Marker Substrate Research Articles

Abstract 1381: The effect of proteasome inhibitor velcade on tumor microenvironment

Abstract It has been reported that the therapeutic effect of the proteasome inhibitor Velcade is due to its selective interference in the hypoxia pathway. We studied the effect of Velcade on tumor microenvironment and examined the underlying molecular mechanisms. We have generated a human colorectal cancer xenograft model (#C53) in which the hypoxia-inducible dual reporter fusion gene (HSV1-TK and eGFP) was under the control of hypoxia-response-element (HRE). In vitro, #C53 cells were treated with Velcade in normoxic and hypoxic conditions, and the following assays were performed in comparison with controls: eGFP (flow cytometry), CA9 (western blot), VEGF (ELISA), and TK activity (trapping of the marker substrate 14C-FIAU). In vivo, #C53 xenografts were treated with Velcade and various assays performed, including a) Dynamic contrast-enhanced (DCE) MRI pre and post treatment; b) dual hypoxia marker (pimonidazole and EF5) administration pre and post treatment; c) fluorescence microscopy of Hoechst 33342 (perfusion), eGFP, HIF-1α, and CA9; and d) plasma VEGF level (ELISA), Where applicable data from control and treated tumors were compared. Our results showed that in both in vitro and in vivo experiments Velcade treatment increased the level of HIF1α, but decreased those of hypoxia-induced eGFP, TK, CA9 and VEGF. Interestingly, in the dual hypoxia marker study there were significant EF5-positive regions that did not co-localize with pimonidazole-positive regions, suggesting de novo hypoxia and perhaps another novel effect of Velcade on tumor microvasculature. Consistent with these results, DCE MRI demonstrated decrease global tumor blood flow with Velcade treatment. Our data suggest that Velcade suppresses the hypoxia response by disrupting the HIF1 transcriptional activity. In addition, our results suggest a novel function of Velcade in modifying the tumor microenvironment and decreasing tumor perfusion as noninvasively detected by DCE MRI. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1381.

109 HEPATITIS B VIRUS RESTRAINS ACTIVATION OF SPECIFIC INTERFERON RESPONSIVE GENES IN HUMAN HEPATOCYTES REPOPULATING THE LIVER OF UPA/SCID MICE

had no effect of infectivity of HBV. Macropinocytosis could be ruled out as entry pathway of HBV since uptake of the pinocytic marker substrate horseradish-peroxidase (HRP) was efficiently blocked with Ethylisopropylamiloride (EIPA), but had no impact on HBV infection. Agents that prevent endosomal acidification, e.g. Bafilomycin and NH4Cl, were not able to inhibit HBV infection, but blocked infection with SFV very efficiently. Conclusions: These results suggest that in contrast to other viruses, HBV does not use classical clathrinor caveolin-dependent endocytic mechanism for infection, but an unusual endosomal entry pathway that needs to be determined. A low pH within endosomal compartments, important for infection of many other enveloped viruses is not necessary to establish an HBV infection in primary hepatocytes.

110 MODULATION OF HBV REPLICATION AND CELLULAR GENE EXPRESSION BY MICRORNA-1

had no effect of infectivity of HBV. Macropinocytosis could be ruled out as entry pathway of HBV since uptake of the pinocytic marker substrate horseradish-peroxidase (HRP) was efficiently blocked with Ethylisopropylamiloride (EIPA), but had no impact on HBV infection. Agents that prevent endosomal acidification, e.g. Bafilomycin and NH4Cl, were not able to inhibit HBV infection, but blocked infection with SFV very efficiently. Conclusions: These results suggest that in contrast to other viruses, HBV does not use classical clathrinor caveolin-dependent endocytic mechanism for infection, but an unusual endosomal entry pathway that needs to be determined. A low pH within endosomal compartments, important for infection of many other enveloped viruses is not necessary to establish an HBV infection in primary hepatocytes.

Quantitative characterization of direct P-glycoprotein inhibition by St John's wort constituents hypericin and hyperforin.

