Drug Metabolism Letters - Volume 5, Issue 2, 2011

The metabolism of flavone-8-acetic acid (FAA) has been hypothesized to be partly responsible for its potent anticancer activity in mice. The purpose of this study was to identify the mouse enzymes involved in FAA Phase I metabolism and evaluate their possible induction in vivo by FAA. Mouse microsomes metabolized FAA into 6 metabolites: 3',4'-dihydrodiol-FAA, 5,6-epoxy-FAA, 4'-OH-FAA, 3'-OH-FAA, 3',4'-epoxy-FAA and 6-OH-FAA. Using Cyp-specific inhibitors (furafylline, Cyp1a2; α-naphthoflavone, Cyp1b1; tranylcypromine, Cyp2b9; quercetin, Cyp2c29; quinidine, 2d9; diethyldithiocarbamate, Cyp2e1; ketoconazole, Cyp3a11), the formation of 5,6-epoxy-FAA was mainly attributed to Cyps 1a2, 1b1, 2b9, 2c29 and 2e1, whereas the 3',4'-epoxy-FAA was formed by Cyps 2b9 and 3a11. The 4'-OH-FAA was generated by Cyps 1a2, 1b1, 2b9 and 2e1, and the 6-OH-FAA was formed by Cyps 1b1 and 2c9. Using the epoxide scavenger N-acetyl cysteine, 4'-OH-FAA, 3'-OH-FAA and 6-OH-FAA were shown to derive partly from non enzymatic isomerisation of their corresponding epoxides. The specific epoxide hydrolase inhibitor elaidamide allowed the confirmation that 3',4'-dihydrodiol-FAA was formed via the epoxide hydrolase. FAA treatment in vivo in mice led to a significant increase in the hepatic expression of Cyp1a2 (1.9-fold), 2e1 (2.1-fold), 2b10 (3.2-fold), 2d9 (2.3-fold) and 3a11 (2.2-fold), as evaluated by qRT-PCR. In conclusion, several Cyps were shown to be involved in FAA metabolism, particularly Cyps 3a11 and 2b9 which were responsible for the formation of the principal metabolites (5,6-epoxy-FAA, 3',4'-epoxy-FAA), and that FAA could induce the expression of several Cyps after in vivo administration. The possible implication of these enzymes in the in vivo anticancer activity of FAA in mice is discussed.

A comparative pharmacokinetic study of three enrofloxacin injectable solutions was carried out in six healthy Barky rams after intramuscular injection according to a single dose, randomized, crossover experimental design. The three formulations were enrofloxacin 10% (Baytril®), enrofloxacin 10% plus bromhexine 1% (Mucotryl®) and enrofloxacin 10 % solution without bromhexine (Mucotryl without bromhexine). The three formulations were given a single intramuscular dose at a dose rate of 5 mg kg-1 b.wt. The concentrations of the drug in the serum were measured using highperformance liquid chromatography (HPLC) with fluorescence detection. The results indicate that there were no significant differences between the distribution rate constant (kab) and distribution half-life (t1/2ab), the maximum serum concentration (Cmax) and the time to peak concentration (Tmax) between Baytril and the other two formulations. There were significant differences between the elimination half life (t1/2el) and elimination rate constant (kel), for Baytril and enrofloxacin (the exact formulation of Mucotryl without bromhexine). Enrofloxacin was rapidly absorbed following IM administration of 5 mg kg-1 b. wt. The peak serum concentrations (Cmax) were 2.83, 2.45 and 3.12 μg ml-1 and were attained at (tmax) 1.15, 1.41 and 1.26 hours when given Mucotryl, Baytril and enrofloxacin (the exact formulation of Mucotryl without bromhexine) to sheep, respectively. The injectable formulations investigated were bioequivalent after their intramuscular injection to sheep at recommended dose rate. As our results showed that, values of Tmax, Cmax and AUC (Area under time curve concentration) determined for both Baytril, Mucotryl, and enrofloxacin (the exact formulation of Mucotryl without bromhexine ( reference ant test products) are within the acceptable range of 0.80-1.20, this means that the tested products product under investigation was bio-equivalent. These findings showed that the efficacy of the two test formulations was similar or possibly enhanced compared with Baytril based on the pharmacokinetic parameters obtained and due to concentration dependent activity of enrofloxacin

