Characterization of nerve growth factor-induced mechanical and thermal hypersensitivity in rats.

BACKGROUND: Injection of nerve growth factor (NGF) produces mechanical and thermal hypersensitivity in rodents and humans. Treatment with sequestering antibodies demonstrates the importance of NGF in various pain states, with efficacy seen in a number of animal pain models and in painful human conditions. However, these phenomena have not been evaluated in the context of using NGF-induced hypersensitivities as a model of pain. METHODS: NGF-induced behaviours were characterized using von Frey filament, pinprick and thermal endpoints and then pharmacologically evaluated with known reference agents. RESULTS: Intraplantar NGF injection produced a dose-dependent increase in thermal sensitivity that lasted through 24 h post-injection and an immediate long-lasting (2 week) increase in mechanical sensitivity at the injection site, with no effects detected at secondary sites. NGF-induced mechanical sensitivity was pharmacologically characterized at 4 h and 1 week post-NGF injection. The nonsteroidal anti-inflammatory drugs (NSAIDs), celecoxib and diclofenac, were minimally effective against both thermal and mechanical endpoints. Gabapenitn and duloxetine were only moderately effective against thermal and mechanical hypersensitivity. Morphine was effective against thermal and mechanical endpoints at every time point examined. Treatment with the transient receptor potential vanilloid 1 (TRPV1) antagonist A-784168 partially attenuated NGF-induced thermal and mechanical sensitivity at all time points examined. CONCLUSIONS: The results reported here suggest that effects of NGF on thermal and mechanical sensitivity in rats are similar to those reported in human and are partially driven by TRPV1. The rat NGF model may serve as a potential translational model for exploring the effects of novel analgesic agents.

In vitro and in vivo characterization of A-796260: a selective cannabinoid CB2 receptor agonist exhibiting analgesic activity in rodent pain models.

BACKGROUND AND PURPOSE: Selective cannabinoid CB2 receptor agonists have demonstrated analgesic activity across multiple preclinical pain models. AM1241 is an indole derivative that exhibits high affinity and selectivity for the CB2 binding site and broad spectrum analgesic activity in rodent models, but is not an antagonist of CB2 in vitro functional assays. Additionally, its analgesic effects are mu-opioid receptor-dependent. Herein, we describe the in vitro and in vivo pharmacological properties of A-796260, a novel CB2 agonist. EXPERIMENTAL APPROACH: A-796260 was characterized in radioligand binding and in vitro functional assays at rat and human CB1 and CB2 receptors. The behavioural profile of A-796260 was assessed in models of inflammatory, post-operative, neuropathic, and osteoarthritic (OA) pain, as well as its effects on motor activity. The receptor specificity was confirmed using selective CB1, CB2 and mu-opioid receptor antagonists. KEY RESULTS: A-796260 exhibited high affinity and agonist efficacy at human and rat CB2 receptors, and was selective for the CB2 vs CB1 subtype. Efficacy in models of inflammatory, post-operative, neuropathic and OA pain was demonstrated, and these activities were selectively blocked by CB2, but not CB1 or mu-opioid receptor-selective antagonists. Efficacy was achieved at doses that had no significant effects on motor activity. CONCLUSIONS AND IMPLICATIONS: These results further confirm the therapeutic potential of CB2 receptor-selective agonists for the treatment of pain. In addition, they demonstrate that A-796260 may be a useful new pharmacological compound for further studying CB2 receptor pharmacology and for evaluating its role in the modulation of pain.

Orbofiban: an orally active GPIIb/IIIa platelet receptor antagonist.

A key role has been established for platelet activation and thrombus formation in the pathogenesis of acute coronary syndromes, and restenosis after percutaneous interventions. Antiplatelet agents that have a wider spectrum of activity than aspirin, and clopidogrel would be expected to provide improved antithrombotic protection. Preclinical studies were used to predict clinical efficacy of orally active GPIIb/IIIa antagonists such as xemilofiban, sibrafiban, lefradafiban, and orbofiban. While clinical trials have shown potent and sustained platelet inhibition, outcomes of trials with these first generation GPIIb/IIIa compounds have been disappointing. The active moiety of orbofiban is a potent and specific inhibitor of fibrinogen binding to GPIIb/IIIa, leading to inhibition of platelet aggregation to a wide variety of agonists. Studies comparing inhibition of aggregation and bleeding suggest that chronic inhibition of platelet aggregation can be achieved without major bleeding side effects. Thrombus formation is prevented in canine models of thrombosis. Orbofiban is approximately 28% bioavailable with a t(1/2) of 18 hr. The high bioavailability, long half-life, and potential safety suggest orbofiban would be suitable for chronic oral administration. Clinical data demonstrate that orally administered orbofiban has the desired pharmacodynamic effect of inhibiting platelet aggregation but does not demonstrate clinical benefit when examined in large-scale trials.

