David P. Baccanari

Pyrrolo[2,3-d]pyrimidines and pyrido[2,3-d]pyrimidines as conformationally restricted analogues of the antibacterial agent trimethoprim

Conformationally restricted analogues of the antibacterial agent trimethoprim (TMP) were designed to mimic the conformation of drug observed in its complex with bacterial dihydrofolate reductase (DHFR). This conformation of TMP was achieved by linking the 4-amino function to the methylene group by one- and two-carbon bridges. A pyrrolo[2,3-d]pyrimidine, a dihydro analogue, and a tetrahydropyrido[2,3-d]pyrimidine were synthesized and tested as inhibitors of DHFR. One analogue showed activity equivalent to that of TMP against DHFR from three species of bacteria. An X-ray crystal structure of this inhibitor bound to Escherichia coli DHFR was determined to evaluate the structural consequences of the conformational restriction.

X-Ray Crystallographic Studies of Candida Albicans Dihydrofolate Reductase. High Resolution Structures of the Holoenzyme and an Inhibited Ternary Complex.

The recent rise in systemic fungal infections has created a need for the development of new antifungal agents. As part of an effort to provide therapeutically effective inhibitors of fungal dihydrofolate reductase (DHFR), we have cloned, expressed, purified, crystallized, and determined the three-dimensional structure ofCandida albicans DHFR. The 192-residue enzyme, which was expressed in Escherichia coli and purified by methotrexate affinity and cation exchange chromatography, was 27% identical to human DHFR. Crystals of C. albicans DHFR were grown as the holoenzyme complex and as a ternary complex containing a pyrroloquinazoline inhibitor. Both complexes crystallized with two molecules in the asymmetric unit in space group P21. The final structures had R-factors of 0.199 at 1.85-Å resolution and 0.155 at 1.60-Å resolution, respectively. The enzyme fold was similar to that of bacterial and vertebrate DHFR, and the binding of a nonselective diaminopyrroloquinazoline inhibitor and the interactions of NADPH with protein were typical of ligand binding to other DHFRs. However, the width of the active site cleft of C. albicans DHFR was significantly larger than that of the human enzyme, providing a basis for the design of potentially selective inhibitors.

Coupled oxidation of NADPH with thiols at neutral pH

Abstract NADPH and NADH are rapidly oxidized in neutral imidazole chloride buffer at 30 °C in the presence of mercaptoethanol or dithiothreitol. The product of the NADPH reaction has been determined to be enzymically active NADP+. Oxidation of the pyridine nucleotides is coupled to the autooxidation of the thiol and is inhibited by ethylenediamine tetraacetic acid, stimulated by metal ions (FeSO4), and requires oxygen. The rapid oxidation of thiols and NADPH at neutral pH was found to occur only in imidazole and, to a lesser extent, in histidine buffer. Under the conditions employed, 300 μ m dithiothreitol and 30 μ m NADPH are oxidized in 30 min. Both NADPH and thiol oxidations are inhibited by catalase, whereas superoxide dismutase only inhibits the oxidation of NADPH. NADPH oxidation is also inhibited by the hydroxyl radical scavengers formate, mannitol, or benzoate. A reaction mechanism is proposed in which imidazole promotes the metal-catalyzed oxidation of thiols at neutral pH. The superoxide radical generated either by the thiol oxidation or directly oxidizes NADPH or forms hydrogen peroxide and hydroxyl radicals which can oxidize NADPH. Hydrogen peroxide is also involved in the autooxidation of the thiol.

