Phase I study of AG 331, a novel thymidylate synthase inhibitor, in patients with refractory solid tumors.

PURPOSE: This was a phase I study of AG 331 to determine systemic tolerance and pharmacokinetics following single and multiple escalating intravenous doses. METHODS: The study was an open-label phase I trial that was divided into two components. In phase IA (single dose), six dose levels from 12.5 to 225 mg/m2 were administered to 18 patients (3 at each dose level) and serial blood samples were collected for 72 h. Upon achieving satisfactory pharmacologic parameters, the multiple dosing component (phase IB) was initiated. Six dose levels from 50 to 800 mg/m2 per day were administered for 5 consecutive days to 18 patients. Pre- and postdose blood samples were obtained on days 1-4 and serial blood samples were collected over 24 h following dose 5. Nonhematologic and hepatic toxicities were assessed, serum AG 331 concentrations were measured and pharmacokinetic parameters determined. RESULTS: Other than fatigue, no severe toxicities were encountered in phase IA. Liver toxicity was manifested by elevations in transaminase first noted at multiple doses of 200 mg/m2 per day for 5 days. Fever and malaise but no myelosuppression were noted. The mean terminal t1/2 following single doses was significantly shorter than the t1/2 following multiple dosing (6.8 vs 9.9 h) and clearance was significantly faster following single doses than following multiple dosing (81.7 vs 30.4 l/h), but no significant difference in Vd was noted. CONCLUSIONS: The dose-related toxicity profile precludes further clinical development at this time. The pharmacokinetics of AG 331 following single and multiple doses showed significant differences.

Characterization of the selectivity and mechanism of human cytochrome P450 inhibition by the human immunodeficiency virus-protease inhibitor nelfinavir mesylate.

In vitro studies with human liver microsomes and P450 probe substrates were performed to characterize selectivity and mechanism of cytochrome P450 inhibition by nelfinavir mesylate. At therapeutic concentrations (steady-state plasma concentrations approximately 4 microM), nelfinavir was found to be a competitive inhibitor of only testosterone 6beta-hydroxylase (CYP3A4) with a Ki concentration of 4. 8 microM. At supratherapeutic concentrations, nelfinavir competitively inhibited dextromethorphan O-demethylase (CYP2D6), S-mephenytoin 4-hydroxylase (CYP2C19), and phenacetin O-deethylase (CYP1A2) with Ki concentrations of 68, 126, and 190 microM, respectively. Nelfinavir did not appreciably inhibit tolbutamide 4-hydroxylase (CYP2C9), paclitaxel 6alpha-hydroxylase (CYP2C8), or chlorzoxaxone 6beta-hydroxylase (CYP2E1) activities. The inhibitory potency of nelfinavir toward CYP3A4 suggested the possibility of in vivo inhibition of this isoform, whereas in vivo inhibition of other P450s was considered unlikely. In a one-sequence crossover study in 12 healthy volunteers, nelfinavir inhibited the elimination of the CYP3A substrate terfenadine and the carboxylate metabolite of terfenadine. The 24-hr urinary recoveries of 6beta-hydroxycortisol were reduced by an average of 27% during nelfinavir treatment, consistent with CYP3A inhibition by nelfinavir. Inhibition of CYP3A4 by nelfinavir in vitro was NADPH-dependent requiring the catalytic formation of a metabolite or a metabolic intermediate. The catechol metabolite of nelfinavir (M3) was considered unlikely to be responsible for inhibition as the addition of catechol O-methyl transferase, S-adenosyl methionine, and ascorbic acid to the preincubation mixture did not protect against the loss of testosterone 6beta-hydroxylase activity. Also, the addition of M3 to human liver microsomes did not inhibit CYP3A4. Although incubations with nelfinavir showed a time- and concentration-dependent loss of CYP3A4 activity, the partial or complete recovery of enzyme activity upon dialysis indicated that inhibition was reversible. Microsomal incubations with nelfinavir and NADPH did not result in a loss of spectral P450 content compared with the NADPH control. Glutathione, N-acetylcysteine, and catalase did not attenuate CYP3A4 inhibition by nelfinavir. Collectively, these results suggest that the probable mechanism for CYP3A4 inhibition by nelfinavir is a transient metabolic intermediate or stable metabolite that coordinates tightly but reversibly to the heme moiety of the P450.

Viracept (nelfinavir mesylate, AG1343): a potent, orally bioavailable inhibitor of HIV-1 protease.

Using a combination of iterative structure-based design and an analysis of oral pharmacokinetics and antiviral activity, AG1343 (Viracept, nelfinavir mesylate), a nonpeptidic inhibitor of HIV-1 protease, was identified. AG1343 is a potent enzyme inhibitor (Ki = 2 nM) and antiviral agent (HIV-1 ED50 = 14 nM). An X-ray cocrystal structure of the enzyme-AG1343 complex reveals how the novel thiophenyl ether and phenol-amide substituents of the inhibitor interact with the S1 and S2 subsites of HIV-1 protease, respectively. In vivo studies indicate that AG1343 is well absorbed orally in a variety of species and possesses favorable pharmacokinetic properties in humans. AG1343 (Viracept) has recently been approved for marketing for the treatment of AIDS.

