Drug Metabolism Letters - Volume 5, Issue 3, 2011

This screening aimed to evaluate quantitatively the occurrence of fungal metabolites in Brazil nuts. Nuts were collected from Agroforest production areas in Amazon basin region. A total of 235 mycotoxins (including the most prominent ones) was screened by a multi-mycotoxin method based on HPLC-MS/MS. The recovery of metabolites by the method was between 56 and 136%. Fifteen mycotoxins were detected and quantified, in at least one sample; namely, aflatoxins (AFB1, AFB2, AFG1, and AFM1), sterigmatocystin, methyl-sterigmatocystin, kojic acid, citrinin, cyclosporin A, cyclosporin C, cyclosporin D, cyclosporin H, rugulosin, alternariol-methylether and emodin. This is the first study dealing with the detection of the latter nine metabolites in Brazil nuts. Alternariol-methylether (from 0.75 to 3.2 μg/kg) was the only metabolite detected in all analyzed samples.

During early drug discovery, the synthetic pathways for test compounds are not well defined and impurities in the test compounds are inevitable. Compounds undergo serial screening tests at this stage to assess their biological activities and drug-like properties. Impurities in the test compounds can produce false positive results and therefore complicate the interpretation of data. P450 inhibition is one of the screens used in the early drug discovery process to assess the potential of drug-drug interactions caused by the inhibition of P450 enzymes. The impact of impurities on P450 inhibition has not been investigated. In this study, the impact of impurities on CYP2D6 IC50 values was evaluated using model compounds. Cimetidine was chosen as the test compound. Quinidine, fluoxetine, fluvoxamine, and ibuprofen were chosen to represent impurities as they inhibit CYP2D6 to varying degrees. The IC50 values of these model impurities for CYP2D6 were 0.11 μM, 0.98 μM, 13.4 μM, and >100 μM, respectively. Impurities with potent CYP2D6 inhibition, such as quinidine, can significantly decrease the apparent IC50 value for the mixture. With the addition of only 2% quinidine to cimetidine (mol/mol), the apparent IC50 value of cimetidine decreased from 98 μM to 4.4 μM. With the addition of 10% quinidine, the apparent IC50 decreased to 1.04 μM. Such a significant decrease in apparent IC50 values can produce a false alert and cause the inappropriate elimination of good compounds at an early stage. Impurities with low inhibitory potential, such as fluvoxamine and ibuprofen, did not cause a significant change in apparent IC50 values. An impurity can have a similar effect on the IC50 values for inhibition of other biological activities. The effect of an impurity on apparent IC50 values can be predicted by using a simulation curve if the potency of the impurity is characterized.

CPI-613 is a novel anti-tumor compound with a mechanism-of-action which appears distinct from the current classes of anti-cancer agents used in the clinic. CPI-613 demonstrates both in vitro and in vivo anti-tumor activity. In vitro metabolic studies using liver S9 were performed which demonstrated that CPI-613 undergoes both phase 1 (oxidation) and phase 2 (glucuronidation) transformations. Its metabolic half-life varied between species and ranged from 8 minutes (Hanford minipig) to 47 minutes (CD-1 mouse). We performed metabolite mass assessments using selected in vitro incubation samples and demonstrated that +16 amu oxidation with and without +176 amu glucuronidation products were generated by human and animal liver S9. LC/MS/MS fragmentation patterns showed that an uncommon sulfoxide metabolite was formed and the O-glucuronidation occurred at the terminal carboxyl moiety. We observed that the +192 amu sulfoxide/glucuronide was generated only in human liver S9 and not by any of the other species tested. Synthetic metabolites were prepared and compared with the enzymatically-generated metabolites. Both the chromatographic retention times and the LC/MS/MS fragmentation patterns were similar, demonstrating that the synthetic metabolites were virtually identical to the S9-generated products. CYP450 reaction phenotyping and inhibition data both suggested that multiple CYP isozymes (2C8 and 3A4, along with minor contributions by 2C9 and 2C19) were involved in CPI-613 metabolism and sulfoxide formation. Plasma samples from human subjects dosed with CPI-613 also contained the sulfoxide ± glucuronide metabolites. These results show that the in vitro- and in vivo-generated phase 1 and phase 2 metabolites were in good agreement.

