Apixaban, an oral, direct and highly selective factor Xa inhibitor: in vitro, antithrombotic and antihemostatic studies.

BACKGROUND: Apixaban is an oral, direct and highly selective factor Xa (FXa) inhibitor in late-stage clinical development for the prevention and treatment of thromboembolic diseases. OBJECTIVE: We evaluated the in vitro properties of apixaban and its in vivo activities in rabbit models of thrombosis and hemostasis. METHODS: Studies were conducted in arteriovenous-shunt thrombosis (AVST), venous thrombosis (VT), electrically mediated carotid arterial thrombosis (ECAT) and cuticle bleeding time (BT) models. RESULTS: In vitro, apixaban is potent and selective, with a K(i) of 0.08 nm for human FXa. It exhibited species difference in FXa inhibition [FXa K(i) (nm): 0.16, rabbit; 1.3, rat; 1.7, dog] and anticoagulation [EC(2x) (microm, concentration required to double the prothrombin time): 3.6, human; 2.3, rabbit; 7.9, rat; 6.7, dog]. Apixaban at 10 microm did not alter human and rabbit platelet aggregation to ADP, gamma-thrombin, and collagen. In vivo, the values for antithrombotic ED(50) (dose that reduced thrombus weight or increased blood flow by 50% of the control) in AVST, VT and ECAT and the values for BT ED(3x) (dose that increased BT by 3-fold) were 0.27 +/- 0.03, 0.11 +/- 0.03, 0.07 +/- 0.02 and > 3 mg kg(-1) h(-1) i.v. for apixaban, 0.05 +/- 0.01, 0.05 +/- 0.01, 0.27 +/- 0.08 and > 3 mg kg(-1) h(-1) i.v. for the indirect FXa inhibitor fondaparinux, and 0.53 +/- 0.04, 0.27 +/- 0.01, 0.08 +/- 0.01 and 0.70 +/- 0.07 mg kg(-1) day(-1) p.o. for the oral anticoagulant warfarin, respectively. CONCLUSIONS: In summary, apixaban was effective in the prevention of experimental thrombosis at doses that preserve hemostasis in rabbits.

Leprosy in Israel: an imported disease--the support of histopathological examination for its detection.

Leprosy is rare and non-endemic in Israel. Cases of leprosy are invariably imported by immigrants or foreign workers arriving from endemic areas. In view of the relative rarity of the disease, clinicians and pathologists are not always alert to the possibility of the disease or recognize potential symptoms. A case history is presented of a 31-year-old immigrant presenting symptoms of skin lesions and nodules on the hands and facial region, especially the ear lobe. Confirmation of the infection was provided by histopathology of suspected lesions stained for acid-fast bacilli (modified Fite-Faraco staining).

Design and synthesis of a series of (2R)-N(4)-hydroxy-2-(3-hydroxybenzyl)-N(1)- [(1S,2R)-2-hydroxy-2,3-dihydro-1H-inden-1-yl]butanediamide derivatives as potent, selective, and orally bioavailable aggrecanase inhibitors.

A pharmacophore model of the P1' site, specific for aggrecanase, was defined using the specificity studies of the matrix metalloproteinases and the similar biological activity of aggrecanase and MMP-8. Incorporation of the side chain of a tyrosine residue into compound 1 as the P1' group provided modest selectivity for aggrecanase over MMP-1, -2, and -9. A cis-(1S)(2R)-amino-2-indanol scaffold was incorporated as a tyrosine mimic (P2') to conformationally constrain 2. Further optimization resulted in compound 11, a potent, selective, and orally bioavailable inhibitor of aggrecanase.

Discovery of macrocyclic hydroxamic acids containing biphenylmethyl derivatives at P1', a series of selective TNF-alpha converting enzyme inhibitors with potent cellular activity in the inhibition of TNF-alpha release.

SAR exploration at P1' using an anti-succinate-based macrocyclic hydroxamic acid as a template led to the identification of several bulky biphenylmethyl P1' derivatives which confer potent porcine TACE and anti-TNF-alpha cellular activities with high selectivity versus most of the MMPs screened. Our studies demonstrate for the first time that TACE has a larger S1' pocket in comparison to MMPs and that potent and selective TACE inhibitors can be achieved by incorporation of sterically bulky P1' residues.