The effect of ethyl acetate extract of pomelo fruit on systemic exposure of verapamil was explored in rabbits. Two groups each of 8 locally-inbred New Zealand male rabbits were used. The first group was used for single-dose treatment (both verapamil and pomelo extract). The second group was used for multiple-dose treatment, pomelo extract (once daily for 14 days) and verapamil single doses (at days 7 and 14). A verapamil dose of 30 mg/kg and a pomelo extract dose of 45 mg/kg were used. Single-dose treatment with pomelo extract resulted in a minor change in mean Cmax of verapamil in plasma, while a decrease of 37.8% in AUC0-24 and 28.3% in AUC0-∞ was observed but did not reach statistical significance. After the first period of multiple dose treatment (pomelo extract for 7 days), the combination increased the concentration of verapamil in plasma with a significant increase in mean Cmax, AUC0-24 and AUC0-∞ by 461.9%, 299.7%, and 261.1%, respectively (p values were 0.005, 0.002, and 0.006, respectively). In contrast, after the second period (day 14 of pomelo extract use), the combination decreased the concentration of verapamil in the plasma with a substantial decrease in mean Cmax, AUC0-24, and AUC0-∞, by 68.2%, 69.7% and 58.3%, respectively. This decrease did not reach statistical significance (p values were 0.073, 0.081 and 0.083, respectively). The Tmax was not affected significantly in both studies. The study illustrates a complex time-dependent interaction between verapamil and the ethyl acetate extract of pomelo mix. More intensive studies are needed to further understand the nature of the interaction.

Twenty adult male rabbits (Oryctolagus cuniculus) received sildenafil (0, 1, 3, 6, 9 mg/kg/day) for 4 weeks to investigate the testicular histological alterations induced by overdoses of this drug. Exposure to overdoses of sildenafil had provoked tubular and interstitial histological alterations. Abnormality in the germinal epithelium of the seminiferous tubules included spermatocytes karyopyknosis, spermatocytes degeneration, desquamation, spermatid giant cells and arrest of spermatogenesis. Additionally, increased Leydig cells cellularity, tubular degeneration, thickening of the interstitium were also observed. The encountered histological findings indicate that chronic exposure to sildenafil overdoses produces significant morphological and histological alterations in the testes which finally might lead to complete arrest of spermatogenesis.

The availability of a reliable in vitro assay to evaluate time-dependent inhibition (TDI) of cytchrome P450 enzymes by novel compounds is essential for the identification of candidate medicines. We have evaluated three assay methods, making use of 59 marketed compounds and 28 novel GSK compounds. Recombinant bactosomes expressing the CYP3A4 isozyme were used with two fluorescence-based methods: a “Re-addition” assay and a “30 min” assay. The third method evaluated used pooled human liver microsomes (PHLM) with LC-MS/MS detection (the data for GSK compounds were evaluated in this study, whereas data for marketed drugs were reported recently). Our evaluation showed that the Re-addition method is comparable to the LC-MS/MS method in terms of predictivity and reproducibility. In conclusion, Re-addition method is inexpensive, and provides a simple assessment of the risk of TDI for novel compounds. This assay is particularly appropriate for use during the early stages of drug discovery.

Vernakalant hydrochloride is a novel antiarrhythmic drug for the rapid conversion of atrial fibrillation to sinus rhythm and prevention of relapse. In this open-label, parallel design study, 8 healthy men received single 240-mg doses of [14C]vernakalant hydrochloride, given as a 10-minute intravenous (IV) infusion on day 1, and as an oral gel capsule on day 22. Plasma, urine, and fecal samples were collected for 7 days after dosing to measure vernakalant and its metabolites and to estimate mass balance of total [14C] recovery. The disposition and metabolic profile of vernakalant after both IV and oral administration, depended on cytochrome P450 (CYP)2D6 genotype. Vernakalant underwent rapid and extensive distribution during infusion, which resulted in similar maximum plasma concentrations in extensive metabolizers (EMs) and poor metabolizers (PMs) for IV but not oral administration. Vernakalant was metabolized rapidly and extensively to a 4-O-demethylated metabolite with glucuronidation in EMs; direct glucuronidation predominated in PMs. Slower clearance in PMs contributed to 3- and 6-fold higher drug exposure (AUC0-∞; IV and oral dosing, respectively). Urinary recovery of unchanged vernakalant was higher in PMs as well. Total [14C] was recovered predominantly in urine, while lower levels were recovered in feces. Mass balance was achieved, with a mean recovery of 99.7% of the IV dose and 98.7% of the oral dose, in EMs, and 89.2% and 88.2% of the IV and oral doses, respectively, in PMs. Vernakalant was well tolerated. The pharmacokinetics and metabolism of vernakalant depend on CYP2D6 genotype with more pronounced effects on exposure following oral administration; however, the differences between EMs and PMs are unlikely to be clinically significant following short-term IV infusion.

