot elastic modulus). ^b Analysis was performed using thromboelastography (TEG) on human whole blood as described in 'Experimental Procedures'. Parameters deduced from this analysis included R (time to clot initiation), a(angle), MA (maximum amplitude) and G (shear elastic modulus).

Figure 10. A) shows the fonriation of occlusive platelet-rich thrombus in the carotid artery of mice using a 3.5% FeCl ₃ solution with two doses of Sb04L, i.e. 100 μg and 1 ,000 μg. B) demonstrates a dose-dependent decrease in coagulation with a complete inhibition of clot formation at a dose of 1 ,000 μg of Sb04L. The number shown in brackets shows the fraction of mice which showed complete thrombotic plug formation.

DETAILED DESCRIPTION

The invention is related to a family of low molecular weight lignin compounds (e.g., MW ranging from 5000 to 15000) defined by a generic sulfated beta-04 lignin (Sb04L) scaffold having the general chemical structure:

where Y = S0 ₃ M (M = Na ⁺ , Ca ² \ NH ₄ ⁺ and other equivalent positively charged ions) or H; n can be 0 - 50 (e.g., 4-6, 5- 10, 5-25, 10-25, 15-25, 10-20, 1 -50 as well as other ranges therebetween);

R, - CH ₂ OY;

R ₂ , R ₃ , R ₅ , and R ₆ can be hydrogen (-H), CI -10 linear alkyl (e.g., -CH ₃ , -CH ₂ CH ₃ , etc.), C I -10 branched alkyl (e.g., -CH(CH ₃ ) ₂ , -CH ₂ CH(CH ₃ ) ₂ , etc.), Cl -10 and O1-10 oxyalkyl (e.g., -OCH3, -OCH2OCH3, etc.), electron-donating (e.g., -OH, -OR (R = CI -10 alky], aryl, allyl, and benzyl), -NH ₂ , -NHR (R = C I- 10 alkyl, aryl, allyl, and benzyl), electron- withdrawing (e.g., -NO2, -SO3H, -S0 ₃ M (M = Na ⁺ , Ca ²⁺ , NH ₄ ⁺ and other equivalent positively charged ions) or halogens (-F, -Br, -CI and -I), and can be the same or different; and chiral centers denoted by (*) are either R or S, and can be the same or different.

The low molecular weight sulfated beta-04 lignin(s) (Sb04L) are oligomer(s) which can be prepared using a fully chemical system and they possess highly specific and potent thrombin inhibitory activity. Hence, Sb04L lignins have excellent human plasma and blood anticoagulant potential. A defining feature is that Sb04L is an oligomeric lignin molecule that contains only one type of inter-monomeric residue linkage i.e. the β-0-4 linkage. This reduces heterogeneity dramatically, so much so that the agent can be assessed for purity using standard

chromatographic tools. The anticoagulant is easily prepared in few simple steps, suggesting that the new anticoagulant is likely to be inexpensive. Further, industrial scale preparation of Sb04Ls is possible, thereby increasing its utility.

The synthesis of the Sb04Ls can be achieved in three easy steps using, for example, alpha-bromo-l ,3-ketoester and aryl phenol. The first step involves the polymerization step utilizing a base such as potassium carbonate.

Example of Polymerization: In a flask containing the monomer ethyl 2-bromo-3-(4-hydroxy-3- methoxyphenyl)-3-oxo-propanoate (0.97 g) (1) (or other appropriate monomer) in anhydrous DMF (5mL), anhydrous K ₂ C0 ₃ (0.58 g) was added and stirred under nitrogen. After 24 h the reaction mixture was poured onto ice-water mixture ( 120 ml) and the pH adjusted to -2.5 with 2M HC1. The precipitated polymer (2) was filtered, washed with water and lyophilized to remove moisture. In the Example of Polymerization Rj = -COOEt.

NMR characterization showed the following corresponding broad peaks for Ή-NMR (DMSO-d6): 7.46-7.78 (C2-H and C5-H), 6.91 -7.14 (Cp-H), 6.52-6.66 (C6-H), 4.19-4.20 (- OCH2CH3), 3.79-3.81 (-OCH3), 1.12- 1 .22 (-OCH2CH3).

¹³ C-NMR (DMSO-d6) 188.53, 1 89.08 (Ca), 165.81 , 166.02 (Cy), 153.16, 150.93, 149.48, 149.01 , 147.64, 128.33, 125.51 , 124.75, 123.73, 1 15. 1 7, 1 14.08, 1 13.91 , 1 13.09, 1 12.51 , 1 12.30 (aromatic carbons), 78.54, 78.37 (Cp), 61.80, 61.67 (-OCH2CH3), 55.78, 55.58 (-OCH3), 13.77 (-OCH2CH3).

After polymerization, the second step is a reduction of the polymer which can be achieved with methanol.

Example of Reduction: The solid (600 mg) (2) was suspended in methanol ( 10 ml) at 50 °C followed by careful addition of NaBH ₄ (683 mg) while maintaining 50 °C. The mixture gradually turned into a clear solution, which was stirred for 24 h. The solution was then neutralized with acetic acid and poured into 0.5M HCl (200 mL) to precipitate the reduced polymer, which was isolated by centrifugation, washed with water and lyophilized. Dissolution and preferential precipitation using 1 ,4-dioxane and diethyl ether helped remove low molecular weight chains of the polymer and precipitate the high molecular weight chains. The precipitate (3) was filtered and dried using vacuum. NMR characterization showed the following corresponding broad peaks for Ή-NMR

(DMSO-de): 4.75 (Cct-H), 4.28 (C-β-Η), 3.20-3.71 (Cy-H and - CH ₃ ), 7.02 (C2-H), 6.94 (C5-H), 6.85 (C6-H). l ³ C-N R (DMSO-de): 70.8, 71 .4 (Co), 83.6, 84.4 (Cp), 59.8, 59.9 (Cy), 55.4 (-OCH ₃ ), 134.7- 135.0 (C I ), 1 1 1 .3-1 1 1 .7 (C2), 148.9 (C3), 146.7- 147.0 (C4), 1 14.5- 1 15.0 (C5), 1 19.4- 1 19.7 (C6).

The final step is sulfation of the free hydroxy groups.