Synthesis and pharmacology of modified amidine isoxazoline glycoprotein IIb/IIIa receptor antagonists.

Selective antagonism of the platelet GPIIb/IIIa receptor represents an attractive mechanism for the prevention and treatment of a number of thrombotic disease states. The antiplatelet activity of the oral GPIIb/IIIa receptor antagonists DMP 754 and DMP 802 have been disclosed. In this paper, the synthesis and biological evaluation of a series of potent N-substituted benzamidine isoxazolines are explored. The effect of benzamidine substitution on the duration of antiplatelet efficacy in dog is presented.

Design, synthesis, and structure-activity relationships of macrocyclic hydroxamic acids that inhibit tumor necrosis factor alpha release in vitro and in vivo.

To search for TNF-alpha (tumor necrosis factor alpha) converting enzyme (TACE) inhibitors, we designed a new class of macrocyclic hydroxamic acids by linking the P1 and P2' residues of acyclic anti-succinate-based hydroxamic acids. A variety of residues including amide, carbamate, alkyl, sulfonamido, Boc-amino, and amino were found to be suitable P1-P2' linkers. With an N-methylamide at P3', the 13-16-membered macrocycles prepared exhibited low micromolar activities in the inhibition of TNF-alpha release from LPS-stimulated human whole blood. Further elaboration in the P3'-P4' area using the cyclophane and cyclic carbamate templates led to the identification of a number of potent analogues with IC(50) values of

Regulation of gene expression and cell growth in vivo by tetracycline using the hollow fiber assay.

The hollow fiber assay presents a potentially unique tool to study the effects of regulated gene expression in cell lines that do not form tumors in vivo. The hollow fibers allow small molecules to pass freely through while keeping the cells within the fibers and segregated from host cells. OSp16.1 cells, derived from the U24 clone of the U2-OS osteogenic sarcoma tumor line, express the p16INK4a tumor suppressor under the regulation of tetracycline (tet) (Mitra J et al. Mol Cell Bio 19:3916, 1999). The in vitro induction of p16 in the OSp16.1 cell line is regulated by tet. The hollow fiber assay was used to determine whether the regulation of the p16 gene could be achieved in vivo, since these cells did not grow in the xenograft model. There were no differences in the in vivo growth pattern of U24 cells loaded into the hollow fibers with and without tet: 807% and 839% net growth, respectively. OSp16.1 cells in fibers in mice receiving 3.33 mg/kg/day tet had a 644% net growth after 21 days. There was a 194% net growth without tet. Immunoblotting of extracts prepared from the hollow fibers confirmed that p16 was induced in the absence of tet. These data demonstrate this assay is a useful tool for studying the effects of regulated gene expression in vivo.

Recent origin and spread of a common Lithuanian mutation, G197del LDLR, causing familial hypercholesterolemia: positive selection is not always necessary to account for disease incidence among Ashkenazi Jews.

G197del is the most prevalent LDL receptor (LDLR) mutation causing familial hypercholesterolemia (FH) in Ashkenazi Jew (AJ) individuals. The purpose of this study was to determine the origin, age, and population distribution of G197del, as well as to explore environmental and genetic effects on disease expression. Index cases from Israel (n=46), South Africa (n=24), Russia (n=7), The Netherlands (n=1), and the United States (n=1) were enlisted. All trace their ancestry to Lithuania. A highly conserved haplotype (D19S221:104-D19S865:208-D19S413:74) was identified in G197del chromosomes, suggesting the occurrence of a common founder. When two methods were used for analysis of linkage disequilibrium (LD) between flanking polymorphic markers and the disease locus and for the study of the decay of LD over time, the estimated age of the deletion was found to be 20 +/- 7 generations (the 95% confidence interval is 15-26 generations), so that the most recent common ancestor of the mutation-bearing chromosomes would date to the 14th century. This corresponds with the founding of the Jewish community of Lithuania (1338 a.d.), as well as with the great demographic expansion of AJ individuals in eastern Europe, which followed this settlement. The penetrance of mutation-linked severe hypercholesterolemia is high (94% of heterozygotes have a baseline concentration of LDL cholesterol (LDL-C) that is >160 mg/dl), and no significant differences in the mean baseline lipid level of G197del carriers from different countries were found. Polymorphisms of apolipoprotein E and of scavenger-receptor class B type I were observed to have minor effects on the plasma lipid profile. With respect to determinative genetic influences on the biochemical phenotype, there is no evidence that could support the possibility of a selective evolutionary metabolic advantage. Therefore, the founder effect in a rapidly expanding population from a limited number of families remains a simple, parsimonious hypothesis explaining the spread of G197del-LDLR-linked FH in AJ individuals.