Time-dependent or mechanism-based CYP3A4 inhibition is an important adverse drug property that should be carefully managed during drug development. Evaluation of time-dependent inhibition is traditionally performed using liver microsomes or recombinant P450 isoforms. We report here higher throughput approaches to evaluate time-dependent CYP3A4 inhibition assay using cultured cryopreserved human hepatocytes. The assays were performed in human hepatocytes cultured in 96-well plates, with luciferin-IPA as the CYP3A4 specific substrate. The advantages of the approach are as follows: 1. The use of 96-well plates minimizes the quantity of human hepatocytes and test materials required for the assays. 2. The use of luciferin-IPA allows CYP3A4 activity to be quantified rapidly using a plate reader, thereby avoiding the need for LC/MS that is required for traditional substrates such as testosterone and midazolam. 3. The use of cultured (plated) hepatocytes allows effective removal of treatment medium and washing of the cells without the laborious centrifugation step that is required for hepatocytes in suspension. Two assays were developed: 1. IC50 shift assay; and 2. enzyme kinetic assay. The IC50 shift assay is intended for general screening purpose with which a time-dependent CYP3A4 inhibitor would be identified by an increase in inhibitory potency (quantified as a decrease in IC50) upon a 30 min. pre-incubation of hepatocytes with the inhibitor at 37 deg. C. Results with model inhibitors showed that the IC50 assay readily distinguished the time-dependent inhibitors (1-aminobenzotriazole, erythromycin) from the non-timedependent inhibitor (ketoconazole). The enzyme kinetic assay is used for the derivation of the kinetic parameters KI and kinact. With this assay, time and concentration dependent inhibition of CYP3A4 were observed for 1-aminobenzotriazole and erythromycin. With hepatocytes from 4 donors, KI and kinact values were calculated to be 22.0 to 70.7 μM, and 0.09 to 0.51 min-1, respectively, for 1-aminobenzotriazole; and 47.3 to 75.1 μM, and 0.26 to 1.48, respectively, for erythromycin. DMSO (tested up to 2% v/v) was found to significantly attenuate the time-dependent inhibitory effects of 1- aminobenzotriazole, and had no apparent effects on erythromycin. Acetonitrile and methanol at 1% v/v had significantly less effects. The higher throughput assays describe here can to be used routinely for the evaluation of time-dependent CYP3A4 inhibitory potential of drug candidates during early phases of drug development.

Background: Oxytocin (OT) is one of the important paracrine factors that prostate synthesizes. OT maintains its resting tone and stimulates its contractile activity. However, the involvement of OT in modulating cell proliferation of the prostate is being investigated. In fact, alterations in OT concentrations accompany both benign prostatic hyperplasia/ hypertrophy and carcinoma of the prostate. The enzyme Insulin-regulated aminopeptidase (IRAP) is the main responsible of OT levels regulation through its catabolism. To date, the long-acting selective α1-adrenergic receptor antagonist doxazosin is widely used to the treatment of BPH. Thus, our aim was to analyze the effects of doxazosin on IRAP specific activity and its putative effects on prostate OT regulation and functions. Methods: Fifteen male Wistar rats were treated subcutaneously with 10 mg/Kg doxazosin during 15 days and fifteen controls were treated with the vehicle only. After the treatment period, prostate was removed to obtain soluble and membrane-bound fractions. Soluble and membrane-bound IRAP specific activities were assayed fluorometrically using leucyl-β-naphthylamide as substrate. Prostate OT content was assayed by enzyme immunoassay. Results: Doxazosin treatment significantly increased membrane-bound IRAP specific activity in rat prostate by 59.4%, whereas no changes were observed in the soluble fraction. Treatment with doxazosin also significantly increased OT concentration by 26.3%. Conclusions: In vivo administration of doxazosin to male rats modify both prostatic IRAP activity and OT levels. Because there is now evidence that OT plays a physiological role in the regulation of growth and muscular contractility within the gland, more attention should be paid to IRAP activity, which could represent a new target for the regulation of the functions of OT under physiological or pathological conditions such as BPH and prostate cancer.

Alkaline phosphatase (ALP) belongs to hydrolase group of enzymes. It is responsible for removing phosphate groups from many types of molecules, including nucleotides and proteins. Cimetidine (trade name Tagamet) is an antagonist of histamine H2-receptor that inhibits the production of gastric acid. Cimetidine is used for the treatment of gastrointestinal diseases. In this study the inhibitory effect of cimetidine on mouse renal ALP activity was investigated. Our results showed that cimetidine can inhibit ALP by uncompetitive inhibition. In the absence of inhibitor the Vmax and Km of the enzyme were found to be 13.7 mmol/mg prot.min and 0.25 mM, respectively. Both the Vmax and Km of the enzyme decreased with increasing cimetidine concentrations (0- 1.2 mM). The Ki and IC50 of cimetidine were determined to be about 0.5 mM and 0.52 mM, respectively.

We recently established a method for quantitative determination of human catalytically active UDPglucuronosyltransferases (UGTs) other than UGT2A1 and UGT2B28 by real-time reverse transcription-polymerase chain reaction (RT-PCR), and applied the method to an exhaustive analysis of localization in various human tissues. We report here an additional quantitative determination method targeting UGT2B28. To date, there have been no reports on the distribution of UGT2B28 mRNA expression in human tissues based on quantitative determination. Human UGT2B28 was clearly detected in the breast and adipose tissue. UGT2B28 expression in the breast was comparatively low, about 1.6% of GAPDH mRNA levels, and was less than 5% of normalized (against GAPDH) UGT2B7 and 2B10 mRNA expression levels in the liver. Although the UGT2B28 has 97% identity with UGT2B11 at the cDNA sequence level, the primers constructed for UGT2B28 did not detect UGT2B11. In addition, significant expression of UGT2B11 was detected in the liver, breast and kidney, and was clearly different from the distribution of UGT2B28. Therefore, we conclude that the real-time RT-PCR method established here is very specific for human UGT2B28 isozyme.