The ATP-binding cassette transporter P-glycoprotein (P-gp) exerts a critical role in the systemic disposition of, and exposure to, lipophilic and amphipathic drugs, carcinogens, toxins and other xenobiotics. The ability of P-gp to transfer a wide variety of structurally unrelated compounds from the cell interior across the membrane bilayer remains intriguing. Since natural product chemicals in the widely consumed St John's wort appear to exert antidepressant effects by an unknown mechanism, the constituents are frequently studied for interactions with various biomacromolecules as well as cytotoxins or isolated cells. The drug interactions caused by this widely used herbal remedy are under-appreciated. Various clinical interactions have been observed upon the co-administration of St John's wort, and P-gp and CYP3A4 have been indicted as the cause. We characterized several St John's wort constituents for their interaction with P-gp and their specific effects on the P-gp export activity of several marker substrates. Two of these constituents, hyperforin and hypericin, inhibit the active efflux of the fluorescent markers daunorubicin (IC(50) approximately 30 microM) and calcein-AM. Herein, we show in-vitro results that can both explain the competing clinical observations of initial elevated exposure of P-gp substrate drugs (P-gp inhibition) followed by under-exposure (P-gp induction) when St John's wort is co-administered, and provide a further warning against unchecked co-administration of drugs with St John's wort.

Qualitative Analysis of the Role of Metabolites in Inhibitory Drug‐Drug Interactions: literature evaluation based on the Metabolism and Transport Drug Interaction Database

FDA guidance is specific in the characterization of metabolites of new drugs that may be involved in toxicities, but does not address the potential for metabolites to cause inhibitory drug‐drug interactions. This work was conducted to evaluate what fraction of inhibitors of common drug metabolizing enzymes (CYPs 1A2, 2E1, 2D6, 2C9, 2C19, 2C8, 2B6 and 3A4) have circulating metabolites that may contribute to in vivo interactions. A literature analysis was conducted using the Metabolism and Transport Drug Interaction Database to identify all precipitants (i.e. inhibitors) that cause >20% increase in the AUC of marker substrates. Of 130 inhibitors identified, 106 were confirmed to have metabolites that circulate in plasma. Only 7% of the inhibitors did not have circulating metabolites. Of the 21 potent inhibitors, 17 had circulating metabolites; the remaining 4 were all extensively metabolized. Based on available in vitro data, 21 of the inhibitors were mechanism‐based inactivators of P450 enzymes while 111 were reversible inhibitors. In vitro evaluation of inhibition potential was conducted for only 32% of the circulating metabolites of the inhibitors. We conclude that circulating metabolites are often present with inhibitors of P450 enzymes suggesting a need for increased efforts to characterize the inhibitory potency of metabolites of candidate drugs, and for newer models for in vitro‐in vivo extrapolations.

Qualitative Analysis of the Role of Metabolites in Inhibitory Drug−Drug Interactions: Literature Evaluation Based on the Metabolism and Transport Drug Interaction Database

Guidance from the Food and Drug Administration on drug interaction studies does not include a specific section on contributions of metabolites to observed inhibitory drug-drug interactions, and the quantitative role of drug metabolites in inhibitory drug-drug interactions is not presently known. The current work was undertaken to evaluate what fraction of inhibitors of common drug-metabolizing enzymes [cytochrome P450 (P450) 1A2, 2E1, 2D6, 2C9, 2C19, 2C8, 2B6, and 3A4] have circulating metabolites that may contribute to observed in vivo interactions. A literature analysis was conducted using the Metabolism and Transport Drug Interaction Database to identify all precipitants (i.e., inhibitors) that cause more than a 20% increase in the area under the plasma concentration-time curve (AUC) of marker substrates. The database, PubMed, and product labels were then used to determine whether circulating metabolites were present after administration of these inhibitors. Of the total of 129 inhibitors identified, 106 were confirmed to have metabolites that circulate in plasma. An additional 14 inhibitors were identified that are extensively metabolized but whose metabolites either have not been identified or have not been investigated. Hence, only 7% of the inhibitors did not have circulating metabolites. Of the 21 potent inhibitors (>or=5-fold increase in AUC) currently known, 17 had circulating metabolites, and the remaining four were all extensively metabolized. On the basis of available in vitro data, 24 of all of the inhibitors were mechanism-based inactivators of P450 enzymes, while 105 were characterized as reversible inhibitors. In vitro evaluation of inhibition potential was conducted for only 32% of the circulating metabolites of the inhibitors. In conclusion, circulating metabolites are often present with inhibitors of P450 enzymes, suggesting a need for increased efforts to characterize the inhibitory potency of metabolites of candidate drugs and for newer models for in vitro to in vivo extrapolations.