Synthesis and SAR of benzamidine factor Xa inhibitors containing a vicinally-substituted heterocyclic core.

The selective inhibition of coagulation factor Xa has emerged as an attractive strategy for the discovery of novel antithrombotic agents. Here we describe highly potent benzamidine factor Xa inhibitors based on a vicinally-substituted heterocyclic core.

Discovery of 1-[3-(aminomethyl)phenyl]-N-3-fluoro-2'-(methylsulfonyl)-[1,1'-biphenyl]-4-yl]-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (DPC423), a highly potent, selective, and orally bioavailable inhibitor of blood coagulation factor Xa.

Factor Xa (fXa) plays a critical role in the coagulation cascade, serving as the point of convergence of the intrinsic and extrinsic pathways. Together with nonenzymatic cofactor Va and Ca2+ on the phospholipid surface of platelets or endothelial cells, factor Xa forms the prothrombinase complex, which is responsible for the proteolysis of prothrombin to catalytically active thrombin. Thrombin, in turn, catalyzes the cleavage of fibrinogen to fibrin, thus initiating a process that ultimately leads to clot formation. Recently, we reported on a series of isoxazoline and isoxazole monobasic noncovalent inhibitors of factor Xa which show good potency in animal models of thrombosis. In this paper, we wish to report on the optimization of the heterocyclic core, which ultimately led to the discovery of a novel pyrazole SN429 (2b; fXa K(i) = 13 pM). We also report on our efforts to improve the oral bioavailability and pharmacokinetic profile of this series while maintaining subnanomolar potency and in vitro selectivity. This was achieved by replacing the highly basic benzamidine P1 with a less basic benzylamine moiety. Further optimization of the pyrazole core substitution and the biphenyl P4 culminated in the discovery of DPC423 (17h), a highly potent, selective, and orally active factor Xa inhibitor which was chosen for clinical development.

The design and synthesis of noncovalent factor Xa inhibitors.

Thrombosis is a major cause of mortality in the industrialized world. Therefore, the control of blood coagulation has become a major target for new therapeutic agents. One attractive approach is the inhibition of factor Xa (fXa), the enzyme directly responsible for thrombin generation. In this review we describe our approaches in the design and synthesis of small molecule, noncovalent fXa inhibitors. Rational drug design and selective screening of our GPIIb/IIIa library afforded several lead compounds for our fXa program. Following-up the leads in the isoxazoline series led to potent fXa inhibitors such as SF303 and SK509 with only one basic group. The isoxazole series was then designed to remove the chiral center in the isoxazoline ring, and this effort led to SA862 which has subnanomolar fXa affinity. Optimizing the core structure generated a series of novel five-membered ring heterocycles substituted with benzamidine, which are potent fXa inhibitors. Further optimization in the pyrazole series resulted in the discovery of fXa inhibitors such as SN429 with picomolar fXa affinity. Efforts to improve the oral bioavailability by lowering the basicity of these compounds, while simultaneously maintaining potency against fXa, culminated in the discovery of DPC 423. DPC 423 was selected for clinical evaluation as a potent and orally bioavailable fXa inhibitor.

Design, synthesis, and biological evaluation of potent and selective amidino bicyclic factor Xa inhibitors.