Protective effect of oral xemilofiban in arterial thrombosis in dogs: increased activity in combination with aspirin.

BACKGROUND: Inhibition of platelet aggregation by preventing the binding of fibrinogen to glycoprotein (GP) IIb/IIIa on activated platelets results in antithrombotic activity. We report on the antithrombotic effect of xemilofiban (SC-54684A), an oral GP IIb/IIIa antagonist, administered alone or with aspirin (ASA) in an acute thrombosis model. METHODS AND RESULTS: Conscious dogs were treated with xemilofiban (1.25, 2.5, 5.0, or 6 mg/kg, n=6); low-dose (LD, 81 mg) ASA, n=7; high-dose (HD, 162 mg) ASA, n=6; xemilofibran 1.25 mg/kg plus LD ASA, n=6; xemilofibran 1.25 mg/kg plus HD ASA, n=6; or placebo, n=7. Dogs were anesthetized 60 minutes later, and the effects of the treatments were evaluated after electrolytic injury (250 microA for 180 minutes) in the left circumflex coronary artery. Bleeding time (BT) was assessed in a separate study. Incidence of thrombosis was reduced (P<0.05) by xemilofiban > or =2.5 mg/kg, HD ASA, or xemilofiban 1.25 mg/kg plus HD ASA compared with placebo. Xemilofiban > or =2.5 mg/kg or xemilofiban 1.25 mg/kg plus HD ASA significantly increased time to occlusion, inhibited ex vivo platelet aggregation to collagen >90%, and prevented or decreased (P<0.05) cyclic flow variations (CFVs) compared with placebo. BT was increased (P<0.05) with xemilofiban > or =2.5 mg/kg but not with xemilofiban 1.25 mg/kg plus HD ASA. CONCLUSIONS: Xemilofiban > or =2.5 mg/kg, HD ASA, or xemilofiban 1.25 mg/kg plus HD ASA significantly reduced the incidence of thrombosis. These doses of xemilofiban or xemilofiban 1.25 mg/kg plus HD ASA increased time to occlusion, inhibited ex vivo platelet aggregation by >90%, and prevented or reduced CFVs. Xemilofiban > or =2.5 mg/kg but not xemilofiban 1.25 mg/kg plus HD ASA significantly increased BT.

Assessment of platelet function assays.

Optical aggregometry, traditionally used to assess platelet function, is highly dependent on sample preparation and technical procedure; as a result, data from various laboratories can be quite variable. In a study designed to assess the sources of variation, it was determined that the total standard deviation ranged from 3.6% to 7.7%. The assay variation by one analyst on one aggregometer on a single day ranged from 1.7 to 4.6. Day-to-day variation contributed 42% to 63% of the total variation, between-operator variation contributed 1% to 33% of the total variation, and within [between repeat measurements for a given sample by a given operator on a single day] variation contributed 22% to 36% of the total variation. Because of the disadvantages associated with optical aggregometry, alternate platelet function assays were considered and their correspondence to optical aggregometry was evaluated: activated clotting time, whole blood aggregometry, platelet count ratio, Platelet Function Analyzer (PFA-100, Dade), and Rapid Platelet function Assay (Accumetrics). Of those assays evaluated, activated clotting time and PFA-100 are assays that measure aspects of coagulation and hemostasis, whereas whole blood aggregometry, platelet count ratio, and RPFA are more closely related to platelet function. Each assay has value in monitoring various aspects of the coagulation process. The best method for monitoring safety and efficacy of various inhibitors of platelet function will ultimately be determined by clinical trials.

