Increased local expression of coagulation factor X contributes to the fibrotic response in human and murine lung injury.

Uncontrolled activation of the coagulation cascade contributes to the pathophysiology of several conditions, including acute and chronic lung diseases. Coagulation zymogens are considered to be largely derived from the circulation and locally activated in response to tissue injury and microvascular leak. Here we report that expression of coagulation factor X (FX) is locally increased in human and murine fibrotic lung tissue, with marked immunostaining associated with bronchial and alveolar epithelia. FXa was a potent inducer of the myofibroblast differentiation program in cultured primary human adult lung fibroblasts via TGF-beta activation that was mediated by proteinase-activated receptor-1 (PAR1) and integrin alphavbeta5. PAR1, alphavbeta5, and alpha-SMA colocalized to fibrotic foci in lung biopsy specimens from individuals with idiopathic pulmonary fibrosis. Moreover, we demonstrated a causal link between FXa and fibrosis development by showing that a direct FXa inhibitor attenuated bleomycin-induced pulmonary fibrosis in mice. These data support what we believe to be a novel pathogenetic mechanism by which FXa, a central proteinase of the coagulation cascade, is locally expressed and drives the fibrotic response to lung injury. These findings herald a shift in our understanding of the origins of excessive procoagulant activity and place PAR1 central to the cross-talk between local procoagulant signaling and tissue remodeling.

Novel P2Y12 adenosine diphosphate receptor antagonists for inhibition of platelet aggregation (II): pharmacodynamic and pharmacokinetic characterization.

Antiplatelet drugs are used to prevent aberrant platelet activation in pathophysiologic conditions such as myocardial infarction and ischemic stroke. The key role that ADP plays in this process has led to the development of antiplatelet drugs that target the P2Y12 receptor. The aim of this study was to characterize the pharmacodynamic (PD) and pharmacokinetic (PK) properties of the novel P2Y12 receptor antagonists, BX 667 and BX 048. BX 667 blocks ADP-induced platelet aggregation in human, dog and rat blood (IC50=97, 317 and 3000 nM respectively). BX 667 had nominal effects on collagen-induced aggregation and weakly inhibited arachidonic acid-induced aggregation. BX 667 has an active metabolite, BX 048, that also potently inhibits ADP-induced aggregation (IC50=290 nM) in human blood. BX 667 was shown to have high oral bioavailability in both dog and rat unlike BX 048. Administration of BX 667 resulted in a rapid and sustained inhibition of platelet aggregation where the extent and duration of platelet inhibition was directly proportional to circulating plasma levels. This report describes the PK/PD properties of BX 667 showing that it has the properties required for a potential antiplatelet therapeutic agent.

Novel P2Y12 adenosine diphosphate receptor antagonists for inhibition of platelet aggregation (I): in vitro effects on platelets.

ADP plays a key role in platelet aggregation which has led to the development of antiplatelet drugs that target the P2Y12 receptor. The aim of this study was to characterize the effects of two novel P2Y12 receptor antagonists, BX 667 and its active metabolite BX 048, on platelets. BX 667 and BX 048 block the binding of 2MeSADP to platelets and antagonize ADP-induced platelet aggregation in human, dog and rat washed platelets. Both compounds were shown to be reversible inhibitors of platelet aggregation. BX 048 prevents the decrease in cAMP induced by treatment of platelets with ADP. The specificity of BX 667 and BX 048 was demonstrated against cell lines expressing P2Y1 and P2Y6 as well as against a panel of receptors and enzymes. Taken all together these data show that both BX 048 and BX 667 are potent P2Y12 antagonists with high specificity which, in the accompanying paper are demonstrated to behave predictably in vivo.

A dual-labeling method to quantify unscheduled DNA synthesis in primary cells.

Unscheduled DNA synthesis (UDS) has been used as an endpoint for measuring DNA damage in vitro and in vivo. Determination of UDS is regarded as a reliable genotoxicity assay by regulatory agencies including US FDA and EPA. In this study, we have developed an improved UDS assay to detect DNA damage and repair processes upon chemical exposure. We utilized a dual-labeling procedure in which fluorescent DAPI stained nuclei of live cells and [(3)H]thymidine labeled cells undergoing new DNA synthesis. The occurrence of UDS in cells was quantified by either manual nucleus counting or net intensity approaches. This assay was validated by testing known genotoxic compounds 2-acetylaminofluorene (2-AAF), ethylmethanesulfonate (EMS), and N-nitrosodimethylamine (NDL) in primary rat hepatocytes as well as in confluent human mammary epithelial cells. In addition, fluorescent labeling of nuclei DNA helped to distinguish apoptotic cells from non-apoptotic cells. Chemical effects on cell functions were also examined by conducting the cytotoxicity assay along with the UDS assay. To conclude, the dual-labeling UDS assay offers advantages of reduced subjective bias, increasing sensitivity and reproducibility. The assay is suitable for testing compounds in higher capacity format with much less compound needed.

A novel P2Y(12) adenosine diphosphate receptor antagonist that inhibits platelet aggregation and thrombus formation in rat and dog models.