Thrombotic diseases are a major cause of death and morbidity. Factor Xa (fXa) plays a vital role in the regulation of normal homeostasis and abnormal intravascular thrombus development in the blood coagulation cascade. A novel series of fXa inhibitors incorporating an amidino 6,5-fused bicyclic moiety at the P1 position has been designed and synthesized based on molecular modeling studies. Structure-activity relationship (SAR) studies have led to selective subnanomolar fXa inhibitors. The most potent fXa inhibitor in this series (72, SE170) has a potent inhibition constant (K(i) = 0.3 nM), is 350-fold selective for fXa over trypsin, and also shows good in vivo efficacy in a rabbit arterio-venous thrombosis model (ID(50) = 0.14 micromol/kg/h). An X-ray crystal structure of 72 complexed to bovine trypsin was completed, and a binding mode of 72 with fXa has been proposed based on modeling with human des-Gla-fXa.

Nonpeptide factor Xa inhibitors II. Antithrombotic evaluation in a rabbit model of electrically induced carotid artery thrombosis.

SK549 (mol. wt. 546 Da) is a synthetic, selective inhibitor of human coagulation factor Xa (fXa) (K(i) = 0.52 nM). This study compared the antithrombotic effects of SK549 and a series of benzamidine isoxazoline fXa inhibitors with aspirin, DuP 714 (a direct thrombin inhibitor), recombinant tick anticoagulant peptide, or heparin in a rabbit model of electrically induced carotid arterial thrombosis. Compounds were infused i.v. continuously from 60 min before electrical stimulation to the end of the experiment. Values of ED(50) (dose that increases the carotid blood flow to 50% of the control) were 0.12 micromol/kg/h for SK549, 0.56 micromol/kg/h for aspirin, 0.14 micromol/kg/h for DuP 714, 0.06 micromol/kg/h for recombinant tick anticoagulant peptide, and >100 U/kg/h for heparin. The EC(50) (plasma concentration that increased blood flow to 50% of the control) for SK549 was 97 nM. Unlike aspirin and heparin, SK549 was efficacious and, at 1.5 micromol/kg/h i.v. (n = 9), maintained carotid blood flow at 87 +/- 6% of control level for greater than 90 min. Unlike heparin, SK549 inhibited ex vivo fXa activity but not ex vivo thrombin activity. There was a highly significant correlation between K(i) (fXa) and ED(50) of a series of fXa inhibitors (r = 0. 85, P <.001). Therefore, these results suggest that SK549 is a novel, potent, and effective antithrombotic agent in a rabbit model of arterial thrombosis. It is likely that SK549 exerts its antithrombotic effect through selective inhibition of fXa. Furthermore, SK549 may be clinically useful for the prevention of arterial thrombosis.

Synthesis and activity studies of conformationally restricted alpha-ketoamide factor Xa inhibitors.

Conformationally restricted borolysine compounds containing a 2-(2-cyanophenylthio) benzoyl in the P3 position unexpectedly led to enhanced factor Xa inhibition. In an effort to improve both the potency and selectivity of this series by extending into the S' domain, we have replaced the boronic acid with alpha-ketoamides, utilizing a novel process that was developed in our labs.

The hollow fiber assay: continued characterization with novel approaches.

The hollow fiber assay, a unique in vivo model, permits the simultaneous evaluation of compound efficacy against multiple cell lines in two physiological compartments. This assay has been used to characterize in vivo activity of cytotoxic compounds. The purpose of the present study was to characterize and optimize this assay for compounds with a defined mechanism of action, specifically cell cycle inhibition. Two human tumor cell lines and one normal human cell line were loaded into polyvinylidene fluoride hollow fibers at two or more cell concentrations and grown in mice for 3-10 days. The data demonstrate the importance of characterizing the initial loading density of various cell lines in the evaluation of compounds. All studies were performed with cells in the linear part of the cell growth curves. Initial loading densities of 1-2 x 10(4) cells/fiber gave the greatest opportunity for growth in the three human cell lines tested (HCT116 colon carcinoma, NCI-H460 non-small cell carcinoma, and AG 1523 normal fibroblast). Utilizing the MTT assay, standard curves were constructed to correlate the final number of cells with optical density (OD) readings at 540 nm in order to calculate cell numbers in the fibers. Insights into the mechanism of action of cisplatin have been gained using Western blot analysis of the cell cycle markers PCNA (a protein present throughout the cell cycle) and Rb (a protein that acts as a tumor suppressor gene product) from the hollow fiber cells. In cisplatin-treated NCI-H460 cells both PCNA and Rb phosphorylation decreased, suggesting the arrest of the cells prior to the S phase. Standard therapeutic agents, cisplatin, racemic flavopiridol, cyclophosphamide and mitomycin C, were evaluated independently in the hollow fiber assay and the xenograft model. The data demonstrate that compounds active in the hollow fiber assay are also active in the xenograft.