Aged Garlic Extract Stimulates P-Glycoprotein and Multidrug Resistance Associated Protein 2 Mediated Effluxes

The growing concomitant consumption of drugs and herbal preparations such as garlic, and the numerous reports about the influence of herbal preparations on intestinal transport, led us to evaluate the influence of aged garlic extract on the transport function and electrophysiological parameters of the small intestinal mucosa. Aged garlic extract induced increase of the absolute value of the transepithelial potential difference and of the short-circuit current in both permeability models tested (rat jejunum, Caco-2 cell monolayers) without affecting transepithelial electrical resistance. It also caused a significant increase of the P-glycoprotein and multidrug resistance associated protein 2 mediated effluxes through rat jejunum of marker substrates Rhodamine 123 and 2,4-dinitrophenyl-S-glutathione, respectively. Rhodamine 123 efflux through the Caco-2 cell monolayers was not altered by aged garlic extract, whereas the efflux of 2,4-dinitrophenyl-S-glutathione increased significantly. So altered activity of the important transport proteins could significantly change the pharmacokinetic properties of conventional medicines taken concomitantly with aged garlic extract.

In vitro assessment of P450 induction potential of novel chemopreventive agents SR13668, 9-cis-UAB30, and pentamethychromanol in primary cultures of human hepatocytes

Several compounds, including 2,10-dicarbethoxy-6-methoxy-5,7-dihydroindolo[2,3- b]carbazole (SR13668), (2E,4E,6Z,8E)-8-(3′,4′-dihydro-1′(2′H)-napthalen-1′-ylidene)-3,7-dimethyl-2,4,6-octatrienoic acid (9-cis-UAB30), and 2,2,5,7,8-pentamethyl-6-chromanol (PMCol), were selected as promising chemopreventive agents and have entered preclinical trials for cancer prevention. The potential for adverse drug events resulting from interactions with other administered drugs, food components, or food additives presents an important question. Among the most important drug–drug interactions (DDI) is the potential of a new chemical entity (NCE) to induce cytochrome P450 enzymes (P450). Drug induction of P450 enzymes can lead to adverse drug interactions by increasing the metabolism of other drugs that are substrates for the induced isoform. Currently, sandwich cultured primary human hepatocytes are the standard for predicting human P450 enzyme induction in vitro as these cells retain the ability to respond to prototypical P450 inducers with the same specificity and potency exhibited in vivo. Therefore, a select panel of inducible P450 target genes ( CYP1A2, CYP2B6, and CYP3A4) and their induction activity (measured by LC–MS/MS of respective marker substrate metabolites) were monitored in cultured hepatocytes following treatment with SR13668, 9-cis-UAB30, or PMCol to predict clinically significant drug-induced expression. The concentration ranges of the NCE used were selected to maximize the clinical relevance of these results. All responses were evaluated according to major prototypical P450 inducers (i.e., 3-methylcholanthrene, 3-MC; phenobarbital, PB; rifampicin, RIF) and increases ≥40% of the respective positive control(s) were considered an indication of demonstrable induction. Herein, we report that there is low potential for DDI with SR13668 and PMCol due to enzyme induction of CYP1A2, CYP2B6, and CYP3A4 expression at the concentrations examined. Similarly, the study results suggested that 9-cis-UAB30 has low potential to induce CYP1A2 and CYP3A4 expression at the concentrations examined. However, 9-cis-UAB30 was shown to significantly induce CYP2B6 enzyme activity at 10 μM suggesting the potential for DDI as a result.