3 :: = -CH ₂ OH

Et ₃ N:S0 ₃ , Me ₃ N, DMF, 65°C

4 :: = -CH ₂ OY, Y = -H or -S0 ₃ Na

Example of Sulfation: The reduced polymer (50 mg) (3) so obtained was made to react with triethylamine-sul ur trioxide complex (200 mg) in anhydrous DMF (5 ml) at 65 °C for 24 h under reflux. Following the reaction, 30% (w/v) aqueous sodium acetate (35 ml) was added and the mixture stirred overnight. The solution was then poured into ice-cold ethanol (100 ml) to precipitate crude Sb04L LMWL, which was filtered and washed twice with ice-cold ethanol (10 ml each). The recovered solid was desalted using FloatALyzer G2 (Spectrum Labs) dialysis tubes (MWCO 0.1-0.5 Da). Lyophilization of the dialyzed solution gave Sb04L (4). NMR characterization showed the following corresponding broad peaks for Ή-NMR (DMSO-d6): 6.77 (aromatic protons), 3.47-4.27 (C -H, Cp-H, Cy-H, -OCH ₃ ).

^,3 C-NMR could not give sufficient signal even at high concentrations of the sample, possibly due to the presence of a wide variety of sulfation patterns and molecules of different chain lengths.

The final step in the exemplary synthesis procedure is sulfation of the free hydroxy groups. Purification of the final product can be performed by either aqueous dialysis or by ultrafiltration. The Sb04L so synthesized was studied for thrombin, factor Xa, factor IXa, factor XIa, factor Vila and factor Xlla inhibition and found to exhibit potent inhibition of thrombin alone. It prevented human plasma and whole blood clotting at pharmaceutically relevant concentrations and was active in in vivo models. The molecular weight of the Sb04Ls is low and can range from 5000-15000 Dalton and may be more precisely controlled to e.g., 8000 to 12,000 Da, or 9000±500 Da. For exemplary purposes with regarding molecular weight measurements, gel permeation chromatography (GPC) of Sb04L using Phenogel 5micron (Phenomenex, Torrance, CA, 7.6 mm i.d. ^χ 300 mm) column and 0.1 N NaOH mobile phase was performed at a constant flow rate of 0.7 mL/rnin and gave a characteristic broad elution profile by monitoring absorbance at 254 nm. Polystyrene standards of different molecular weights and ferulic acid were used for calibration purposes. The relationship between logarithm of the molecular weight and the elution volume (V) of the standards was found to be linear with a correlation co-efficient of 0.99. The Sb04L chromatogram was sliced into 1000 time periods providing 1000 average molecular weights with their corresponding absorbances. These values were used to calculate M _W , MN, and Mp values. The molecular weight of the final sulfated lignins were measured using aqueous phase GPC- HPLC and found to have a number average molecular weight M of 9,200 Da and weight average molecular weight Mw of 12,300 Da. This procedure is similar to that used earlier for sulfated low molecular weight lignins.

As demonstrated below, the Sb04L compounds, or mixtures thereof, may be useful as an anticoagulant either in vitro, ex vivo or in vivo. They appear to act direct, allosteric inhibition of clotting enzymes. These Sb04L lignin compounds may be combined with a pharmaceutical carrier. In some formulations, the Sb04L lignins can be the same or different. In some formulations, the Sb04L lignins can be combined with other lignins or other selected materials. Suitable pharmaceutically acceptable carriers are known to those of skill in the art. Typically, such compositions are prepared either as liquid solutions or suspensions, however solid forms such as tablets, pills, powders and the like are also contemplated. Solid forms suitable for solution in, or suspension in, liquids prior to administration for in vivo applications, or for addition to blood or plasma in in vivo, in vitro, and ex vivo applications may also be prepared. The preparation (e.g., a composition containing one or more Sb04L compounds) may also be emulsified. The active ingredients may be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredients. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol and the like, or combinations thereof. In addition, the composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and the like. If it is desired to administer an oral form of the composition, various thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders and the like may be added. The composition of the present invention may contain any such additional ingredients so as to provide the composition in a form suitable for administration. The final amount of Sb04L in the formulations may vary. However, in general, the amount in the formulations will be from about 1 to about 99% by weight, (e.g., 5% or 10% to 20%, 30%, 40%, 50%, or 60% of one or more Sb04L compounds with the remainder being pharmaceutical carriers, excipients and/or other ingredients).

The Sb04L compositions can be added directly to blood or plasma in an in vitro or ex vivo application. For in vivo applications, the Sb04L compositions (preparations) may be administered by any of the many suitable means which are well known to those of skill in the art, including but not limited to by injection, inhalation, orally, intranasally, by ingestion of a food product containing the antagonist, topically, as eye drops, via sprays, etc. In preferred antigoagulant embodiments, the mode of administration may be intravenous provisioning or topical application. In addition, the compositions may be administered in conjunction with other treatment modalities such as other medicaments, other types of therapy, and the like.

The amount of Sb04L that is administered to an individual in an in vivo application (who is usually a mammal, typically a human) will vary based on several factors, as will be understood by those of skill in the art. For example, the dose and frequency of administration may vary according to the gender, age, weight, general physical condition, ethnic background, etc. of the individual, as well as whether or not the individual has other diseases or conditions that might impinge on the treatment. Generally, the dose will be in the range of from about 0.01 to about 10000 mg/kg of body weight (e.g., 1 to 50, 100, 250 or 500 mg/kg, etc.).

Exemplary applications of the Sb04L lignins include, but are not limited to 1) as anticoagulant and/or antithrombotic agents in cardiovascular disease therapy; 2) as anti-HSV-1 , anti-HSV-2, and anti-HIV-1 agents; 3) as agents against other viruses include dengue, varicella, etc.; 4) as regulators of angiogenesis with implications in cancer; 5) regulator of stem cell growth (either inhibition or proliferation). Their anticoagulant activity can be mediated by already available antidote protamine which is FDA approved for heparin toxicity. Sb04L compounds are less heterogeneous than heparin and low molecular weight heparins. Sb04L compounds are potent inhibitors of coagulation with high selectivity. Mechanistically, they possess novel mode of action and thus are radically different from all known anticoagulants. The Sb04L are readily synthesized, and are not isolated from animal sources and hence less prone to prion borne diseases. EXAMPLES

A non-saccharide, synthetic heparin mimetic that exhibits potent plasma and blood anticoagulation through selective, direct and allosteric thrombin inhibition