The protective dose of the potent GPIIb/IIIa antagonist SC-54701A is reduced when used in combination with aspirin and heparin in a canine model of coronary artery thrombosis.

BACKGROUND: Fibrinogen receptor antagonists block the fibrinogen-platelet interaction at the GPIIb/IIIa receptors and inhibit thrombus formation. SC-54701 is the active metabolite of SC-54684A, an orally fibrinogen receptor antagonist. We compared the efficacy of SC-54701A (SCa, hydrochloride salt) with that of aspirin (ASA) or heparin and with combination therapy in a canine model of continuous current injury. METHODS AND RESULTS: Sixty-six dogs were used (6 per treatment). SCa (15-minute loading dose followed by [//] infusion [microgram/kg per minute]: (0.87//0.39=1 X SCa; 0.52//0.23=0.6 X SCa; and 0.425//0.20= 0.5 X SCa), ASA (2.8 mg/kg), heparin (200 U/kg plus 1000 U/h), or saline (0.1 mL/kg) was administered intravenously. Experimental time was 180 minutes of current. Time to occlusion was increased (P < .05) by SCa (T=incidence of thrombosis) (1 X SCa, >180 minutes [T=0]; 0.6 X SCa, 158 +/- 15 minutes [T=2]; 0.5 X SCa, 130 +/- 22 minutes [T=4]), heparin (114 +/- 16 minutes [T=5]), and ASA plus heparin (130 +/- 11 minutes [T=5]) relative to saline (58 +/- 7 minutes [T=6]). Time to occlusion for the SCa treatments was increased compared with ASA (64 +/- 7 minutes [T=6]). When 0.5 X SCa was administered with ASA plus heparin, time to occlusion was >180 minutes [T=0]. SCa provided complete protection at > or = 90% inhibition of ex vivo collagen-induced platelet aggregation. Cyclic flow variations were minimal with SCa or any treatment involving 0.5 X SCa and ASA. CONCLUSIONS: SCa has dose-dependent antithrombotic efficacy and inhibits ex vivo platelet aggregation. ASA, heparin, or saline was ineffective in this model. SCa (0.5X) plus ASA and heparin maximized the antithrombotic effect of this lower dose of SCa.

SC-54684A: an orally active inhibitor of platelet aggregation.

BACKGROUND: Intravenous therapy has been shown to be beneficial in the prevention of acute platelet-associated thrombotic events. However, orally active agents would be advantageous for chronic therapy. Fibrinogen receptor antagonists block the fibrinogen/platelet interaction and thus inhibit a step required for thrombus formation. To date, no orally active fibrinogen binding inhibitors have been characterized. SC-54684A, now in clinical trial, is the orally active prodrug of a potent and specific fibrinogen binding antagonist. METHODS AND RESULTS: We measured inhibition of 125I-fibrinogen binding to activated platelets and inhibition of aggregation in platelet-rich plasma to selected agonists and showed IC50s of 1.0 x 10(-8) and 3 to 7 x 10(-8) mol/L, respectively. Specificity of the active moiety was determined by studying its effect on the binding of (1) neutrophils to interleukin (IL)-1 beta-stimulated endothelial cells, (2) endothelial cells to fibronectin, and (3) vitronectin to isolated vitronectin and fibrinogen receptors. No effect was observed on the binding neutrophils to IL-stimulated endothelial cells or endothelial cell binding to fibronectin. There was a fivefold separation between binding to isolated receptors of vitronectin and fibrinogen. Collagen-induced aggregation was inhibited by 80%, and bleeding time was increased approximately 2.5-fold when the active moiety was infused to steady state at 0.2 micrograms/kg per minute in dogs. When the ester prodrug was given orally and the active moiety was given intravenously, the oral systemic activity was approximately 20%. Pharmacokinetic analysis after intravenous infusion of the prodrug or active moiety showed that the prodrug was rapidly converted to the active moiety; the active moiety had a t1/2 of 6.5 hours. When the prodrug was administered both orally and intravenously, the systemic availability of the active moiety was 62%. CONCLUSIONS: SC-54684A, an orally active antiplatelet drug now in clinical trial, is shown to be a potent, specific fibrinogen binding inhibitor that blocks platelet aggregation to a wide variety of known stimuli and has good bioavailability in animals.

