Maria T. Stranieri

L-374,087, an efficacious, orally bioavailable, pyridinone acetamide thrombin inhibitor

Replacement of the amidinopiperidine P1 group of 3-benzylsulfonylamino-6-methyl-2-pyridinone acetamide thrombin inhibitor L-373,890 (2) with a mildly basic 5-linked 2-amino-6-methylpyridine results in an equipotent compound L-374,087 (5, Ki = 0.5 nM). Compound 5 is highly selective for thrombin over trypsin, is efficacious in the rat ferric chloride model of arterial thrombosis and is orally bioavailable in dogs and cynomolgus monkeys. The structural basis for the critical importance of both methyl groups in 5 was confirmed by X-ray crystallography.

Pharmacokinetic optimization of 3-amino-6-chloropyrazinone acetamide thrombin inhibitors. Implementation of P3 pyridine N-oxides to deliver an orally bioavailable series containing P1 N-benzylamides.

In this manuscript we demonstrate that a modification principally directed toward the improvement of the aqueous solubility (i.e., introduction a P3 pyridine N-oxide) of the previous lead compound afforded a new series of potent orally bioavailable P1 N-benzylamide thrombin inhibitors. An expedited investigation of the P1 SAR with respect to oral bioavailability, plasma half-life, and human liver microsome stability revealed 5 as the best candidate for advanced evaluation.

Enhancement of recombinant tissue plasminogen activator-induced reperfusion by recombinant tick anticoagulant peptide, a selective factor Xa inhibitor, in a canine model of femoral arterial thrombosis

Tick anticoagulant peptide (TAP) is a 60 amino acid protein originally isolated from the soft tick, Ornithodoros moubata, which exhibits potent anticoagulant properties due to its selective inhibition of blood coagulation factor Xa. We evaluated a recombinant version of TAP (rTAP) for its ability to accelerate recombinant tissue plasminogen activator (rt-PA) -mediated lysis of an occlusive thrombus and prevent acute reocclusion in a canine model of femoral artery thrombosis. An occlusive thrombus was formed by insertion of a thrombogenic copper coil into the femoral artery of anesthetized dogs. Blood flow velocity was monitored directly and continuously by Doppler flowmetry. 60 min after occlusion, dogs received an i.v. infusion of either saline or rTAP (0.5, 2.5 or 8.0 μg/kg/min), followed 45 min later by rt-PA (0.8 mg/kg, i.v. over 90 min; n=8/group). The saline and rTAP infusions were discontinued 1 h after stopping the rt-PA. All dogs achieved reperfusion, with a time to reperfusion in the saline-treated (vehicle) group (administered rt-PA alone) of 61 ±7 min. The time to reperfusion was slightly decreased in the 0.5ug/kg/min rTAP group (47±4min, p=NS). In the groups administered rTAP at 2.5 and 8.0 ug/kg/min, significant reductions in the time to reperfusion were observed (28±4 and 32±5mins, respectively, p<0.05). Following termination of the rt-PA, all vehicle dogs reoccluded in 37±11 min. The lowest dose of rTAP, 0.5 ug/kg/min, had no effect on either the reocclusion incidence or time (8/8 in 39±7min). In contrast, the two higher doses of rTAP maintained vessel patency in all dogs during the rTAP infusion period and dramatically delayed the time to reocclusion. However, 7/8 dogs eventually reoccluded in 117±12 and 140±9min for the groups receiving rTAP infusions of 2.5 and 8.0 ug/kg/min, respectively. Maximal elevations in activated partial thromboplastin time or template bleeding time associated with the rTAP administration were only 1.3- and 1.1-fold of baseline values, respectively. The dramatic effect of factor Xa inhibition on the efficacy of rt-PA-mediated reperfusion and acute reocclusion following rt-PA suggests that factor Xa inhibition may represent a potentially useful therapeutic adjunct to thrombolytic therapy.

Simple high-performance liquid chromatographic method to analyze serum creatinine has several advantages over the Jaffé picric acid reaction as demonstrated with a cimetidine dose response in rhesus monkeys.