Abstract

Sulfated low molecular weight (LMW) lignins which are non-saccharide mimetics of LMW heparins exhibit potent plasma and blood anticoagulation through direct, allosteric inhibition of clotting enzymes. Sulfated β-04 lignin (Sb04L) is an advanced molecule that is easily synthesized in only three steps and is homogeneous in terms of inter-monomer linkages. Sb04L was found to potently inhibit human thrombin and plasmin (/C50 = 15-34 nM), while related coagulation factors or heparin-binding serine proteases were inhibited with orders of magnitude weaker potency. Plasma antithrombin did not affect Sb04L inhibition of thrombin, while activation of protein C by thrombin-thrombomodulin complex was inhibited with 20-times poorer potency by Sb04L. Kinetic studies showed reduction in maximal rate of substrate hydrolysis, but not in Michaelis constant, indicating a direct allosteric mechanism of action. Competitive inhibition studies suggested that Sb04L interacted with heparin-binding exosite 2 of thrombin. Human plasma and whole blood coagulation assays showed that Sb04L's

anticoagulation effect was comparable to that observed with a clinically used LMW heparin. Protamine rapidly and quantitatively reversed Sb04L inhibition of thrombin. Overall, Sb04L is a non-polysaccharide, non-animal derived, very readily synthesizable, thrombin-selective, direct allosteric anticoagulant that can truly challenge the wide-spread clinical use of heparins. Introduction

Thrombotic disorders such as pulmonary embolism, deep vein thrombosis, and disseminated intravascular coagulation are a major cause of mortality in humans. Heparin and warfarin, two key anticoagulants that treat pro-thrombotic conditions, were discovered in the first half of the 20 ^th century and are still used in essentially the same form. ¹ Yet, both suffer from serious bleeding risks and adverse reactions. ^" Low molecular weight (LMW) heparins have a better safety profile, but are not devoid of bleeding events. ⁴ ' ⁶ Clinical data collected over the past decade shows that newer agents introduced in the clinic including bivalirudin, argatroban, and fondaparinux do not significantly improve anticoagulation therapy over heparins, ^7"9 while initial indications with the recently introduced drug, dabigatran, are that it too suffers from bleeding issues. ^{10,1 1} Ximelagatran, introduced in 2003, had to be quickly withdrawn because of significant hepatotoxicity. Initial reports with rivaroxaban, introduced in Europe in 2008 and in the US in July 201 1 , indicate significant benefit with some bleeding risks, but a conclusive statement on its safety record will take time. ^12,13 Mechanistically, anticoagulants can be either active site- or allosteric site-targeting agents. Examples of the enzyme active site inhibitor class include argatroban, melagatran, rivaroxaban, DX9065a, apixaban, and numerous others in various stages of clinical trials. In contrast, no molecule has been introduced in the clinic that inhibits a coagulation enzyme on an exclusively allosteric basis. Allosteric regulation of coagulation enzyme's function offers a couple of major advantages. Allosterism offers the possibility of fine control over an enzyme's activity. Whereas the efficacy of competitive inhibitors is typically 100%, that for allosteric inhibitors may be tunable so as to achieve 'regulation', i.e., less than quantitative inhibition. ¹⁴ ' ⁵ In principle, allosteric thrombin regulation can strike a delicate balance between pro- and anti-coagulant activities. Allosteric regulation can also better realize specificity of function. Coagulation enzymes are all trypsin-like, homologous enzymes with considerable similarity in their active site geometry.' ⁶ In contrast, significantly greater differences are found in their exosite geometries. For example, exosite 2 of thrombin, which binds heparin, bears some homology to a corresponding site in factor Xa, but display little correspondence to similar sites in factors IXa and XIa. ¹⁷ In effect, allosteric regulation promises exquisite control over both specificity of recognition and efficacy of inhibition.

Recently, our laboratory designed the first few examples of exclusively allosteric coagulation enzyme inhibitors. These include the sulfated LMW lignins and sulfated

benzofurans. ^17-22 Sulfated LMW lignins were originally designed to mimic the allosteric interaction of heparin with antithrombin, a key regulator of coagulation. ^23,24 Yet, detailed studies pointed to direct inhibition of thrombin, factor Xa and factor Xla as the major mechanism of action. ^{17,22 25} Interestingly, these molecules were found to bind in the heparin-binding site of these enzymes and induce allosteric inhibition. ^" ' ^"

Sulfated LMW lignins are similar to unfractionated heparin (UFH) or LMW heparins in terms of structural polydispersity and heterogeneity (Figure 1 ). The molecules are composed of oligomer! c chains of varying lengths, which can be 5-15 units long, and inter-monomeric linkages, which include β-04, β-5, β-β, 5-5 and others. ^" Theoretically, more than 36,000 hexameric sequences are possible from these variations. Yet, sulfated LMW lignins are completely unlike heparins with regard to their backbone. In contrast to the polysaccharide backbone of the heparins, sulfated LMW lignins possess a highly aromatic scaffold. In fact in terms of structure, sulfated LMW lignins are unlike any other class of anticoagulant investigated to-date, including the heparins, the coumarins, the hirudins, the peptidomimetics and the small molecule direct inhibitors. in this Example, we report on the development of a sulfated β-04 lignin (Sb04L), a chemically synthesized lignin, which exhibits highly selective and potent inhibition of human thrombin (and plasmin). Sb04L is homogeneous with regard to its inter-monomer linkages. It inhibits thrombin through an allosteric process by binding in exosite 2. Human whole blood studies demonstrate that Sb04L-based anticoagulation that is comparable to that induced by enoxaparin. Most importantly, Sb04L is synthesized in a few simple steps from readily available small molecules, which bodes well for a relatively inexpensive alternative to UFH and LMW heparin. Additionally, Sb04L inhibition of thrombin is quantitatively and rapidly reversed by protamine, which should greatly increases confidence in its usage. Thus, this Example describes a family of completely synthetic, non-polysaccharide, non-animal derived, thrombin-selective, allosteric anticoagulants which may be used in treating human and animal subjects. Experimental Procedures

Proteins and Chemicals. Human proteases were from either Haematologic Technologies (Essex Junction, VT), Sigma-AIdrich (St. Louis, MO), or Elastin Products Company

(Owensville, MO). Chromogenic substrates were obtained either from Sekisui Diagnostics (Stamford, CT), Sigma-AIdrich or Diapharma (West Chester, OH). HirP, a Tyr63 -sulfated hirudin-(54-65) peptide labeled with 5-(carboxy)fiuorescein, i.e., [5F]-Hir[54-65](S0 ₃ ^' ), was from the Bock laboratory. ²⁶ Monomer used in Sb04L preparation was synthesized using

7

literature protocol. ^" Plasma and whole blood clotting reagents were from Fisher Diagnostics (Middletown, VA) or Haemoscope Corporation (Niles, IL).