Effects of Nicotine Exposure on In Vitro Metabolism of Chlorpyrifos in Male Sprague-Dawley Rats

The routine use of tobacco products may modify key metabolizing systems, which will further impact the metabolism of environmental contaminants. The objective of this study was to quantify the effect of repeated in vivo exposures to nicotine, a major pharmacologically active component of cigarette smoke, on in vitro metabolism of chlorpyrifos (CPF). CPF is an organophosphorus (OP) insecticide that is metabolized by cytochrome P-450 (CYP450) to its major metabolites, chlorpyrifos-oxon (CPF-oxon) and 3,5,6-trichloro-2-pyridinol (TCP). Male Sprague-Dawley rats were dosed subcutaneously with 1 mg nicotine/kg for 1, 7, or 10 d. Rats were sacrificed 4 or 24 h after the last nicotine treatment, and liver microsomes were prepared. The microsomes were incubated with varying concentrations of CPF and the production of the metabolites CPF-oxon and TCP were measured. The metabolism of CPF to the active oxon metabolite did not show significant changes following repeated nicotine treatments, evidenced by the unchanged pseudo first-order clearance rate of V max/K mapp. The V max describing the metabolism of CPF to the inactive metabolite, TCP was increased in 24-h postdosing groups, after both single and repeated treatments of nicotine. In contrast, the metabolism to TCP was unchanged in groups evaluated at 4 h (single or repeated) post nicotine dosing. Some basic marker substrate activities were also investigated to ensure that nicotine exerted effects on CYP450 activities. Total P450 reduced spectra were not altered by nicotine treatment, but marker substrate activities for CYP1A and CYP2E1 were increased at 24 h after the single treatment, and marker substrate activity for CYP2B was decreased 4 h after 7 d of treatment. Results of this in vitro study suggest that repeated nicotine exposure may result in altered metabolism of CPF. Future in vivo experiments based on these results need to be conducted to ascertain the impact of in vivo nicotine exposures on CPF metabolism in rats.

The ontogeny of drug metabolizing enzymes and transporters in the rat

Knowledge of the ontogeny of the various systems involved in distribution and elimination of drugs is important for adequate interpretation of the findings during safety studies in juvenile animals. The present study was designed to collect information on plasma concentrations of total protein and albumin, enzyme activity and mRNA expression of cytochrome P450 isoenzymes (CYP1A1/2, CYP2B1/2, CYP2E1, CYP3A1/2, and CYP4A1), carboxylesterase and thyroxin glucuronidation (T4-GT) activity in liver microsomes, and mRNA expression of transporters (Mdr1a/b, Mrp1–3 and 6, Bsep and Bcrp, Oct1–2, Oat1–3 and Oatp1a4) in liver, kidney and brain tissue during development in Sprague–Dawley rats. Enzyme activities were determined by measuring the metabolism of marker substrates; expression of mRNAs was assessed using RTq-PCR. There were considerable differences in the ontogeny of the individual cytochrome P450 isoenzymes. In addition, ontogeny patterns of enzyme activity did not always parallel ontogeny patterns of mRNA expression. Ontogeny of the transporters depended on the transporter and the organ studied. Changes in mRNA expression of the various transporters during development are likely to result in altered elimination and/or tissue distribution of substrates, with concomitant changes in hepatic metabolism, renal excretion and passage through the blood–brain barrier. Consideration of the ontogeny of metabolizing enzymes and transporters may improve the design and interpretation of results of toxicity studies in juvenile animals.

