Stephanie Webber

Circulating Metabolites of the Human Immunodeficiency Virus Protease Inhibitor Nelfinavir in Humans: Structural Identification, Levels in Plasma, and Antiviral Activities

ABSTRACT Nelfinavir mesylate (Viracept, formally AG1343) is a potent and orally bioavailable human immunodeficiency virus (HIV) type 1 (HIV-1) protease inhibitor (Ki = 2 nM) and is being widely prescribed in combination with HIV reverse transcriptase inhibitors for the treatment of HIV infection. The current studies evaluated the presence of metabolites circulating in plasma following the oral administration of nelfinavir to healthy volunteers and HIV-infected patients, as well as the levels in plasma and antiviral activities of these metabolites. The results showed that the parent drug was the major circulating chemical species, followed in decreasing abundance by its hydroxy-t-butylamide metabolite (M8) and 3′-methoxy-4′-hydroxynelfinavir (M1). Antiviral assays with HIV-1 strain RF-infected CEM-SS cells showed that the 50% effective concentrations (EC50) of nelfinavir, M8, and M1 were 30, 34, and 151 nM, respectively, and that the corresponding EC50 against another HIV-1 strain, IIIB, in MT-2 cells were 60, 86, and 653 nM. Therefore, apparently similar in vitro antiviral activities were demonstrated for nelfinavir and M8, whereas an approximately 5- to 11-fold-lower level of antiviral activity was observed for M1. The active metabolite, M8, showed a degree of binding to human plasma proteins similar to that of nelfinavir (ca. 98%). Concentrations in plasma of nelfinavir and its metabolites in 10 HIV-positive patients receiving nelfinavir therapy (750 mg three times per day) were determined by a liquid chromatography tandem mass spectrometry assay. At steady state (day 28), the mean plasma nelfinavir concentrations ranged from 1.73 to 4.96 μM and the M8 concentrations ranged from 0.55 to 1.96 μM, whereas the M1 concentrations were low and ranged from 0.09 to 0.19 μM. In conclusion, the findings from the current studies suggest that, in humans, nelfinavir forms an active metabolite circulating at appreciable levels in plasma. The active metabolite M8 may account for some of the antiviral activity associated with nelfinavir in the treatment of HIV disease.

The purification of rat and sheep liver dihydropteridine reductases by affinity chromatography on methotrexate-sepharose

Abstract An efficient procedure employing affinity chromatography has been developed for the isolation of sheep and rat liver dihydropteridine reductases. The affinity matrix is synthesized by the carbodiimide-promoted condensation of methotrexate (MTX) and 1,6-diaminohexane to give 1-aminohexyl-6-amido-MTX, which is subsequently coupled to cyanogen bromide-activated Sepharose. The purification sequence requires (i) chromatography of a dilute acetic acid extract of diced liver on DEAE-cellulose, (ii) two successive passages of the product from the previous step through the affinity matrix, and (iii) filtration through Sephadex G-200. The products, recovered in overall 30% yields, exhibit average specific activities of 47.5 and 63 μmol of NADH oxidized/mg/min and show single bands of mobility 0.35 and 0.19 for the sheep and rat liver sources, respectively. SDS-polyacrylamide electrophoresis before and after titration with dimethyl suberimidate indicates that the enzymes are dimeric with molecular weights of 52,000 (sheep) and 51,000 (rat). Both enzymes show a preference for NADH over NADPH as cofactor. However, differences in the extinction coefficient at 280 nm, the isoelectric point, and NADH binding constants suggest that significant variations in physical characteristics exist between the two proteins.

Comparative activity of rat liver dihydrofolate reductase with 7,8-dihydrofolate and other 7,8-dihydropteridines

The various interactions of rat liver dihydrofolate reductase with two unconjugated 7,8-dihydropteridines, 7,8-dihydrobiopterin and 6-methyl-7,8-dihydropteridine, have been compared with those of 7,8-dihydrofolate and folate. Of particular interest was the reactivity demonstrated by 7,8-dihydrobiopterin because of the potential physiological significance of this reaction both in the regeneration of tetrahydrobiopterin, a cofactor for various biological hydroxylations, and as a step in the biosynthesis of this compound from GTP. Kinetic experiments gave Km values of 0.17, 6.42, and 10.2 microM for 7,8-dihydrofolate, 7,8-dihydrobiopterin, and 6-methyl-7,8-dihydropteridine, respectively, with Vmax = 6.22, 2.39, and 1.54 mumol min-1 mg-1. With folate the enzyme showed high affinity (Km = 0.88 microM) but low Vmax (0.20 mumol min-1 mg-1). The natural cofactor was NADPH and a Km of approximately 0.7 microM was measured with each substrate. The enzyme was activated by both p-hydroxymercuribenzoate and urea when assayed with 7,8-dihydrofolate but was inhibited when 7,8-dihydrobiopterin was the substrate. The pH optimum for dihydrofolate reduction was 4 with enhancement at pH greater than or equal to 5.5 in the presence of 1 M NaCl. Peak activity with 7,8-dihydrobiopterin occurred at pH 4.8; this was shifted to pH 5.3 but was not enhanced by 1 M NaCl. Inhibition with methotrexate was similar whether the enzyme was assayed with either the conjugated or unconjugated 7,8-dihydro derivatives. The rat liver enzyme, highly unstable after purification, was stabilized in the presence of the nonionic detergent, Tween-20 (0.1%); however, the comparative properties toward the conjugated and unconjugated substrates were not altered by this treatment.