Chemical Synthesis of Sulfated β-04 Lisnin (SbQ4L). The synthesis of Sb04L was developed from reported polymerization and sulfation strategies. Briefly, ₂ C0 ₃ and ethyl 2- bromo-3-(4-hydroxy-3-methoxyphenyl)-3-oxo-propanoate were stirred in anhydrous DMF under nitrogen for 24 h, poured into ice-water mixture and the pH adjusted to -2.5 with 2M HCI. The precipitated polymer was filtered and lyophilized. The solid was suspended in methanol at 50°C and NaBH ₄ carefully added. After 24 h, the solution was neutralized with acetic acid, poured into 0.5M HCI and precipitated polymer was isolated by centri ugation. The solid was then dissolved in 1 ,4-dioxane and high molecular weight chains precipitated in diethyl ether. The reduced polymer was sulfated with triethylamine-sulfur trioxide complex in anhydrous DMF at 65°C for 24 h, ^30,3 ' dissolved in 30% (w/v) aqueous sodium acetate and poured into ice-cold ethanol to precipitate crude Sb04L. The recovered solid was desalted using FloatALyzer G2 (Spectrum Labs) dialysis tubes (MWCO 0.1 -0.5 kDa) and lyophilized.

Molecular Weights of SbO L. The number- (M _N ), weight- (Mw), and peak- (Mp) average molecular weights of Sb04L were measured as described earlier. ²³ Briefly, gel permeation chromatography (GPC) of Sb04L was performed on a Phenogel 5μ column (Phenomenex, Torrance, CA) using 0.1 N NaOH mobile phase flowing at 0.7 ml/min with detection at 254 nm. The Sb04L chromatogram was sliced into 1000 time periods providing 1000 average molecular weights with their corresponding absorbances, which were used to calculate M _\v , M _N , and Mp values. ²³

Direct Inhibition of Proteases. Inhibition of coagulation enzymes (thrombin, factors Xa, IXa, XIa and Vila) by Sb04L was measured using chromogenic substrate hydrolysis assays in standard manual 3 ml cuvettes, as described previously, ' while inhibition of other proteases was measured using a 96-well microplate format. This protocol was also adopted for studying thrombin inhibition in the presence of antithrombin or in the presence of serum albumin. The buffers, conditions and chromogenic substrates used in these experiments were derived from the literature. Briefly, a Sb04L in an appropriate buffer was incubated with a target protease for 10 min, followed by addition of chromogenic substrate and rapid measurement of the initial rate of substrate hydrolysis (A405). The ratio of the initial rate in the presence and absence of Sb04L gave the residual protease activity at each inhibitor concentration from which the /C50 was calculated using logistic equation 1 , u _{1 + 1 0} Utog[S/>O4i] _o -lo _S /C _jll )x//S} ^* » In this equation, Y is the residual protease activity; YM and Yo are the maximum and minimum residual activities, respectively; IC$o is the concentration of the inhibitor that results in 50% inhibition of enzyme activity; and HS is the Hill slope.

Inhibition ofThrombin-Thrombomodulin-Mediated Activation of Protein C by SbQ4L. The protocol for measuring the activation of protein C by thrombin-rTM complex essentially followed the literature. ³² Briefly, 5 μΙ of Sb04L (2.3 ng/ml to 2.3 mg/mlj was added to 70 μΐ of 50 mM Tris-HCl buffer, pH 7.4, containing 100 mM NaCl, and 1 mM CaC5 ₂ at 37°C, followed by addition of 5 μΐ of thrombin (240 11M) and 5 μΐ of rTM (400 nM). To this mixture, 5 μΙ of protein C (10 μΜ) was added followed by incubation for another 10 min. At the end of this period, APC generation was quenched with 5 μΐ of 10 mM EDTA and 16 μΜ argatroban, and quantified from the initial rate of hydrolysis of 400 μΜ S-2366. The apparent IC50 of Sb04L inhibition of activation of protein C was calculated using equation 1.

Michaclis-Menten Kinetics and Competitive Binding Studies. The kinetics of substrate hydrolysis by thrombin in the presence of Sb04L and competitive studies with exosite 1 and exosite 2 ligands followed our protocols for sulfated LMW lignins, ^17"19 ' ²² Plasma and Whole Blood Clotting Assays. The protocol for human plasma activated partial thromboplastin (APTT) and prothrombin time (PT), and human whole blood

thromboelastography (TEG) and hemostasis analysis (HAS) in the presence and absence of Sb04L followed our earlier studies with sulfated LMW lignins. ^18,19,21 ' ²³

Reversal ofSb04L Activity by Protamine. Protamine (4.9 μg l to 14.8 g/1) was added to a solution of Sb04L-thrombin complex in a 20 mM Tris-HCl buffer, pH 7.4, containing 100 mM NaCl, 2.5 mM CaCl ₂ and 0.1 % PEG8000 at 37°C followed by measuring the residual thrombin activity from substrate hydrolysis, as described above. In the absence of protamine, Sb04L gave -50% inhibition of thrombin at 37°C. Protamine alone did not affect the activity of thrombin as assessed by appropriate controls. Results

Synthesis and Characterization of Sb04L. Of the several inter-residue linkages present in lignins, the β-5 and β-04 linkages are most abundant. ³³ Earlier work had shown that sulfated LMW lignins devoid of the β-5 linkage, and hence enriched in β-04 linkage, generated significant anticoagulant activity. ²² ' ²³ Based on this, we reasoned that a lignin containing only β- 04 linkage may exhibit much higher anticoagulation potential. Hence, we resorted to a wholly chemical process of alkaline polymerization of ethyl 2-bromo-3-(4-hydroxy-3-methoxyphenyl)- 3-oxo-propanoate followed by sodium borohydride reduction and sulfation using tri ethyl amine- sulfur trioxide complex (Figure 1 ). Each of these steps have been reported earlier for a variety of other reactants and were achieved in fairly high yields. ^{28' 1}