Irreversible platelet inhibitors, such as aspirin and clopidogrel, have limited anti-thrombotic efficacy in the clinic due to their bleeding risk. We have developed an orally active reversible P2Y(12) receptor antagonist, BX 667. The aim of this study was to determine if the reversible antagonist BX 667 had a greater therapeutic index than the irreversible P2Y(12) receptor antagonist clopidogrel. Since BX 667 is rapidly converted to its active metabolite BX 048 in rats, we first injected BX 048 intravenously (iv) in a rat arterial venous (A-V) shunt model of thrombosis. BX 048 dose- and concentration-dependently attenuated thrombosis. When administered orally, BX 667 and clopidogrel had similar efficacy, but BX 667 caused less bleeding than clopidogrel. In a rat model of a platelet-rich thrombus induced by vessel injury with FeCl(2), both BX 667 and clopidogrel exhibited higher levels of thrombus inhibition after oral administration compared to their potency in the A-V shunt model. Again, BX 667 caused less bleeding than clopidogrel. In a dog cyclic flow model, iv injection of either BX 667 or clopidogrel dose-dependently reduced thrombus formation with lower bleeding for BX 667 than clopidogrel. Inhibition of thrombosis was highly correlated with inhibition of ADP-induced platelet aggregation in these animal models. In dogs pre-treated with aspirin, BX 667 maintained its wider therapeutic index, measured by inhibition of platelet aggregation over bleeding, compared to the aspirin-clopidogrel combination. These data demonstrate that the reversible P2Y(12) receptor antagonist, BX 667, has a wider therapeutic index than clopidogrel in experimental models of thrombosis.

Cell-based assays for profiling activity and safety properties of cancer drugs.

INTRODUCTION: Our goal was to establish versatile and high capacity assays to characterize activity and toxicity of chemotherapeutics. The major anti-cancer activity indicators of these agents included inhibition of proliferation and induction of apoptosis to cancer cells. In addition, cytotoxicity and myelosuppressive activity to normal cells were parameters to evaluate toxicity of these drugs. METHODS: Using a panel of cell-based assay systems, we investigated activity and toxicity properties of selected cancer drugs. Drug effects on a number of normal and cancer cell types from human origin were evaluated. RESULTS: Topoisomerase inhibitors (camptothecin, doxorubicin and etoposide) and microtubule inhibitors (colchicine and paclitaxel) showed anti-proliferation activity and induced apoptosis in MDA-231 cancer cells. Except for doxorubicin, these drugs had relatively low toxicity to normal cells because the dosage required for cytotoxicity EC(50) was >200-folds higher than the dosage for anti-proliferation EC(50) in cancer cells (MDA-231, HL60). However, these drugs were potent inducers of myelotoxicity in human bone marrow progenitor cells. In comparison, the DNA alkylating agents (cisplatin and carboplatin) were less potent proliferation inhibitors (EC(50)>10 microM) in MDA-231 cells and they were also less myelosuppressive. DISCUSSION: Using marketed drugs as examples, our study established a multiple assay platform for profiling in vitro properties of cancer drugs. In drug discovery, such a platform will help to expedite lead selection at early stage.

Human primary renal cells as a model for toxicity assessment of chemo-therapeutic drugs.

Chemo-therapeutic drugs act on cancerous and normal cells non-selectively and often cause organ impairments during treatment. Improving safety or reducing toxicity becomes an important challenge for developing better anticancer drugs. In the present study, effects of selected anticancer drugs (camptothecin, doxorubicin, colchicine, paclitaxel, cisplatin, and carboplatin) on cell viability and proliferation was investigated. The anti-proliferative activity of each drug on cancer cells (human hepatoma HepG2) and human primary renal proximal tubule cells (hRPTECs and LLC-PK1) was determined with the [(3)H]thymidine incorporation assay. Results indicated all six drugs blocked cell proliferation in cancer and normal cells. When the anti-proliferation potency was ranked in hRPTECs based on EC50 values, camptothecin is the most potent, followed by doxorubicin, paclitaxel, colchicine, cisplatin and carboplatin. Cytotoxicity of drugs to hRPTECs was assessed with the ATP bioluminescence assay. Doxorubicin and cisplatin were known to induce nephrotoxicity in vivo and they were indeed cytotoxic to hRPTECs in our study with EC50 values at 11.2 and 39.6 microM. All other drugs are not cytotoxic in the concentrations tested. These drugs typically displayed separation of EC50s between potency (anti-proliferation) and cytotoxicity. The dose separation provides a concentration range for each drug to act on cell proliferation without induction of significant cytotoxicity. Our results suggest that hRPTEC system can serve as an in vitro model for assessing potential nephrotoxicity of chemo-therapeutic drugs.

Human in vitro pharmacodynamic profile of the selective Factor Xa inhibitor ZK-807834 (CI-1031).

ZK-807834 (also known as CI-1031) is a small molecule that potently and selectively inhibits Factor Xa. Studies in animals have shown that ZK-807834 attenuates thrombosis and thrombus progression after fibrinolysis and exhibits an increased antithrombotic-to-bleeding risk ratio compared with conventional agents. The present study describes the human in vitro anticoagulant and pharmacodynamic profile ZK-807834. Consistent with its selective inhibition of Factor Xa, ZK-807834 in the range of 0.3-0.5 microM prolonged prothrombin time (PT) and activated partial thromboplastin time (aPPT) twofold without affecting thrombin time (TT). Intersubject variability of in vitro anticoagulant activity was nominal and gender-independent. ZK-807834 inhibited Factor Xa in clot-bound prothrombinase with an average IC50 of 10+/-7 (S.D.) nM. ZK-807834 exhibited no direct effect on ADP- or collagen-induced platelet aggregation. Based on the potency and specificity, ZK-807834 may represent an important advance in the development of selective and safe antithrombotics.