5-Ethynyluracil (776C85): Protection from 5-fluorouracil-induced neurotoxicity in dogs

5-Ethynyluracil (776C85) is a potent mechanism-based inactivator of dihydropyrimidine dehydrogenase (DPD), the enzyme that catalyzes the rapid catabolism of 5-fluorouracil (5-FU). Because catabolism is the major route for 5-FU clearance, we studied the effect of 5-ethynyluracil on the pharmacokinetics and toxicity of continuous i.v. 5-FU infusion in the dog. 5-FU at 40 mg/kg/24 hr resulted in a steady-state plasma 5-FU concentration of 1.3 microM and was fatal with dogs dying from apparent neurotoxicity. 5-Ethynyluracil lowered the total clearance of 5-FU from 9.9 to 0.2 L/hr/kg and enabled 1.6 mg/kg/24 hr 5-FU to achieve a steady-state plasma 5-FU concentration of 2.4 microM with no apparent toxicity. 5-FU at 4 mg/kg/24 hr achieved a steady-state plasma 5-FU concentration of 5.3 microM and produced only mild gastrointestinal disturbances in 5-ethynyluracil-treated dogs. Thus, a catabolite of 5-FU appears to be responsible for the 5-FU-induced neurotoxicity in dogs.

Dihydropyrimidine dehydrogenase inactivation and 5-fluorouracil pharmacokinetics: allometric scaling of animal data, pharmacokinetics and toxicodynamics of 5-fluorouracil in humans

Abstract The pharmacokinetics of 5-fluorouracil (5-FU) in different animal species treated with the dihy-dropyrimidine dehydrogenase (DPD) inactivator, 5-ethynyluracil (776C85) were related through allometric scaling. Estimates of 5-FU dose in combination with 776C85 were determined from pharmacokinetic and toxicodynamic analysis. Method: The pharmacokinetics of 5-FU in the DPD-deficient state were obtained from mice, rats and dogs treated with 776C85 followed by 5-FU. The pharmacokinetics of 5-FU in humans were then estimated using interspecies allometric scaling. Data related to the clinical toxicity for 5-FU were obtained from the literature. The predicted pharmacokinetics of 5-FU and the clinical toxicity data were then used to estimate the appropriate dose of 5-FU in combination with 776C85 in clinical trials. Results: The allometric equation relating total body clearance (CL) of 5-FU to the body weight (B) (CL=0.47B0.74) indicates that clearance increased disproportionately with body weight. In contrast, the apparent volume of distribution (Vc) increased proportionately with body weight (Vc=0.58 B0.99). Based on allometric analysis, the estimated clearance of 5-FU (10.9 l/h) in humans with DPD deficiency was comparable to the observed values in humans lacking DPD activity due to genetic predisposition (10.1 l/h), or treatment with 776C85 (7.0 l/h) or (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVdUrd, 6.6 l/h). The maximum tolerated dose (MTD) of 5-FU in combination with 776C85 was predicted from literature data relating toxicity and plasma 5-FU area under the concentration-time curve (AUC). Based on allometric analysis, the estimated values for the MTD in humans treated with 776C85 and receiving 5-FU as a single i.v. bolus dose, and 5-day and 12-day continuous infusions were about 110, 50 and 30 mg/m2 of 5-FU, respectively. Discussion: The pharmacokinetics of 5-FU in the DPD-deficient state in humans can be predicted from animal data. A much smaller dose of 5-FU is needed in patients treated with 776C85.

α-Fluoro-β-alanine: Effects on the antitumor activity and toxicity of 5-fluorouracil

We have shown previously that (R)-5-fluoro-5,6-dihydrouracil (FUraH(2)) attenuates the antitumor activity of 5-fluorouracil (FUra) in rats bearing advanced colorectal carcinoma. Presently, we found that alpha-fluoro-beta-alanine (FBAL), the predominant catabolite of FUra that is formed rapidly via FUraH(2), also decreased the antitumor activity and potentiated the toxicity of FUra. In rats treated with Eniluracil (5-ethynyluracil, GW776), excess FBAL, in a 9:1 ratio to FUra, produced similar effects when administered 1 hr before, simultaneously with, or 2 hr after FUra. FBAL also decreased the antitumor activity of FUra in Eniluracil-treated mice bearing MOPC-315 myeloma at a 9:1 ratio with FUra, but not at a 2:1 ratio. FBAL did not affect the antitumor activity of FUra in mice bearing Colon 38 tumors. We also evaluated the effect of thymidylate synthase (TS) and thymidine kinase (TK) from tumor extracts after FUra +/- Eniluracil +/- FBAL treatment. The activity of TK was similar among the three groups at both 18 and 120 hr. There was also no difference in TS inhibition ( approximately 35%) at 18 hr. However, significantly more TS inhibition was observed in the Eniluracil/FUra group than in the FUra-alone group at 120 hr. FBAL did not alter the effect of Eniluracil/FUra in TS inhibition. Neither FUraH(2) nor FBAL affected the IC(50) of FUra in culture. Thus, the effect of FBAL did not result from direct competition with FUra uptake or immediate anabolism. Either another downstream catabolite that is not formed in cell culture is the active agent, or the effect requires the complexity of a living organism or an established tumor.