The M _N , M\v and Mp of Sb04L were measured using aqueous GPC-HPLC, as described earlier, ²³ and found to be 9200, 12300, and 9100, respectively. This implies that on an average Sb04L oligomer is nearly 23 residues long. In comparison, an average full-length heparin chain is ~50 saccharides in length, whereas an average LMW heparin chain has 16 residues. The GPC- HPLC chromatograms also indicated absence of species with molecular weight less than 1000 suggesting an enriched preparation of longer oligomers that constituted composition greater than 95%. The polydispersity, i.e., the ratio of Mw to M _N , of Sb04L was found to be 1.336, which is similar to that of UFH used in the clinic. The sulfation density calculated on the basis of the difference in Mw between Sb04L and its unsulfated precursor was ~2 sulfate groups per monomer. Elemental analysis measurements indicated a composition of C 35.39, H 4.1 and S 1 1.54, which match a hypothetical Sb04L chain of 23 residues containing 27 sulfate groups and calculated composition of C 35.20, H 4.02, S 1 1.08. This indicates an average of 1 .2 sulfate groups per monomer. These measurements compare favorably with that of UFH and LMW heparins, which show an average of ~1.6 anionic groups per monomer. Additional purity and consistency of Sb04L preparation studies were performed using reversed-phase ion pairing UPLC-MS, and in vitro thrombin inhibition tests on three independent batches of synthetic Sb04L. The three batches demonstrated essentially identical UPLC-MS fingerprints and indistinguishable IC50 for thrombin inhibition. Thus overall, the elemental analysis measurements in combination with UPLC-MS fingerprints indicate the presence of uniform β-£?4 linkage in Sb04L preparation in high purity (>95%) in comparison to sulfated LMW lignins studied earlier. Sb04L is a Selective, Direct Inhibitor of Human Thrombin. The inhibition of coagulation factors Ila, Xa, IXa, Xla and Vila was studied using spectrophotometric measurement of the residual protease activity in the presence of varying levels of Sb04L. Figure 2A shows the decrease in residual protease activity as the concentration of Sb04L was varied over ~10 ⁵ -fold. Previous studies have shown that UFH, LMW heparin and fondaparinux do not inhibit these enzymes at concentrations as high as 100 /ml. ^" The change in activity was fitted using the dose-response equation 1 to calculate !Cso (Table 1).

Table 1. Parameters for sulfated β-04 lignin (Sb04L) inhibition of coagulation proteases. " Protease tog {/C ₅ o (g/ml)} /C ₅ « ^g/ml) Y _M K ₀ HS

Thrombin -6.8 ± 0,1 ^Λ 0.17 ±0.01 99 ± 1 29+1 1.610.1

Factor Xa -3.3 ±0.6 500 ±300 100 ±4 ~1 0.5 ±0.2

Factor IXa NI ^C NI _ ( - -

Factor Xla -4.1 ±0.1 89 ±9 98 ±3 41 ± 2 1.0 ±0.2

Factor Vila -6.3 ±0.1 0.54 ±0.05 103 ±3 31 ±3 1.1 ±0.1

Factor Vila TF --5.0" >1Γ 100 ±2 -38 ^e -0.5 ^e

Plasmin -6.4 ±0.1 0.38 ±0.04 103 ± 1 31 ± 2 1.1 ±0.1

Thrombin w/ AT ' -6.7 ±0.1 ^b 0.20 ±0.01 64 ± 1 6± 1 1.9 ±0.4

Thrombin/rTM - PC -5.4 ±0.1 4.20 ±0.08 98 ±2 3 ±5 0.96 ±

: 0.14

"The IC5 , HS, Y _M , YQ values were obtained following non-linear regression analysis of direct inhibition of the protease (see 'Experimental Methods' for details). Errors represent ± 1 S. E. c ^' No inhibition was observed upto concentrations as high as 1 mg/ml. ''Not applicable. 'Estimated values. ½ the presence of 200 nM AT. inhibition of activation of protein C by thrombin - thrombomodulin complex. Sb04L inhibits thrombin with an /C50 of 0.17 ug/ml, which corresponds to -14 nM (Table 1 ). Intrinsic pathway proteases factors Xa and XIa were also inhibited by Sb04L, however the /C50 values were in the range of 90-500 μ£/πιΙ corresponding to 10-55 μΜ. With regard to factor IXa, essentially no inhibition was noticeable at concentrations as high as 28.7 μ^παΐ (Figure 2A). Thus, Sb04L inhibited thrombin nearly 590-2940-fold better than these coagulation enzymes.

With regard to the extrinsic pathway, Sb04L was found to inhibit factor Vila with an ΛΓ50 of 0.54 μ^ηιΐ (Figure 2A, Table 1 ), only 3-fold higher than that for thrombin. Yet, in the presence of recombinant tissue factor (rTF), the potency decreased by more than 55-fold. Factor VIIa-rTF complex is the more relevant physiologic protease and this suggests that Sb04L is likely to primarily target the propagation step of coagulation.

To assess the selectivity features of Sb04L in more detail, we studied a small group of heparin-binding serine proteases including cathepsin G, HLE, kallikrein, PPE, APC, plasmin, chymotrypsin, and trypsin. The inhibition of these proteases by sulfated LMW lignins, parent molecules from which Sb04L was designed, was studied earlier. ²⁵ The activity of each protease was measured in a spectrophotometric assay in the presence of Sb04L at 0.37 (Figure 2B) and 1.62 mg/ml, which represent - 1850- and 8100- fold higher concentrations than the /C50 of thrombin inhibition. Except for human plasmin, none of the other proteases studied were found to be appreciably inhibited by Sb04L. For plasmin, a dose-response study led to an /C50 of 0.38 μ^πιΐ (Figure 2A, Table 1 ) suggesting potency similar to that against thrombin. In toto, results indicate that Sb04L is a selective inhibitor of human thrombin and plasmin, despite the considerable structural similarity between these trypsin-like proteases. Such high selectivity has not been observed earlier. ²³ ' ²⁵

SbQ4L Inhibition of Thrombin is Unaffected by the Presence ofAntithrombin. Polymeric heparin is a powerful antagonist of thrombin because of its ability to bind plasma antithrombin and bridge with thrombin. ¹ ' ^3'4 In contrast, sulfated LMW lignins were found to bind the serpin with reasonable affinity, yet loose direct inhibition potential. ²⁴ To assess the influence of antithrombin on Sb04L activity, direct inhibition of thrombin was studied in the presence of 200 nM serpin under otherwise identical conditions. A decrease in thrombin activity with increasing Sb04L levels was observed, which was identical to that measured in the absence of the serpin, except for the differences in maximal (YM) and minimal (Y ₀ ) residual thrombin activities. The YM and Yo values decreased by 35% and 23%, respectively, due to the reaction of thrombin with antithrombin. However these changes did not affect Sb04L inhibition of thrombin (/Cso = 0.2 and 0.17 μ^ιηΐ with and without antithrombin, respectively (Table 1 )). From this result it can be inferred that antithrombin does not retard or stimulate Sb04L's inhibitory activity. This is a significant point of variance from the parent sulfated LMW lignins, for which the serpin- mediated pathway was found to be a competing side reaction. ²⁴ The results highlight an interesting structural aspect that a specific scaffold, i.e., the β-04-linked scaffold, of the many possible in sulfated LMW lignins does not appear to bind the serpin with high affinity, which improves mechanistic selectivity.