High-performance liquid chromatographic method for the determination of nelfinavir, a novel HIV-1 protease inhibitor, in human plasma.

Nelfinavir mesylate, a potent and orally bioavailable inhibitor of HIV-1 protease (Ki=2 nM), has undergone Phase III clinical evaluation in a large population of HIV-positive patients. A high-performance liquid chromatography analytical method was developed to determine the pharmacokinetic parameters of the free base, nelfinavir, in these human subjects. The method involved the extraction of nelfinavir and an internal standard, 6,7-dimethyl-2,3-di-(2-pyridyl)quinoxaline, from 250 microl of human plasma with a mixture of ethyl acetate-acetonitrile (90:10, v/v). The analysis was via ultraviolet detection at 220 nm using a reversed-phase C18 analytical column and a mobile phase consisting of 25 mM monobasic sodium phosphate buffer (adjusted to pH 3.4 with phosphoric acid)-acetonitrile (58:42, v/v) that resolved the drug and internal standard peaks from non-specific substances in human plasma. The method was validated under Good Laboratory Practice (GLP) conditions for specificity, inter- and intra-assay precision and accuracy, absolute recovery and stability. The mean recovery ranged from 92.4 to 83.0% for nelfinavir and was 95.7% for the internal standard. The method was linear over a concentration range of 0.0300 microg/ml to 10 microg/ml, with a minimum quantifiable level of 0.0500 microg/ml for nelfinavir.

Preclinical pharmacokinetics and distribution to tissue of AG1343, an inhibitor of human immunodeficiency virus type 1 protease.

AG1343, a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) protease (Ki = 2 nM), was designed by protein structure-based drug design techniques. AG1343 has potent antiviral activity (95% effective dose = 0.04 microgram/ml) against a number of HIV-1 strains in acute and chronic models of infection. As part of its preclinical development, the oral bioavailability of AG1343 in rats, dogs, monkeys, and marmosets was determined and its tissue distribution in rats was evaluated. There were no major interspecies differences in AG1343 pharmacokinetics. Following intravenous administration, the elimination half-life of AG1343 ranged from 1 to 1.4 hr. The total volume of distribution (2 to 7 liters/kg) exceeded the volume of total body water, indicating extensive tissue distribution. Systemic clearance of AG1343 (1 to 4 liters/kg) in the different species corresponded to hepatic blood flow, suggesting possible hepatic involvement in the elimination of AG1343. Following oral administration, peak levels in plasma ranged from 0.34 microgram/ml after treatment with 10 mg/kg of body weight in the dog to 1.7 micrograms/ml after dosing with 50 mg/kg in the rat. Because of the slow absorption of AG1343, plasma concentrations of AG1343 exceeding that required for 95% inhibition of HIV-1 replication were maintained for up to 7 h after a single oral dose in all species evaluated. Average oral bioavailability of AG1343 ranged from 17% in the marmoset to 47% in the dog. Studies of distribution to tissue in the rat after oral administration of 14C-AG1343 established extensive distribution with concentrations in most tissues exceeding that found in plasma. Of particular significance were high levels of AG1343 equivalent in mesenteric lymph nodes (32.05 micrograms/g) and spleen tissue (9.33 micrograms/g). The major excretory route for AG1343 was via feces, with 100% of the dose recovered by 48 h. Results from these studies demonstrate that AG1343 is orally bioavailable and that levels in plasma in the therapeutic range are achievable and are maintained for prolonged periods in the animal models tested. On the basis of these and other findings, AG1343 was developed for further testing in human subjects.

AG337, a novel lipophilic thymidylate synthase inhibitor: in vitro and in vivo preclinical studies.

