The discovery of 4-(3-pentylamino)-2,7-dimethyl-8-(2-methyl-4-methoxyphenyl)-pyrazolo-[1 ,5-a]-pyrimidine: a corticotropin-releasing factor (hCRF1) antagonist.

Structure activity relationship studies led to the discovery of 4-(3-pentylamino)-2,7-dimethyl-8-(2-methyl-4-methoxyphenyl)-pyrazo lo-[1,5-a]-pyrimidine 11-31 (DMP904), whose pharmacological profile strongly supports the hypothesis that hCRF1 antagonists may be potent anxiolytic drugs. Compound 11-31 (hCRF1 Ki = 1.0+/-0.2 nM (n = 8)) was a potent antagonist of hCRF1-coupled adenylate cyclase activity in HEK293 cells (IC50= 10.0+/-0.01 nM versus 10 nM r/hCRF, n = 8); alpha-helical CRF(9-41) had weaker potency (IC50 = 286+/-63 nM, n = 3). Analogue 11-31 had good oral activity in the rat situational anxiety test; the minimum effective dose for 11-31 was 0.3 mg/kg (po). Maximal efficacy (approximately 57% reduction in latency time in the dark compartment) was observed at this dose. Chlordiazepoxide caused a 72% reduction in latency at 20 mg/kg (po). The literature compound 1 (CP154526-1, 30 mg/kg (po)) was inactive in this test. Compound 11-31 did not inhibit open-field locomotor activity at 10, 30, and 100 mg/kg (po) in rats. In beagle dogs, this compound (5 mg/kg, iv, po) afforded good plasma levels. The key iv pharmacokinetic parameters were t1/2, CL and Vd,ss values equal to 46.4+/-7.6 h. 0.49+/-0.08 L/kg/h and 23.0+/-4.2 L/kg, respectively. After oral dosing, the mean Cmax, Tmax t1/2 and bioavailability values were equal to 1260+/-290 nM, 0.75+/-0.25 h. 45.1+/-10.2 h and 33.1%, respectively. The overall rat behavioral profile of this compound suggests that it may be an anxiolytic drug with a low motor side effect liability.

4-(1,3-Dimethoxyprop-2-ylamino)-2,7-dimethyl-8-(2, 4-dichlorophenyl)pyrazolo[1,5-a]-1,3,5-triazine: a potent, orally bioavailable CRF(1) receptor antagonist.

Structure-activity studies in the pyrazolo[1,5-a]-1,3,5-triazine series led to the discovery that compound 11i (DMP696) is a potent hCRF(1) receptor antagonist (K(i) = 1.7 nM vs 7.5 nM for alpha-hel-CRF(9-41), hCRF(1) adenylate cyclase IC(50) = 82 nM vs 286 nM for alpha-hel-CRF(9-41)). Compound 11i has excellent oral pharmacokinetic profiles in rats and dogs (37% and 50% oral bioavailabilities, respectively). This compound displays good activity in the rat situational anxiety model (MED = 3 mg/kg (po)), whereas a literature standard 1 (CP154526-1) was inactive (MED > 30 mg/kg (po)). Analogue 11i reduced stereotypical mouth movements in rhesus monkeys by 50% at 21 mg/kg (po) using the human intruder paradigm. Overall, the profile of pyrazolotriazine 11i indicates that hCRF(1) receptor antagonists may be anxiolytic agents, which have reduced motor side effect profiles.

Protein binding of brequinar in the plasma of healthy donors and cancer patients and analysis of the relationship between protein binding and pharmacokinetics in cancer patients.