Isoxazolines and isoxazoles as factor Xa inhibitors.

3,4,5-Trisubstituted isoxazolines (2) and isoxazoles (3) were prepared and evaluated for their in vitro and in vivo antithrombotic efficacy. They were compared to 3,5,5-trisubstituted isoxazolines (1) for Factor Xa selectivity and potency. They were also compared in an arterio-venous (A-V) shunt model of thrombosis.

Ring constrained analogues of beta-alanine-containing GPIIb/IIIa receptor antagonists.

A series of ring constrained analogues of the GPIIb/IIIa receptor antagonist XR299 (1) was investigated as potential inhibitors of glycoprotein IIb/IIIa, a platelet receptor that plays a key role in platelet aggregation and platelet adhesion. Ring size was found to have a large effect on in vitro potency. Selected compounds showed good in vitro activity, a preference for binding to activated platelets, and modest duration of action when dosed i.v. as a racemate in a canine model.

The de novo design and synthesis of cyclic urea inhibitors of factor Xa: optimization of the S4 ligand.

In this report refinements to the S4 ligand group leads to compound 19, an inhibitor of fXa with good potency in vitro and an improved pharmacokinetic profile in rabbit. The X-ray crystallographic study of a representative analogue confirms our binding model for this series.

Isoxazolines as potent antagonists of the integrin alpha(v)beta(3).

Starting with lead compound 2, we sought to increase the selectivity for alpha(v)beta(3)-mediated cell adhesion by examining the effects of structural changes in both the guanidine mimetic and the substituent alpha to the carboxylate. To prepare some of the desired aminoimidazoles, a novel reductive amination utilizing a trityl-protected aminoimidazole was developed. It was found that guanidine mimetics with a wide range of pK(a)'s were potent antagonists of alpha(v)beta(3). In general, it appeared that an acylated 2-aminoimidazole guanidine mimetic imparted excellent selectivity for alpha(v)beta(3)-mediated adhesion versus alpha(IIb)beta(3)-mediated platelet aggregation, with selectivity of approximately 3 orders of magnitude observed for compounds 3g and 3h. It was also found in this series that the alpha-substituent was required for potent activity and that 2,6-disubstituted arylsulfonamides were optimal. In addition, the selective alpha(v)beta(3) antagonist 3h was found to be a potent inhibitor of alpha(v)beta(3)-mediated cell migration.

Disubstituted indazoles as potent antagonists of the integrin alpha(v)beta(3).

A new series of indazole-containing alpha(v)beta(3) integrin antagonists is described. Starting with lead compound 18a, variations in a number of structural features were explored with respect to inhibition of the binding of beta(3)-transfected 293 cells to fibrinogen and to selectivity for alpha(v)beta(3) over GPIIbIIIa, another RGD-binding integrin. Indazoles attached to a 2-aminopyridine or 2-aminoimidazole by a propylene linker at the indazole 1-position and to a diaminopropionate derivative via a 5-carboxylate amide provided the best potency with moderate selectivity. Several differences in the SAR of the diaminopropionate moiety were observed between this series and a series of isoxazoline-based selective GPIIbIIIa antagonists. Compound 34a (SM256) was a potent antagonist of alpha(v)beta(3) (IC(50) 2.3 nM) with 9-fold selectivity over GPIIbIIIa.