Biological activity of a novel rationally designed lipophilic thymidylate synthase inhibitor

AG-331 {N6[4-(N-morpholinosulfonyl)benzyl]-N6-methyl-2,6-diamino-benz[cd]indole glucuronate} is a novel lipophilic thymidylate synthase (TS) inhibitor. The properties of this compound were investigated in H35 rat hepatoma cells and in three variant cell lines resistant to antifolates by differing mechanisms. There was no evidence for any intracellular effect of AG-331 on dihydrofolate reductase (DHFR); however, the low degree of cross-resistance found for the H35FF line, which has elevated TS levels, suggested that TS may not be the sole locus of action of AG-331 in hepatoma cells. TS-directed effects of AG-331 were suggested by the pattern of its inhibition of deoxyuridine incorporation into DNA and the lesser effects on purine incorporation. In addition, H35 cells treated with 10 μM AG-331 were shown to accumulate in the S phase of the cell cycle, and this effect could be reversed by coadministration of thymidine. However, when treatments were conducted at a 5-fold higher concentration of AG-331, no S-phase block was apparent, suggesting the loss of a TS-directed effect at high inhibitor concentrations. Thymidine and folinic acid also failed to protect cells against AG-331 cytotoxicity, suggesting an alternate mode of action. Similar results were also obtained in protection experiments with a human hepatoma cell line, HEPG2, although previous results obtained in colon- and breastcancer cell lines have suggested TS specific effects for AG-331. The possibility that biotransformation of AG-331 to other toxic species may occur in liver-derived cell lines has yet to be investigated.

Absence of reproductive and developmental toxicity in rats following oral dosing with nelfinavir

The potential for nelfinavir mesylate (VIRACEPT) to produce reproductive toxicity was evaluated in rats administered oral doses of 200, 500, or 1000mg/kg/day. In the fertility and early embryonic development to implantation study, male rats were treated beginning 28 days prior to mating until necropsy and females for 2 weeks prior to mating and through gestation day (GD) 7. In the pre- and postnatal development study, pregnant rats were treated from GD 6 through lactation day (LD) 20. Selected F(1) pups from this study were evaluated in sensory and behavioral tests and were subsequently mated. Pregnant F(1) females were euthanized on GD 20 and their F(2) fetuses were examined. F(1) animals were not directly dosed with the drug. No treatment-related effects were observed on any male reproductive parameters. Administration of nelfinavir did not produce adverse effects on fertility, pregnancy, embryo-fetal development, parturition, or lactation in the F(0) generation. Similarly, no adverse effects of nelfinavir treatment were observed on pre- and postnatal growth, development, reproductive performance and embryo-fetal development in the F(1) offspring. Based on the results of this study, the no-observed-adverse-effect-level (NOAEL) for developmental and reproductive toxicity in rats was considered to be 1000mg/kg/day, the highest dose tested.

Synergy between 5,10-dideaza-5,6,7,8-tetrahydrofolic acid and methotrexate in mice bearing L1210 tumors

SummaryIn vivo studies with 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (DDATHF), an inhibitor of glycinamide ribonucleotide transformylase, indicate that at doses ranging from 2.5 to 10 mg/kg, it prolongs the survival of mice implanted with L1210 tumors. Lower doses of this agent have no effect. Parallel studies with methotrexate indicate that DDATHF is not as potent or as efficacious as methotrexate in this animal model. Low methotrexate can cause a significant increase in the survival of L1210 tumor-bearing mice, suggesting synergism between these two antifolates.

Absence of embryo-fetal toxicity in rats or rabbits following oral dosing with nelfinavir.