Sb04L Inhibits Activation of Protein C by Thrombin-rTM Complex. Thrombin's procoagulant activity is drastically modulated by cell surface TM, which transforms it into an anticoagulant protease with specificity for protein C. ^35,36 To assess whether TM binding to thrombin alters the activity of Sb04L, we studied the efficacy of protein C activation. The level of proteolytically active protein C formed by thrombin-rTM complex can be measured spectrometrically through hydrolysis of S-2366 ³² following suppression of thrombin's native proteolytic activity using argatroban (~1 OOxK.]) as its specific inhibitor. Measurement of APC levels in the presence of varying Sb04L levels led to a sigmoidal profile on a semi-log plot (Figure 3) suggesting that as the Sb04L inhibited the protein C activation potential of thrombin- rTM complex. The / ₅ o was measured to be 4.2 μ /ml (Table 1 ), which was -20-fold higher than that measured for Sb04L inhibiting the procoagulant activity of thrombin alone. Interestingly, the efficacy of inhibition of protein C activation was found to be nearly 100% (Table 1 ), which is significantly higher than that for thrombin alone (-60%, Table 1 ). The results indicate that Sb04L reduces the procoagulant protential of thrombin much more than the anticoagulant potential.

Sb04L is an Allosteric Inhibitor of Thrombin. To understand the basis of thrombin inhibition, we measured the kinetics of Spectrozyme TH hydrolysis at pH 7.4 in the presence for Sb04L. Plots of the initial rates versus substrate concentration were hyperbolic, as expected (Figure 4). As the concentration of Sb04L was increased from 1.2 ng/ml to 2.3 μ /πιΐ, maximal velocity of hydrolysis, J^MAX, decreased steadily. Fitting the data using the standard Michaelis- Menten equation gave an essentially invariant KM _, W of 1.5 μΜ. This suggests that Sb04L does not affect small molecule chromogenic substrate binding to the active site of thrombin. The FMAX decreased from 27.5 to 7.4 mAbsU/min (Figure 4) corresponding to a decrease of more than 70%. Thus, Sb04L appears to not sterically hinder the interaction of thrombin substrate, but brings about changes in the active site that reduce the catalytic rate. This implies that Sb04L is a non-competitive, allosteric inhibitor of human thrombin.

Allostei Arises from Binding in Exosite 2 of Thrombin and Not in Exosite 1, To decipher the origin of allostery, we measured the effect of HirP, a hirudin-based dodecapeptide, on the Sb04L inhibition of thrombin. Earlier work has shown that HirP binds in exosite 1 with an affinity of 28 nM and increases the catalytic efficiency of thrombin. ²⁶ Thus, thrombin inhibition by Sb04L was studied in the presence of the exosite 1 competitor (Figure 5A), As the

concentration of the dodecapeptide was increased from 0 to 3.1 -times its affinity, the apparent /C ₅ o of thrombin inhibition remained essentially invariant at 0.15 μg/ml (Table 2).

Table 2. Inhibition of human thrombin by Sb04L in the presence of HirP, an exosite 1 ligand, and UFH, an exosite 2 ligand, at pH 7.4 and 25 °C."

/Cso ( g/ml) HS

[HirP] nM

0 0.15 ± 0.01* 102 + 14 24 + 1 1.2 + 0.1

8.6 0.17 ± 0.02 103 ± 2 23 ± 1 1.2 ± 0.1

86 0.14 + 0.02 102 ± 3 17 + 2 1.2 ± 0.2

[UFH] μΜ

0.2 0.22 ± 0.01 99 ± 1 26 + 1 1.6 ± 0.1

2.0 0.34 ± 0.02 98 ± 1 26 + 1 1.8 + 0.1

20 0.66 ± 0.05 96 ± 1 24 + 1 1.6 ± 0.2

100 1.25 ± 0.14 100 ± 1 28 + 2 0.9 + 0.1

"The /C50, HS, YM, YO values were obtained following non-linear regression analysis of direct inhibition of human thrombin in 20 mM Tris-HCl buffer, pH 7.4, containing 100 mM NaCI, 2.5 mM CaCl ₂ , and 0.1 % polyethylene glycol (PEG) 8000 at 25 °C. Inhibition was monitored through spectrophotometric measurement of residual thrombin activity. ''Errors represent± 1 S. E. These results imply that the interaction of HirP with thrombin does not affect Sb04L inhibition to a significant extent suggesting that the allosteric inhibitor does not engage exosite I .

To assess whether Sb04L binds in exosite 2 of thrombin, we studied competition with UFH. Figure 5B shows the change in the dose-response profiles of Sb04L inhibiting thrombin in the presence of UFH at pH 7.4. As the concentration of UFH increased from 0.2 to 100 μΜ, the /C ₅ (, increased from 0.22 to 1 .25 /ml (Table 2) indicating that Sb04L competes with UFH for binding to human thrombin.

The efficacy of competition can be gauged by using the Dixon-Webb relationship (Eq. 2), which estimates the ideality of competition between two ligands. In this equation, ATUFH is the dissociation constant of thrombin-UFH complex, which was measured to be 1 5.6+3.1 μΜ under otherwise identical conditions. Analysis using equation 2 showed that the observed IC _$0 was equivalent to that predicted on the basis of ideal competition. For example at 100 μΜ UFH, the predicted /C _{5 ()} is 1 .1 μ¾ ιτ.1, while the observed ICso was 1.25 μg/ml. Thus, Sb04L binds in or near anion-binding exosite 2. ICn\pmtiaed = ^/C 5o + ~) Ε<¾· ²

VFU

Sb04L is a Potent Anticoagulant in Human Plasma. Prothrombin (PT) and activated partial thromboplastin times (APTT) are traditional measures of the anticoagulation state of human plasma. Figure 6A shows the variation in plasma PT and APTT in the presence of Sb04L. A significant concentration-dependent prolongation of clotting times was observed suggesting good anticoagulation potential. A 2-fold increase in PT required 68 g ml, corresponding to 7.5 μΜ, which is significantly lower than that needed for a generic LMW heparin ( 342 g ml, 3 1 .6 μΜ) and a clinically used LMW heparin (enoxaparin, 339 μg/ml or 75 μΜ). ²¹ Likewise, a two-fold increase in APTT required 20 μg/ml (2.2 μΜ) of Sb04L, which compares favorably with a concentration of 5.9 μ /ml ( 1.3 μΜ) for generic LMW heparin and 5.4 μ^ιηΐ ( 1 .2 μΜ) for enoxaparin. ²¹ These results show that Sb04L is nearly as potent as LMW heparins in inducing plasma anticoagulation.