Phenstatin and its derivatives are potential anticancer drug candidates according to their inhibitory properties on tubulin polymerization, cell growth and antivascular activity. However, at the present time, neither pharmacological nor metabolic studies have been conducted in order to strengthen the relevance of phenstatine as a drug discovery candidate. In the present work, the metabolic fate of phenstatin in rat and human microsomal preparations was studied to investigate the stability of this tubulin polymerization inhibitor and any effects of the metabolites on tubulin polymerization and on PC3 cancer cell proliferation. The metabolites were separated by high-performance liquid chromatography and, after their synthesis, characterized by simultaneous LC-DAD-UV and LC-ESI-MS analyses. Thus, eight metabolites were identified. The major biotransformation pathways are carbonyl reduction, O-methylation at C-3', O-methylation after aromatic hydroxylation at the position C-2' on phenyl B ring and O-demethylation on A ring. Four of the identified metabolites were as active or more active, than phenstatin in vitro. Moreover, the better stability of phenstatin versus CA-4 and the lack of quinone formation could justify the design of new analogues which could include various substituents on phenyl rings or linker group in order to modulate the metabolism of phenstatin toward even more active metabolites and so up-regulate the pharmacological activity.

Dietary tyramine is associated with hypertensive crises because of its ability to induce the release of catecholamines. The roles of monoamine oxidase (MAO), flavin-containing monooxygenase (FMO), and cytochrome P450 2D6 (CYP2D6) were studied in terms of the enzymatic elimination of tyramine in vitro at a substrate concentration of 1.0 μM, which is relevant to in vivo serum concentrations. Tyramine elimination by human liver supernatant fractions was decreased by ∼70% in the absence of NADPH. Pargyline, an MAO inhibitor, decreased tyramine elimination rates by ∼30%. Among recombinant P450 and FMO enzymes, CYP2D6 had a high activity in terms of tyramine elimination. Tyramine elimination rates were inhibited by quinidine and significantly correlated with bufuralol 1'-hydroxylation activities (a CYP2D6 marker). Liver microsomes genotyped for CYP2D6*10/*10 and CYP2D6*4/*4 showed low and undetectable activities, respectively, compared with the wild-type CYP2D6*1/*1. The present results suggest that tyramine is eliminated mainly by polymorphic CYP2D6. Tyramine toxicity resulting from differences in individual metabolic elimination is thus genetically determined.

Here we describe a high capacity and high-throughput, automated, 384-well CYP inhibition assay using wellknown HLM-based MS probes. We provide consistently robust IC50 values at the lead optimization stage of the drug discovery process. Our method uses the Agilent Technologies/Velocity11 BioCel 1200 system, timesaving techniques for sample analysis, and streamlined data processing steps. For each experiment, we generate IC50 values for up to 344 compounds and positive controls for five major CYP isoforms (probe substrate): CYP1A2 (phenacetin), CYP2C9 ((S)- warfarin), CYP2C19 ((S)-mephenytoin), CYP2D6 (dextromethorphan), and CYP3A4/5 (testosterone and midazolam). Each compound is incubated separately at four concentrations with each CYP probe substrate under the optimized incubation condition. Each incubation is quenched with acetonitrile containing the deuterated internal standard of the respective metabolite for each probe substrate. To minimize the number of samples to be analyzed by LC-MS/MS and reduce the amount of valuable MS runtime, we utilize timesaving techniques of cassette analysis (pooling the incubation samples at the end of each CYP probe incubation into one) and column switching (reducing the amount of MS runtime). Here we also report on the comparison of IC50 results for five major CYP isoforms using our method compared to values reported in the literature.

This is with reference to the paper entitled: “Comparative Pharmacokinetics and Metabolism Studies in Lean and Diet-Induced Obese Mice, An Animal Efficacy Model for 11β-Hydroxysteroid Dehydrogenase Type 1 (11β-HSD1) Inhibitors”, authored by Mengmeng Wang, Xianbin Tian, Louis Leung, Jianyao Wang, Nicole Houvig, Jason Xiang, Eddine Saiah, Seung Hahm, Vipin Suri, and Xin Xu, published in Drug Metabolism Letters, Vol. 5, Issue 01, 2011. Due to an oversight on the part of the authors, the name of one of the co-authors, “Dr. Zhao-Kui Wan” was overlooked in the author list of the article. Now the new reference as follows: Wang, M.; Tian, X.; Leung, L.; Wang, J.; Houvig, N.; Xiang, J.; Wan, Z.-K.; Saiah, E.; Hahm, S.; Suri, V.; Xu, X. Comparative Pharmacokinetics and Metabolism Studies in Lean and Diet-Induced Obese Mice: An Animal Efficacy Model for 11β-Hydroxysteroid Dehydrogenase Type 1 (11β-HSD1) Inhibitors. Drug Metab. Lett., 2011, 5, 55-63.