Extended inhibition of platelet aggregation with the orally active platelet inhibitor SC-54684A.

BACKGROUND: Platelet aggregation is important in thrombotic events, and platelets play a major role in the etiology of several cardiovascular diseases. Platelet aggregation requires the binding of fibrinogen (fgn) to activated platelets. Synthetic peptides modeled after the RGD binding sequence on the fgn alpha-chain block the platelet glycoprotein (GP) IIb/IIIa receptor for fgn and effectively inhibit aggregation. SC-54684A (SCp, orally active prodrug of the active moiety SC-54701, SCa) is a mimetic of the RGD-containing peptide sequence that is recognized by the platelet GPIIb/IIIa receptor. SCa blocks the binding of fgn to the platelet and therefore prevents platelet aggregation in response to all agonists. METHODS AND RESULTS: SCp was administered orally at 1.25, 2.5, 5.0, and 7.5 mg/kg in a single-dose, dose-ranging study. Blood samples were taken periodically for 24 hours, and platelet-rich plasma was prepared and tested for inhibition of ex vivo collagen-induced platelet aggregation. The plasma concentration of active moiety was determined by bioassay. The time, inhibition, and concentration data were used to predict two doses that would result in minimum daily inhibition levels of 30% and 70% when administered twice daily (0.6 and 2.4 mg/kg, respectively). SCp was administered orally to conscious dogs twice daily for 14 days (after dose adjustment). Blood samples were obtained at daily peak and trough plasma levels (predicted from dose-ranging study). Inhibition of ex vivo collagen-induced platelet aggregation and concentration of active moiety in the plasma were determined. Average inhibition of platelet aggregation and plasma concentration of active moiety amounted to approximately 21% and 14 ng/mL at 1.5 mg/kg BID and 75% and 24 ng/mL at 2.4 ng/kg BID at daily minimum plasma levels (trough) in steady state. Platelet counts in the 2.4-mg/kg group declined from 3.2 x 10(5)/microL to 2.5 x 10(5)/microL in the first 9 days of dosing, with no further decline despite continued administration of compound. No changes were observed in the animals receiving 1.5 mg/kg. CONCLUSIONS: The results of the dose-ranging study show that oral administration of SCp results in dose-dependent inhibition of platelet aggregation. As shown in the 14-day administration, this dose-dependent inhibition can be maintained for an extended period while exhibiting no adverse effects. SCp is a leading candidate for development and is currently in clinical trials.

A new method for measurement of plasma concentration of orally active glycoprotein IIb/IIIa antagonists.

A bioassay for determining concentrations of antiplatelet compounds in plasma or aqueous solution has been developed. The method uses an aliquot of plasma from treated animals to inhibit collagen-induced platelet aggregation in pooled platelet-rich plasma (PRP) obtained from donor dogs. The concentration in plasma from treated animals was estimated using a standard curve of inhibition established using plasma from untreated animals which had been spiked with known amounts of compound. For independent validation, plasma concentrations of certain compounds were determined in identical dog plasma samples by both bioassay and HPLC. Results from the two methods were concordant. The bioassay provides an accurate and sensitive method for measuring antiplatelet activity without the need for extraction of plasma samples and may be used to measure activity in any solution which is compatible with PRP. This assay is routinely used to provide an estimate of absorption of prodrugs and systemic conversion to active compound after oral dosing. Some of the compounds of interest are ester-acid pairs with the inactive ester prodrug being cleaved to the active acid following administration. Compounds were administered orally (ester) or IV (acid) and blood samples were taken periodically for 24 hours. Plasma concentration of active moiety was determined for each time point and the area under the curve (AUC) of concentration vs. time was calculated. Comparing the AUCs for oral and IV routes of administration yielded the Oral Systemic Activity (OSA), a measure of active compound available after oral dosing.

In vitro and in vivo effects of a peptide mimetic (SC-47643) of RGD as an antiplatelet and antithrombotic agent.