The protein binding of weakly acidic and basic drugs has been shown to be altered in cancer patients. Brequinar is a weakly acidic, low-clearance, and highly protein-bound (> 98% bound) antitumor agent. The pharmacokinetic parameters of brequinar are subject to large interpatient variability. This large interpatient variability may be related to brequinar's plasma protein-binding capacity (assuming no change in the intrinsic clearance of the unbound drug). The objectives of this study, therefore, were (a) to characterize brequinar's protein binding in the plasma of healthy donors and cancer patients and (b) to examine the relationships between brequinar's plasma protein binding and its pharmacokinetics in patients. Brequinar protein binding was determined in human serum albumin (HSA) solution, drug-free donor plasma, and brequinar-free, predose plasma samples obtained from a phase I cancer trial. Pharmacokinetic results from this study were used to examine relationships between plasma protein binding and drug disposition. In HSA solution and healthy donor plasma, brequinar's protein binding as determined using spiked samples was concentration-dependent. The unbound brequinar fraction increased by a factor of 3 (from 0.3% to 0.9% free) in 4% HSA solution and by a factor of 4 (from 0.4% to 1.6% free) in donor plasma as the brequinar concentrations increased from 0.1 to 2.3 mM in the HSA solution and from 0.076 to 1.5 mM in the donor plasma. Analysis of brequinar binding characteristics using the binding ratio and Rosenthal binding plots showed that albumin was the primary protein for brequinar binding in human plasma. The addition of various concentrations of alpha 1-acid glycoprotein to 4% HSA solution did not affect the protein binding of brequinar to HSA. The protein binding determined in the plasma of cancer patients was not quantitatively different, except for variability, from that observed in the plasma of healthy donors. Examination of relationships between the unbound brequinar fraction and pharmacokinetics suggested that plasma protein binding was not a major determinant of brequinar disposition in cancer patients.

Phase I and pharmacokinetic study of brequinar sodium (NSC 368390).

Brequinar sodium is a quinoline carboxylic acid derivative that has shown antitumor activity in a number of in vivo murine and human tumor xenograft models. Its mechanism of action is blockade of de novo pyrimidine biosynthesis by inhibition of dihydroorotic acid dehydrogenase. In vitro and in vivo studies demonstrate the superiority of prolonged drug exposure in achieving tumor growth inhibition. This phase I study evaluated the administration of brequinar sodium by short, daily i.v. infusion for 5 days repeated every 4 weeks. Fifty-four subjects were enrolled in the study and received drug in doses ranging from 36-300 mg/m2. The dose-limiting toxicities were mucositis and diffuse skin rash. Other toxicities included myelosuppression, nausea, vomiting, malaise, and burning at the infusion site. The maximum tolerated dose on the "daily times 5" schedule was 300 mg/m2. The recommended phase II dose is 250 mg/m2. Pharmacokinetic analysis of the day 1 drug clearance curves in 51 subjects showed slight nonlinearity in the relationship between dose and area under the clearance curve (AUC). The dose versus AUC relationship was well described using a Michaelis-Menten model of brequinar elimination kinetics with Vmax = 45 (micrograms/ml)/h and Km = 123 micrograms. Analysis of the day 5 drug clearance curves revealed a diminution in Vmax to 30 (micrograms/ml)/h. As a consequence of the reduction in Vmax brequinar plasma concentrations on day 5 were higher than predicted from day 1 drug kinetics. Pharmacodynamic analysis of the day 1 kinetic parameters and the toxicities occurring during the first cycle of drug therapy revealed significant correlations between mucositis and dose, AUC, and peak brequinar concentration; between leukopenia and AUC and peak drug concentration; and between thrombocytopenia and beta elimination rate.

Determination of unlabeled and 13C6-labeled moricizine in human plasma using thermospray liquid chromatography-mass spectrometry.

Moricizine hydrochloride is an orally effective antiarrhythmic agent currently marketed in the Soviet Union and undergoing clinical testing in the United States. To facilitate the simultaneous analysis of unlabeled and 13C6-labeled moricizine in human plasma, a specific and sensitive method employing liquid-liquid extraction followed by thermospray liquid chromatography-mass spectrometry (LC-MS) was developed. Plasma samples, after addition of [2H11]moricizine as an internal standard, were extracted into methylene chloride under alkaline conditions. Extracts were evaporated, reconstituted with mobile phase, and chromatographed on an ODS column. The LC mobile phase consisted of methanol-0.1 M ammonium acetate containing 0.2% triethylamine (65:35) and it was used at a flow-rate of 1.5 ml/min. Under these conditions, moricizine and [13C6]moricizine coeluted at 1.2 min, while [2H11]moricizine eluted slightly earlier. The MS system consisted of a Finnigan 4600 TSQ and a Vestec thermospray interface. Selected ions at m/z 428, 434, and 439 were scanned at 0.2 s per ion. Over a plasma concentration range of 10-800 ng/ml, intra-day precision (n = 3) ranged from 1.8 to 13.3% and intra-day accuracy ranged from 1.9 to 15.8%. This method was successfully used to assay human plasma samples from a pilot moricizine bioavailability study in which tablets and solution containing moricizine hydrochloride and [13C6]moricizine, respectively, were simultaneously administered.