3,4-Dihydro-2-amino-6 methyl-4-oxo-5-(4-pyridylthio)-quinazoline dihydrochloride (AG337) is a water-soluble, lipophilic inhibitor of thymidylate synthase (TS) designed using X-ray structure - based methodologies to interact at the folate cofactor binding site of the enzyme. The aim of the design program was to identify TS inhibitors with different pharmacological characteristics from classical folate analogs and, most notably, to develop non-glutamate-containing molecules which would not require facilitated transport for uptake and would not undergo intracellular polyglutamylation. One molecule which resulted from this program, AG337, inhibits purified recombinant human TS with a Ki of 11 nM, and displays non-competitive inhibition kinetics. It was further shown to inhibit cell growth in a panel of cell lines of murine and human origin, displaying an IC50 of between 0.39 microM 6.6 microM. TS was suggested as the locus of action of AG337 by the ability of thymidine to antagonize cell growth inhibition and the direct demonstration of TS inhibition in whole cells using a tritium release assay. The demonstration, by flow cytometry, that AG337-treated L1210 cells were arrested in the S phase of the cell cycle was also consistent with a blockage of TS, as was the pattern of ribonucleotide and deoxyribonucleotide pool modulation in AG337-treated cells, which showed significant reduction in TTP levels. The effects of AG337 were quickly reversed on removal of the drug, suggesting, as would be expected for a lipophilic agent, that there is rapid influx and efflux from cells and no intracellular metabolism to derivatives with enhanced retention. In vivo, AG337 was highly active against the thymidine kinase-deficient murine L5178Y/TK-lymphoma implanted either i.p. or i.m. following i.p. or oral delivery. Prolonged dosing periods of 5 or 10 days were required for activity, and efficacy was improved with twice-daily dose administration. Dose levels of 25 mg/kg delivered i.p. twice daily for 10 days, 50 mg/kg once daily for 10 days, or 100 mg/kg once daily for 5 days elicited 100% cures against the i.p. tumor. Doses required for activity against the i.m. tumor were higher (100 mg/kg i.p. twice daily for 5 or 10 days) but demonstrated the ability of AG337 to penetrate solid tissue barriers. Oral delivery required doses of > or = 150 mg/kg twice daily for periods of 5-10 days to produce 100% cure rates against both i.m. and i.p. implanted tumors. These results were consistent with the pharmacokinetics parameters determined in rats, for which oral bioavailability of 30-50% was determined, together with a relatively short elimination half life of 2h. Clinical studies with AG337 are currently in progress.

High-performance liquid chromatographic method for the determination of a novel thymidylate synthase inhibitor, AG 331, in human serum.

AG 331 is a novel thymidylate synthase inhibitor currently in Phase I clinical trial. To determine the pharmacokinetic parameters of AG 3331 in human subjects, a suitable analytical method was developed using high-performance liquid chromatography. Serum and urine samples were prepared using both solid-phase extraction and solvent extraction. Either 4,4'-diaminodiphenyl sulfone or benz[cd]indole-2(1H)-one were used as internal standards for the method. A reversed-phase C18 analytical column completely resolved the drug and internal standard peaks from non-specific substances present in biological matrix. The method was validated for precision, accuracy, and reproducibility in serum and was linear over a concentration range of 50-2000 ng/ml, with a limit of detection of 20.0 ng/ml and a quantifiable limit of 50 ng/ml.

Evaluation of hepatic metabolism of salicylic acid in perfused rat liver.

Hepatic metabolism of salicylic acid was studied using a single-pass liver perfusion technique. Livers obtained from male, Sprague-Dawley rats (200-300 g) were perfused with Krebs-Henseleit bicarbonate buffer containing the drug (50-100 micrograms/mL) and glycine (0.5 or 5 mM) or glucose (5.5 mM) or bovine serum albumin (0.33%). Effluent samples over a 30-50 min interval were analyzed for salicylic acid and its metabolites (gentisic acid, salicyluric acid, and salicyl acyl and salicyl phenolic glucuronides). Effluent concentrations of salicylic acid did not differ from those in the perfusate, indicating that the liver is not a site for the metabolism of this drug. Hepatic uptake studies are needed to confirm this finding.

A stability-indicating assay and the preformulation characteristics of the radiosensitizer, 1,2,4-benzotriazin-3-amine 1,4-dioxide.

A stability-indicating LC assay was developed for the analysis of 1,2,4-benzotriazin-3-amine 1,4-dioxide and applied to the preformulation characterization of the drug. The dissociation constants of the drug were determined using UV-vis spectrophotometry. The LC method was used to determine the aqueous stability of the drug under a variety of accelerated conditions, its solubility in a variety of pharmaceutical solvents and its octan-1-ol-water partition coefficient. The preformulation data were used to develop three prototype aqueous formulations of the drug at a concentration of 0.5 mg ml-1 in 5% Dextrose Injection USP, phosphate buffer (pH 7.4) and phosphate buffered mannitol. The 3-month stability of those formulations at room temperature was demonstrated.

Degradation of dacarbazine in aqueous solution.

The effects of initial concentration (0.05-5.0 mg ml-1, 2.5 x 10(-4)-0.025 M) (pH 1-13), buffer concentration (0.01-0.075 M), light, antioxidants and co-solvents on the degradation of dacarbazine in aqueous solution were investigated at 37 degrees C. Liquid chromatography was used to monitor the degradation of dacarbazine as well as the appearance of degradation products. The kinetics of hydrolysis of dacarbazine in the dark were pseudo first-order and independent of the initial concentration of the drug. The degradation of dacarbazine was accelerated by light and at low concentration proceeded by pseudo zero-order kinetics. The pH-rate profiles showed that both the photolytic and the hydrolytic reactions were dependent on the ionization state of the molecule. The main degradation product of both hydrolysis and photolysis was detected by liquid chromatography and confirmed by mass spectrometry to be 2-azahypoxanthine.