The potential for nelfinavir mesylate (VIRACEPT) to induce maternal and embryo-fetal toxicity was evaluated in rats and rabbits following oral administration. The drug was administered by gavage to rats at doses of 200, 500, or 1000mg/kg/day on days 6-17 of gestation and to rabbits at doses of 200, 400, or 1000mg/kg/day on days 7-20 of gestation. Dams and does were euthanized on GD20 and 29, respectively, and the offspring were weighed and examined for external, visceral, and skeletal alterations. Maximum plasma nelfinavir concentrations (C(max)) in rats were comparable to C(max) values in humans and were 3- to 6-fold higher than the reported human trough levels, while plasma nelfinavir levels in rabbits were approximately 0.13-0.17x the human C(max) and 0.25-0.5x the human trough. In rats, no treatment-related maternal or embryo-fetal toxicity was observed at any dose level and the NOAEL for both maternal and fetal toxicity was considered to be 1000mg/kg/day. Two rabbits in the 400mg/kg/day group died prior to scheduled termination. Because no deaths occurred in the high dose group and there were no other treatment-related signs of clinical toxicity in any dose group, these deaths were considered unrelated to nelfinavir. Group mean body weight loss in rabbits was observed at 1000mg/kg/day on gestation days 7-10. Food consumption was also reduced in this treatment group throughout the dosing period. There were no treatment-related findings in other maternal or fetal parameters. Thus, the no-observed-adverse-effect-level (NOAEL) for maternal toxicity in the rabbit was considered to be 400mg/kg/day (based on maternal body weight loss in the high dose group), while the NOAEL for embryo-fetal toxicity in the rabbit was considered to be 1000mg/kg/day. Thus, under the conditions of this study, nelfinavir was not considered to be toxic to the rat or rabbit conceptus.

The effect of specific amino acid modifications on the catalytic properties of rat liver dihydropteridine reductase

Abstract When rat liver dihydropteridine reductase (EC 1.6.99.7) is reacted with p-chloromercuri-benzoate and 5,5′-dithiobis(2-nitrobenzoic acid), six of the eight half-cystine residues, shown to be present by amino acid analysis, can be titrated with the former, and four with the latter reagent. In each case the enzyme is inactivated. The presence of the enzyme cofactor, NADH, affords protection against inactivation by 5,5′-dithiobis(2-nitrobenzoic acid) and reduces to 4 the number of sulfhydryl residues which can be modified by p-chloromercuribenzoate. In contrast, the substrate, “quinonoid” dihydropteridine, and product, tetrahydropteridine, do not prevent enzyme inactivation with either compound. When the reductase is treated with N-bromosuccinimide, eight tryptophan residues react with complete loss of enzymatic activity, however, in the presence of 0.001 m 2-mercaptoethanol the degree of inactivation is reduced to 30%. The relationship of sulfhydryl group modification to reduced enzymatic activity suggests that these residues play a major role in maintaining a functional enzyme tertiary structure.

Isolation of rat liver phenylalanine hydroxylase using a novel pteridine matrix

Abstract Phenylalanine hydroxylase has been isolated quantitatively from rat liver by the following purification sequence: (a) acetic acid extraction, (b) ethanol fractionation, (c) ammonium sulfate precipitation, (d) filtration through Sephadex G-100, and (e) affinity chromatography on a matrix containing 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine bound to Sepharose via a butanediol diglycidyl ether linkage. Steps (d) and (e) were carried out in 0.01 m tris(hydroxymethyl)methylaminopropanesulfonic acid (Taps) buffer solutions, pH 8.5, containing 0.1 m sodium chloride and 0.001 M dithioerythritol. The eluent for step (e) additionally contained 0.04 mg/ml catalase, and enzyme recovery was achieved when 0.1 m phenylalanine was introduced. Enzyme and phenylalanine separation was accomplished by sequential passage through columns of the affinity matrix and Sephadex G-25. The hydroxylase exhibited: a concentration-dependent specific activity of 1 to 6 μmol/min/mg; homogeneity by sodium dodecyl sulfate-polyacrylamide electrophoresis ( M r = 49,000 ± 2000), but heterogeneity (two major bands, M r = 120,000 ± 15,000 and 200,000 ± 20,000) when sodium dodecyl sulfate was omitted; a pH optimum of 6.5, and stability which was markedly enhanced by the presence of the Taps buffer, anaerobic conditions, catalase, and radical scavengers such as 3,5-di- tert -butyl-4-hydroxybenzyl alcohol.

Multiple forms of rat-liver dihydropteridine reductase identified by their differing isoelectric points☆☆☆

Purified rat-liver dihydropteridine reductase is homogeneous by gel filtration (Mr approximately 51,000), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr approximately 25,500), and native polyacrylamide gel electrophoresis, suggesting that the enzyme is composed of two identical subunits. However, analysis by isoelectric focusing has revealed three enzyme forms with approximate isoelectric points of 6.5, 5.9, and 5.7 (designated forms, I, II, and III, respectively). The three forms, isolated in 65% yield by preparative chromatofocusing, are stable in 0.05 M phosphate buffer, pH 6.8, containing 1 mM beta-mercaptoethanol and exhibit similar kinetic constants when the catalytic activities of the isolated forms are compared with quinonoid dihydrobiopterin as substrate. All forms generate complexes with the enzymatic cofactor NADH which are also detectable by IEF. When examined further by IEF under denaturing conditions in 6 M urea the enzyme demonstrates a differing subunit composition for its three forms. Two distinct subunits, designated alpha and beta, can be identified, and additional evidence suggests that the native enzyme forms I, II, and III represent the three differing dimeric combinations alpha alpha (form I), alpha beta (form II), and beta beta (form III).