A simple method for creatinine determination was developed using high-performance liquid chromatography (HPLC) to more accurately monitor serum creatinine levels in experimental animal models when compared to the Jaffé method. The new HPLC procedure will replace the traditional Jaffé method for rhesus monkey kidney function studies. We developed an isocratic method using a polymeric, hydrophilic, silica-based strong cation-exchange bed with a 5.0 mmol/l lithium acetate matrix, pH 4.9, which isolates creatinine with no detectable impurities as determined by three-dimensional ultraviolet-visible spectral analysis. Sample preparation includes deproteination with acetonitrile, evaporation, and resolubilization in mobile phase followed by quantitation with UV detection at 234 nm. Extraction efficiency across the measured range was 96 +/- 2%. From numerous extracted rhesus monkey creatinine curves (n=38) a slope of 251,100 +/- 756 (95% CI) and an intercept of 675.6 +/- 712.7 (95% CI) was calculated. Extraction efficiency and peak purity tests with human plasma were cross-compared with rhesus monkey serum producing equivalent results. An average of 120 samples can be run daily.

C6 modification of the pyridinone core of thrombin inhibitor L-374,087 as a means of enhancing its oral absorption.

1 (L-374,087) is a potent, selective, efficacious, and orally bioavailable thrombin inhibitor that contains a core 3-amino-2-pyridinone moiety. Replacement of the C6 pyridinone methyl group of 1 by a propyl group gave 5 (L-375,052), which retained all the excellent properties of 1, and also yielded higher plasma levels after oral dosing in dogs and rats.

Nonpeptide GPIIb/IIIa inhibitors. 10. Centrally constrained alpha-sulfonamides are potent inhibitors of platelet aggregation

Abstract Potency enhancing features of two series of fibrinogen receptor antagonists were combined to give analogs with improved potency and oral activity. Antagonists containing either alkyl or aryl sulfonamides and a central isoindolinone structural constraint demonstrate high affinity for both activated and unactivated platelet receptors.

Nonpeptide glycoprotein IIb/IIIa inhibitors. 13. design and synthesis of an orally active pyrazolopiperazinone nonpeptide fibrinogen receptor antagonist

Abstract The synthesis and antiplatelet activity of a series of pyrazolopiperazinone nonpeptide fibrinogen receptor antagonists is reported. The sulfonamide analog 6 (L-734, 115), significantly inhibited ex vivo platelet aggregation 24 h after oral administration at doses of 1.0 and 2.0 mg/kg to dogs and rhesus monkeys, respectively.

Non-peptide fibrinogen receptor antagonists. 3. design and discovery of a centrally constrained inhibitor

Abstract Analysis of platelet aggregation inhibition results and rotational isomer preferences has provided an understanding of inhibitory potency for m-phthalic acid analogs 7–7. Constraint of the N-terminal amide led to compound 9, which is potent, selective, and orally active.

Pharmacokinetics and Pharmacodynamics of L-703,014, a Potent Fibrinogen Receptor Antagonist, After Intravenous and Oral Administration in the Dog

The pharmacokinetics and pharmacodynamics of L-703,014, a fibrinogen receptor antagonist, have been examined in the dog. An analytical method which utilizes methanol precipitation of dog plasma proteins followed by HPLC with an automated column switching technique using the chemical analogue L-704,326 as internal standard was developed for the determination of L-703,014 in dog plasma. The compound was not metabolized in the dog and was eliminated in the kidneys and into bile. Of the administered dose, 68.9 ± 1.3% (i.v.) and 80.5 ± 11.9% (p.o.) were recovered in the feces; 20.3 ± 3% (i.v.) and 2.2 ± 0.2% (p.o.) were recovered in the urine by 72 hr. L-703,014 was 23 ± 3.4% bound in dog plasma protein and the mean ratio of plasma/whole blood was 1.22 ± 0.05. The mean terminal half-life was 118 ± 36 min, the mean steady-state volume of distribution was 0.61 ± 0.22 L/kg, and the mean plasma clearance was 8 ± 2 mL/min/kg. Ex vivo platelet aggregation measurements were made by inducing platelet aggregation with 10 µg/ mL collagen in the presence of 1 µM epinephrine as an agonist. The mean C50 was 44.4 ± 6.0 ng/mL, and the mean Hill coefficient was 1.5 ± 0.3. The mean bioavailability was 4.9 ± 1.4% in dogs administered 2.0 mg/kg (p.o.).

Nonpeptide GPIIB/IIIA inhibitors. 16. Thieno[2,3-b]thiophene α-sulfonamides are potent inhibitors of platelet aggregation

Abstract Centrally constrained thieno[2,3- b ]thiophene sulfonamides have provided a potent, selective, orally active series of platelet aggregation inhibitors. Compound 21 showed excellent activity in the dog after a single oral dose of 200 μg/kg.