Despite this high potency (comparable to enoxaparin), Sb04L displays a loss of -100- 340-fold in potency in human plasma from that in in vitro enzyme systems. To assess the basis for this difference, we studied the effect of serum albumin on the effectiveness of Sb04L. Figure 6B shows the change in relative thrombin activity in the presence of fixed concentration of Sb04L and varying concentrations of bovine serum albumin (BSA), a surrogate for its human counterpart. In the absence of BSA, thrombin's hydrolytic activity was 22% ([Sb04L] = 0.42 μ /ιτιι), which was found to increase to 56, 59 and 72% in the presence of 1 , 2.5 and 10 mg/dl BSA, respectively. This suggests that the presence of BSA results in a significant drop in inhibitor potency probably arising from non-specific sequestering of Sb04L. Interestingly, a maximal thrombin activity of -75% is reached suggesting that some Sb04L remains free, and therefore inhibitory, at high enough plasma albumin concentration.

Sb04L is a Potent Anticoagulant of Human Whole Blood As Measured by

Thromboelastographv (TEG*). To evaluate Sb04L as an anticoagulant in whole blood, we employed TEG* ^' , which is quite often used to monitor anticoagulation therapy with LMW heparins. ²¹ TEG ^® assesses the nature of physical forces within a clot, which are dramatically affected by the presence of an anticoagulant in blood. In a nutshell, the clot formation in TEG ^® is recorded as a force transduced on a pin at the center of a blood-containing cup. Several parameters are evaluated from this force measurement including maximum amplitude (MA), the shear elastic modulus (G), the reaction time (R) and the angle a. MA and G are measures of clot stiffness, while R and a are measures of the rate of clotting.

Figure 9 shows the change in R, a, MA and G parameters as a function of the

concentration of Sb04L. Briefly, as the concentration of Sb04L increases from 0 to 152 μg/ml, R increases from 7.7 to 25.9 min, while decreases from 56° for normal blood to 22° indicating that the kinetics of fibrin polymerization and network formation is significantly depressed by the presence of Sb04L. Enoxaparin behaves in a similar manner, except that its effective

concentrations range from 1-5 μg/ml. Likewise, Sb04L reduces MA and G in a manner similar to enoxaparin (Figure 9), except for the ~30-fold better potency of the latter (~ 15-fold molar basis).

Whole Blood Anticoagulation Potency of Sb04L by Hemostasis Analysis (HAS ^1!it ). To further assess the whole blood anticoagulant potential of Sb04L, we utilized HAS™, which evaluates platelet contribution to clot formation (Figure 7). ²¹ This technique evaluates clot structure through the measurement of clot elastic modulus (CEM), which is the ratio of stress induced by platelets to strain arising from the change in clot thickness. The technique also provides information on contractile forces between platelets, i.e., the platelet contractile force (PCF), that adhere to surfaces and restrict relative movement of two cups. PCF depends on the platelet number, their metabolic status, presence of thrombin inhibitors and degree of GPIIb/IIIa exposure. On the other hand, CEM depends on the clot micro-structure, fibrinogen

concentration, and thrombin formation rate. It has been suggested that PCF and CEM changes can be correlated with susceptibility to bleeding and/or thrombotic tendency.

Sb04L affects PCF and CEM in a dose-dependent manner (Figure 9). As the

concentration of Sb04L increases from 0 to 74 μg ml, the PCF and CEM decrease from 8.0 to 0.7 kDynes and 18.3 to 0.6 kDynes/cm ² , respectively. These results parallel those measured through TEG ^® . When comparisons are made with enoxaparin, strikingly similar results are observed except for the range of concentration used for the clinically used anticoagulant.

Whereas PCF value of 0.7 was achieved at 74 μg/ml (8.1 μΜ) for Sb04L, it was achieved at 2.0 μ /ml (444 nM) for enoxaparin suggesting a ~37-fold better potency for the latter on weight basis and 18-fold better potency on molar basis. These results further confirm that Sb04L mimics enoxaparin anticoagulation function fairly well.

SbQ4L Anticoagulantion Can be Reversed by Protamine. A major advantage of heparin therapy is its amenability to protamine-based reversal. It is also one of the reasons why fondaparinux continues to suffer because its iatrogenic bleeding is difficult to reverse rapidly. To test whether Sb04L inhibition of thrombin can be reversed, we studied the recovery of thrombin activity following successive introduction of protamine. Figure 8 shows the thrombin recovery profile with varying levels of protamine after achieving 50% inhibition with Sb04L. The protamine-mediated recovery profile essentially mirrors the Sb04L-induced inhibition profile. More importantly, the level of recovery is quantitative at high enough protamine concentrations. Further, the recovery was instantaneous as no extended incubation was necessary to observe reversal. The concentration of protamine necessary to recover 50% thrombin activity, i.e., RC50, could be calculated using an equation similar to the logistic equation 1 used for inhibition studies and found to be -0.1 μg ml, a concentration equivalent to the /C50 for Sb04L inhibition of thrombin.

In vivo Assays. Two mouse models were used to analyze the anticoagulant potential of Sb04L. FeC carotid artery thrombosis model. The ferric chloride induced injury model is commonly used to analyze the anticoagulant potential of several compounds in mice. Wild type C57B 1 6 mice were anesthetized with 50 mg/kg intraperitoneal (IP) pentobarbital. Sb04L (0, 100, 300, 500 or 1 OOO^tg in 100μ phosphate buffered saline (PBS)) was infused into the right internal jugular vein. Five minutes after infusion the right common carotid artery was exposed and fitted with a Doppler flow probe. Thrombus formation was induced by applying two 1 x 1.5 mm filter papers saturated with FeCl ₃ (3.5% solution) to opposide sides of the artery for three minutes. After washing the site of injury with PBS, flow was monitored for thirty minutes. Mice were sacrificed by pentobarbital overdose after conclusion of the experiment while under anesthesia.

Rose Bengal-laser inury carotid artery thrombosis model. In vivo testing using Rose Bengal thrombosis model is another recognized mouse model used to perform anticoagulant assays. Mice were anesthetized as discussed above and 500 μg of SB04L in 100 μΐ PBS was infused into the right internal jugular vein. Five minutes after infusion, Rose Bengal (75mg/kg) was infused through the internal jugular vein, and the carotid artery was illuminated with a 1.5 mW 540 nm laser (Melles Griot, Carlsbad, CA) positioned 6 cm from the artery. Flow was monitored for 120 minutes. Mice were sacrificed by pentobarbital overdosed after conclusion of the experiment while under anesthesia. The laser light converts the dye to free radicals which result in vessel occlusion with a platelet rich thrombus in 44.7 ± 6.5 minutes in vehicle controls. In mice treated with Sb04L, occlusion occurred in 76±21.3 minutes, demonstrating significant anticoagulant activity.