Objective: Cytochrome P450 (CYP) enzymes exhibit high interindividual variability that is not completely explained by known environmental and genetic factors. To further understand this variability, we hypothesized that microRNAs (miRNAs) may regulate CYP expression. Methods: MiRNA identification algorithms were used to identify the miRNAs that are predicted to regulate twelve major drug metabolizing CYPs and to identify polymorphisms in CYP mRNA 3'-UTRs that are predicted to interfere with normal mRNA-miRNA interactions. Results: All twelve CYPs were predicted to be targets of miRNAs. Additionally, 38 SNPs in CYP mRNA 3'-UTRs were predicted to interfere with miRNA targeting of mRNAs. These predicted miRNAs and SNPs are candidates for future in vitro studies focused on understanding the molecular regulation of these CYP genes. Conclusion: These in silico results provide strong support for a role of miRNA in the regulation and variability of CYP expression.

Therapeutic regimens of intravenous immunoglobulin are currently based on actual body weight. The relationship between immunoglobulin dose and serum IgG level in relation to body size was retrospectively explored in patients on replacement therapy. Data were collected as part of a national audit on immunoglobulin therapy in patients with common variable immunodeficiency. 107 patients received immunoglobulin titrated to optimum effect. Correlations were sought between body mass index, trough IgG levels, infusion frequency and total annual dose. The mean (±SD) trough IgG level was 8.4±1.6 g/L and annual immunoglobulin dose received was 456.8±129.4 g. There was no relationship between annual dose and trough IgG level, regardless of infusion frequency, or adjustment for weight or body mass index. These results support the clinical practice of immunoglobulin prescription by clinical outcome rather than fixed dose by body weight. Future studies exploring immunoglobulin efficacy should include treatment arms with dosages based on both ideal and actual body weight, as ideal body weight-based prescribing would save significant amounts of product.

It is well known that nonsteroidal anti-inflammatory drugs (NSAIDs) delay the elimination of methotrexate. One of the mechanisms is thought to be inhibition of methotrexate uptake via human organic anion transporter 3 (hOAT3, SLC22A8) in the renal proximal tubule by NSAIDs. In this study, we evaluated the inhibitory effects of selective cyclooxygenase-2 inhibitor etoricoxib on hOAT3 by uptake experiments using Xenopus laevis oocytes. The injection of hOAT3 cRNA stimulated the uptake of methotrexate into the oocytes, and its transport was inhibited by etoricoxib. Etoricoxib inhibited estrone sulfate uptake by hOAT3 dose dependently, and the 50% inhibitory concentration was estimated to be 9.8 μM. Eadie-Hofstee plot analysis showed that etoricoxib inhibited hOAT3 in a competitive manner. These findings show that etoricoxib has inhibitory effect on hOAT3, and that the potential is comparable to that of traditional NSAIDs.

Combi-molecules are agents designed to block receptors on their own and to further degrade to bioactive agents. Here we studied the fate of a novel combi-molecule of triazene class termed “ZRS1” in biological medium using multilayer aggregates and mouse tumour models. ZRS1 is a second generation derivative of RB107, a prodrug designed to release an EGFR inhibitor FD105 plus a methyl diazonium species. RB107 contains an acetoxymethyl function that is hydrolyzed too rapidly to generate BJ2000, a monoalkyltriazene that further degrades to FD105 and DNA alkylating methyldiazonium species. Recently, in order to prevent rapid hydrolysis of the acetoxymethylene function in the absence of cells and to delay the release of BJ2000, we designed ZRS1 that contains a more stable acetoxymethyl carbamate function. The results showed that ZRS1 was more stable than RB107 in cell culture medium supplemented with serum, with a rather long half life (>2 h). However, in an experiment where it was allowed to degrade in multilayer aggregates of ovarian cancer cells OV90, it rapidly released BJ2000 and its corresponding metabolite FD105, both in the medium and the multilayer aggregates. Interestingly, the intact ZRS1 could be detected in the multilayer aggregates with a Tmax around 10 min. Studies in vivo, in human DU145 prostate cancer xenograft model, revealed that ZRS1 blocked tumour growth and released FD105 and its acetylated metabolite FD105Ac, the latter being the major metabolite. Likewise, time course analysis in 4T1 mouse syngeneic breast cancer model showed a rapid release of FD105 and FD105Ac in the plasma and in the tumours. In summary, ZRS1 appeared as a good prodrug of the stable EGFR inhibitory metabolites FD105 and FD105Ac. Its ability to generate high concentrations of FD105Ac, a more potent EGFR inhibitor as is its major metabolite, is significant over previous methylating combi-molecules. Furthermore, this study showed that multilayer OV90 aggregates could be developed as an effective model to predict the stability and degradation of ZRS1 in vivo.