Phase I clinical and pharmacokinetic trial of Brequinar sodium (DuP 785; NSC 368390).

Brequinar sodium is a 4-quinolinecarboxylic acid analogue that inhibits dihydroorotate dehydrogenase and subsequent de novo pyrimidine biosynthesis. It has shown dose-dependent antineoplastic activity against several mouse and human tumor models. This trial evaluated Brequinar given as a single daily i.v. bolus over a 5-day period repeated every 28 days. One hundred seven courses of treatment at dosages ranging from 36 to 300 mg/m2/day x 5 were administered to 45 patients (31 male and 14 female) with refractory solid tumors; median age was 58 years (range 30-74); median Southwest Oncology Group performance status was 1 (range, 0-3). Thirty patients had prior cytotoxic chemotherapy. Dose-limiting toxicities were thrombocytopenia and a severe desquamative maculopapular dermatitis. Two of 5 good risk patients at 300 mg/m2 and 3 of 6 poor risk patients at 170 mg/m2 developed a platelet count less than 25 x 10(3)/microliters. Two of 5 good risk patients at 300 mg/m2 and 1 of 6 poor risk patients at 170 mg/m2 developed a severe desquamative dermatitis. Moderate to severe mucositis was usually associated with the thrombocytopenia and/or the dermatitis. Nonhematological drug-related toxicities included nausea and vomiting, malaise, anorexia, diarrhea, phlebitis, reversible transaminase elevation, and mucositis. Other hematological toxicities were anemia, granulocytopenia, and leukopenia. There were no drug-related deaths. There were no objective tumor responses. Plasma and urine levels of Brequinar were quantified by high pressure liquid chromatography in 28 patients. Plasma levels and areas under the curve increased proportionally with increased dose. Brequinar had a harmonic mean terminal t1/2 of 8.1 +/- 3.6 h with a model-independent determined apparent volume of distribution at steady state of 9.0 +/- 2.9 liters/m2 and a total body clearance of 19.2 +/- 7.7 ml/min/m2. Renal excretion was a minor route of elimination for Brequinar. The maximally tolerated dose of Brequinar on a daily x 5 i.v. schedule was 250 mg/m2 for good risk patients. For the daily x 5 i.v. schedule, the recommended dose of Brequinar for phase II evaluation is 250 mg/m2 for good risk patients and 135 mg/m2 for poor risk patients.

Distribution of the novel anticancer drug candidate Brequinar sodium (DuP 785, NSC 368390) into normal and tumor tissues of nude mice bearing human colon carcinoma xenografts.

The distribution of the novel anticancer drug candidate Brequinar Sodium (DuP 785, NSC 368390) was studied in control mice and mice implanted subcutaneously with human colon carcinoma xenografts. Mice were given radiolabeled 14C-Brequinar Sodium intravenously. Brequinar concentrations in blood and various tissues were determined at 1, 6, and 24 h after drug administration. Within 1 h Brequinar distributed to the tumor and all other tissues studied. The tumor-to-blood drug concentration ratios ranged from 0.19 to 0.41. Radioactivity in the liver and small intestine at 1 h accounted for 17% and 13%, respectively, of the dose given. Elimination rates of Brequinar from all tissues were approximately equal to that from blood. Comparison of blood concentrations determined by both radioactivity and HPLC methods suggests that the intact drug is probably the only form in the blood.