Platelet aggregation requires binding of fibrinogen (fgn) to activated platelets and inhibition of this binding blocks platelet aggregation. Synthetic peptides modeled after the platelet binding sequence on fgn block the platelet glycoprotein IIb/IIIa receptor and effectively inhibit aggregation. SC-47643 (SC) is a mimetic of the RGD-containing peptide sequence that is recognized by the platelet IIb/IIIa receptor. SC inhibited fgn binding to activated platelets (IC50: 1.0 x 10(-5) M) and prevented platelet aggregation in response to a variety of platelet agonists in both washed human platelets and platelet rich plasma (IC50's ranging from 4 x 10(-6) to 1 x 10(-5) M, respectively). SC inhibited collagen induced thrombocytopenia in the rat (ED50 0.07 mg/kg and t1/2 36 min). In dogs ex vivo collagen induced platelet aggregation was inhibited 50% after a bolus injection of 1.7 mg/kg. After a steady state infusion (2 hr), the ED50 was 0.03 mg/kg/min, with no effects on blood pressure, heart rate or platelet count. These data demonstrate that SC, a peptide mimetic of the natural fgn binding sequence, is capable of blocking platelet-fgn interactions and platelet aggregation.

Antiplatelet and antithrombotic effects of platelet glycoprotein IIb/IIIa (GPIIb/IIIa) inhibition by arginine-glycine-aspartic acid-serine (RGDS) and arginine-glycine-aspartic acid (RGD) (O-me)Y (SC-46749).

Arginine-glycine-aspartic acid (RGD) is the minimal sequence in fibrinogen that leads to recognition and binding to the glycoprotein IIb/IIIa platelet receptor during aggregation. Analogs of tetrapeptides containing the RGD sequence have been previously shown to block fibrinogen binding to activated platelets in vitro. SC-46749 is an analog of arginine-glycine-aspartic acid-phenylalanine in which the phenylalanine is replaced by O-methyltyrosine. In this study the biological activities of SC-46749 were examined and its actions compared with the tetrapeptide arginine-glycine-aspartic acid-serine (RGDS), one of the natural sequences on the fibrinogen alpha chain that binds to platelets. In vitro, SC-46749 was more potent than RGDS in inhibiting fibrinogen binding (IC50: SC-46749, 27 microM; RGDS, 47 microM), in preventing ADP-induced aggregation in human platelet-rich plasma (IC50: SC-46749, 32 microM; RGDS, 95 microM) and in inhibiting thrombin-induced aggregation in washed human platelets (IC50: SC-46749, 23 microM; RGDS, 64 microM). In rats, SC-46749 prevented collagen-induced thrombocytopenia with an ED50 of 0.87 mg/kg whereas RGDS did not inhibit the response by 50% at doses up to 10 mg/kg. SC-46749 inhibited thrombus formation in an electrically damaged rat carotid artery in a dose-dependent fashion whereas the effects of RGDS were biphasic. RGDS appeared to delay thrombus formation at lower doses but had no effect at higher doses. When infused in dogs for 15 min, SC-46749 prevented ex vivo collagen-induced aggregation at 4 mg/kg/min. These data demonstrate that SC-46749 is a potent inhibitor of platelet aggregation and platelet-dependent thrombus formation.

Simultaneous determination of ventricular function and systemic hemodynamics in the conscious rat.

This study describes a method that utilizes a combination of the Millar transducer and Columbus thermistor in the conscious, spontaneously hypertensive rat and that permits simultaneous evaluation of the effects of cardiovascular agents on ventricular performance and systemic hemodynamics. Left ventricular end diastolic pressure (LVEDP, mmHg) and LV dp/dt (mmHg/sec) were measured with a Millar transducer inserted into the left ventricle of spontaneously hypertensive rats under ether anesthesia. Cardiac output (CO, ml/min) was measured by thermodilution (Columbus Instruments) with a thermocouple in the thoracic aorta via the femoral artery. Mean arterial pressure (MAP, mmHg) was measured via a catheter in the other femoral artery. All cardiovascular parameters measured were shown to attain a steady state within 3 hr after cessation of ether administration. Infusion of the beta adrenergic receptor agonist isoproterenol increased LV max dP/dt, heart rate (HR), and CO, and decreased arterial blood pressure and peripheral resistance. Administration of propranolol resulted in decreased HR, LV dP/dt and CO. The results show that this model was stable over an extended period of time and was responsive to standard inotropic agents. Thus, the combined use of the Millar transducer and the Columbus thermistor appears suitable for the assessment of acute pharmacological interventions on cardiovascular function in the conscious rat.