Figure 10A shows the formation of occlusive platelet-rich thrombus in the carotid artery of mice using a 3.5% FeCI ₃ solution with two doses of Sb04L, i.e. 100 μg and 1 ,000 μg. Figure 10B demonstrates a dose-dependent decrease in coagulation with a complete inhibition of clot formation at a dose of 1,000 μg of Sb04L. The number shown in brackets shows the fraction of mice which showed complete thrombotic plug formation.

Discussion

Despite a massive effort of the past 30 years it has been difficult to find a truly viable alternative to UFH and LMW heparins. The sulfated polysaccharides are very good at resolving thrombotic disorders, while being fairly inexpensive. Poly- or oligo- saccharide variants, e.g., idraparinux and its biotinylated forms, ' fondaparinux conjoined to a direct thrombin inhibitor, and heparin octasaccharides, ^" continue to be developed because of the high anticoagulant efficiency achievable with the saccharide scaffold. Yet, each variant carries a major synthetic burden that cannot be expected to match the much lower cost effectiveness of the heparins. A fundamental challenge, therefore, is to design an anticoagulant that effectively challenges heparins by affording high potency and high selectivity, promising reduced adverse effects, while being very easy to produce.

Sb04L was designed so as to achieve this objective. Sb04L can be synthesized in high yields in only three steps from an appropriately functionalized monomer. This protocol is significantly more efficient than the dozens of steps used in the synthesis of fondaparinux-related anticoagulants ^39"42 as well as in the synthesis of active site directed anticoagulants, e.g., dabigatran, apixaban or rivaroxaban. In fact, the three-step protocol can be completed in a week using a simple synthetic set up, which is expected to put forth a highly cost effective solution to heparins. Process scale up should be possible. The chemistry used in the synthesis of Sb04L ensures that the oligomer is homogeneous with regard to inter-monomeric linkages. This greatly reduces the structural complexity in Sb04L in comparison to its precursor, sulfated LMW lignin,' ⁷ ' ^19,21"25 as well as oligomeric heparins. ¹ ' ³⁴

Sb04L is a highly potent anticoagulant in vitro and ex vivo. Sb04L inhibits thrombin (and plasmin) with high selectivity, while inhibiting factors IXa, Xa and XIa with a potency that is orders of magnitude lower. Factor VIIa-TF complex, the physiologic extrinsic pathway initiator, is inhibited with -10-fold lower potency, while plasma antithrombin does not affect direct action of Sb04L. Competing macromolecule TM, which alters the substrate specificity of thrombin, is likely to be functional only at ~20-times higher concentrations of Sb04L (Table 1).

The in vitro potency against thrombin (0.17 μg/ml) changes to ~20 μg/ml for doubling APTT and -80 μg ml for effective anticoagulation of human whole blood. The primary reason for this significant drop in anticoagulant effect in plasma and blood is likely to be non-specific binding to serum albumin. This is not unusual as nearly all drugs bind to serum proteins, especially albumin. In fact, most agents exhibit significant protein binding resulting in considerable loss in effective potency. Rather binding to serum albumin may actually be an advantage because of the possibility of a slow release mechanism. On weight basis, ~3 μ^ιηΐ enoxaparin induces a blood anticoagulation equivalent to -80 Sb04L suggesting that the non-saccharide anticoagulant is likely to be nearly 20-30-fold weaker in clinical efficiency. This is not necessarily a disadvantage because the more important parameter is the ratio of potency to adverse effects (bleeding, thrombocytopenia, hepatotoxicity, etc.) in vivo, Sb04L inhibits thrombin with an efficacy of -60%, whereas heparins have an efficacy of nearly 100% because their primary effector, antithrombin, is a covalent inhibitor of thrombin and other coagulation enzymes. ¹ ' ³⁴ This implies that even at saturation, the Sb04L-thrombin complex is likely to display a reduced level of pro-coagulant efficacy. This is a specific advantage of allosteric inhibitors and is the reason why these molecules are called regulators. Sb04L appears to possess this advantage and suggests its use as an effective prophylactic agent.

Sb04L allostery arises from binding in exosite 2 of thrombin, which also engages polymeric heparin. Although nearly ideal competition between heparin and Sb04L is indicated by competitive inhibition studies, it is not necessary that the two ligands are strictly mutually exclusive. Exosite 2 is a fairly large area spanning -20*30 A ² and consisting of numerous electropositive residues including Arg93, Argl O l , Argl 65, Arg233, Lys235, Lys236, and Lys240. Of these, polymeric heparin recognizes Lys236, Lys240, Arg93, Argl O l and

Arg233. ^43,4 Sulfated LMW lignins, the precursors of Sb04L, were found to bind to Arg93 and Argl 75. ^{1 7} Thus, the geometries of polymeric heparin and sulfated LMW lignins in exosite 2 of thrombin appear to be significantly different. Structurally, Sb04L is more heparin-like in terms of its sulfation density, but is more LMW lignin-like in terms of its hydrophobic scaffold. Thus, it is not clear whether Sb04L binding in exosite 2 will resemble heparin or sulfated LMW lignins. Yet, this geometry will determine whether Sb04L inhibits thrombin in a fibrin-bound state. ⁴⁵

A key advantage in the use of polymeric heparins is the availability of protamine as an antidote. Sb04L is also a sulfated polymer and its direct inhibition of thrombin is neutralized by protamine. In fact in chromogenic test systems, protamine was found to restore 100% thrombin activity with a RC only 5-fold higher than IC _$ o, Additionally, the effect was instantaneous as no extended incubation was necessary. An average UFH chain contains nearly 1 .7 anionic groups (- OS0 ₃ ^" and -COO ^" ) per monosaccharide, while Sb04L contains nearly two -OSO3 ^" groups, which explains the highly effective protamine reversal. Overall, the present work puts forward a novel designed sulfated β-04 lignin molecule, Sb04L, as a highly selective and potent anticoagulant with considerable promise. Sb04L is exciting because it can be readily synthesized, is not derived from animals, is an allosteric regulator and its anticoagulation is likely to be reversed using the traditional heparin antidote, protamine.

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While the invention has been described in terms of its preferred embodiments, those skilled in the art will recognize that the invention can be practiced with modification within the spirit and scope of the appended claims. Accordingly, the present invention should not be limited to the embodiments as described above, but should further include all modifications and equivalents thereof within the spirit and scope of the description provided herein.