Species-dependent differences in the biochemical effects and metabolism of 5-benzylacyclouridine

The pharmacokinetics and biochemical effects of the uridine phosphorylase (UrdPase) inhibitor 5-benzylacyclouridine (BAU) were investigated in the mouse, rat and monkey. Following i.p. administration of BAU (30 mg/kg) in the mouse and i.v. administration in the rat and monkey, initial BAU plasma half-life values were 36, 36 and 25 min, and the areas under the plasma BAU concentration versus time curves (AUC) were 127, 80 and 76 microM.hr, respectively. Rats were also dosed p.o. and i.v. with BAU at 90 mg/kg, and a comparison of the AUC values showed an oral bioavailability of 70%. Analyses of plasma samples by HPLC indicated that the metabolism of BAU differed in these species. A major BAU metabolite was observed in monkeys. Its concentration was greater than or equal to that of BAU in almost every plasma sample, and its elimination paralleled that of BAU. Urinary recovery of the metabolite was 10-fold higher than the recovery of unchanged drug. The compound was identified as the ether glucuronide of BAU by its UV absorption spectrum, its co-elution with BAU after incubation with beta-glucuronidase, and liquid chromatography/mass spectrum analysis. A different metabolite was detected in rat plasma; its maximum concentration was 15% of the BAU level, and its elution position on the HPLC chromatogram was not affected by the action of beta-glucuronidase. BAU had equivalent potency against UrdPase in liver extracts from the three species, with Ki values of about 0.17 microM. However, the in vivo effects of BAU on plasma uridine concentrations were species dependent. In mice, a 30 mg/kg i.p. dose of BAU increased the plasma uridine concentration to 11 microM from a control level of 1.8 microM. In the rat, a 30 mg/kg i.v. dose of BAU increased plasma uridine to 2.1 from 1.1 microM control levels, and a 300 mg/kg oral dose resulted in a peak plasma uridine concentration of only 6 microM. In the monkey, BAU (30 mg/kg, i.v.) had no effect on plasma uridine despite the presence of 10-100 microM BAU levels in plasma for 1.5 hr. These data show that there are significant differences in the biochemical effects and metabolism of BAU in CD-1 mice, CD rats and cynomolgus monkeys.

Preclinical development of eniluracil: Enhancing the therapeutic index and dosing convenience of 5-fluorouracil

Eniluracil (5-ethynyluracil, GW 776, 776C85) isbeing developed as a novel modulator of 5-fluorouracil (5-FU) forthe treatment of cancer. Eniluracil is an effective mechanism-based inactivator of dihydropyrimidine dehydrogenase (DPD), thefirst enzyme in the catabolic pathway of 5-FU. By temporarilyeliminating this prevalent enzyme, eniluracil providespredictable dosing of 5-FU and enables oral administration of5-FU to replace intravenous bolus and continuously infuseddosing. New DPD is synthesized with a half-life of 2.6 days. Italso eliminates the formation of problematic 5-FU catabolites.Most importantly, in laboratory animals, eniluracil increases thetherapeutic index and absolute efficacy of 5-FU. Accompanyingreports in this journal indicate that eniluracil has promisingclinical potential.