Pharmacokinetics and dosage adjustment in patients with hepatic dysfunction

The liver plays a central role in the pharmacokinetics of the majority of drugs. Liver dysfunction may not only reduce the blood/plasma clearance of drugs eliminated by hepatic metabolism or biliary excretion, it can also affect plasma protein binding, which in turn could influence the processes of distribution and elimination. Portal-systemic shunting, which is common in advanced liver cirrhosis, may substantially decrease the presystemic elimination (i.e., first-pass effect) of high extraction drugs following their oral administration, thus leading to a significant increase in the extent of absorption. Chronic liver diseases are associated with variable and non-uniform reductions in drug-metabolizing activities. For example, the activity of the various CYP450 enzymes seems to be differentially affected in patients with cirrhosis. Glucuronidation is often considered to be affected to a lesser extent than CYP450-mediated reactions in mild to moderate cirrhosis but can also be substantially impaired in patients with advanced cirrhosis. Patients with advanced cirrhosis often have impaired renal function and dose adjustment may, therefore, also be necessary for drugs eliminated by renal exctretion. In addition, patients with liver cirrhosis are more sensitive to the central adverse effects of opioid analgesics and the renal adverse effects of NSAIDs. In contrast, a decreased therapeutic effect has been noted in cirrhotic patients with beta-adrenoceptor antagonists and certain diuretics. Unfortunately, there is no simple endogenous marker to predict hepatic function with respect to the elimination capacity of specific drugs. Several quantitative liver tests that measure the elimination of marker substrates such as galactose, sorbitol, antipyrine, caffeine, erythromycin, and midazolam, have been developed and evaluated, but no single test has gained widespread clinical use to adjust dosage regimens for drugs in patients with hepatic dysfunction. The semi-quantitative Child-Pugh score is frequently used to assess the severity of liver function impairment, but only offers the clinician rough guidance for dosage adjustment because it lacks the sensitivity to quantitate the specific ability of the liver to metabolize individual drugs. The recommendations of the Food and Drug Administration (FDA) and the European Medicines Evaluation Agency (EMEA) to study the effect of liver disease on the pharmacokinetics of drugs under development is clearly aimed at generating, if possible, specific dosage recommendations for patients with hepatic dysfunction. However, the limitations of the Child-Pugh score are acknowledged, and further research is needed to develop more sensitive liver function tests to guide drug dosage adjustment in patients with hepatic dysfunction.

Inhibitory Effects on Cytochrome P450 Enzymes of Pentamidine and Its Amidoxime Pro‐Drug

Pentamidine is an antimicrobial drug, intravenously used in the treatment of trypanosomiasis, leishmaniasis or pneumocystis pneumonia. To elucidate potential drug interactions with pentamidine and N,N'-dihydroxypentamidine, respectively, the cytochrome P450 (CYP450) inhibitory properties of these compounds were determined. The study was performed in vitro by using human liver microsomes and marker substrates of several CYP450 isoenzymes. Marker activities were investigated by high-performance liquid chromatography in presence of known selective inhibitors or at different concentrations of pentamidine and N,N'-dihydroxypentamidine, respectively. No or only minor influence on CYP1A2, 2A6, 2C9, 2C19, 2D6, 2E1 and 3A4 marker activities could be observed, suggesting that neither of the tested substances would exert a significant effect on hepatic CYP450 isoenzymes responsible for the metabolism of co-administrated drugs in vivo. However, in vivo studies are needed before final conclusions can be drawn.

Important Amino Acid Residues that Confer CYP2C19 Selective Activity to CYP2C9

Although CYP2C9 and CYP2C19 display 91% sequence identity at the amino acid level, the two enzymes have distinct substrate specificities for compounds such as diclofenac, progesterone and (S)-mephenytoin. Amino acid substitutions in CYP2C9 were made based on an alignment of CYP2C9, CYP2C19 and monkey CYP2C43 sequences. Mutants of CYP2C9 were expressed in Escherichia coli. Sixteen amino acids, which are common to both CYP2C19 and CYP2C43 but different between CYP2C9 and CYP2C19, were substituted in CYP2C9 (CYP2C9-16aa). Next, the mutated amino acids in CYP2C9-16aa were individually reverted to those of CYP2C9 to examine the effect of each substitution on the enzymatic activity for CYP2C marker substrates. In addition, the role of the F-G loop in CYP2C9 and CYP2C19 was examined for substrate specificity and enzymatic activity. Our results showed: (i) CYP2C9-16aa displays 11% (S)-mephenytoin 4'-hydroxylase and full omeprazole 5-hydroxylase activity compared with that of CYP2C19; (ii) residue 286 is important for conferring CYP2C9-like enzyme activity on CYP2C9-16aa and residue 442 in CYP2C19 may be involved in the interaction with NADPH-P450 reductase; (iii) substitution of the F-G loop in CYP2C9 to that of CYP2C19 enhances tolbutamide p-methyhydroxylase and diclofenac 4'-hydroxylase activities and confers partial (S)-mephenytoin 4'-hydroxylase and omeprazole 5-hydroxylase activities, which are attributed to CYP2C19.

Glucuronidation of the soyabean isoflavones genistein and daidzein by human liver is related to levels of UGT1A1 and UGT1A9 activity and alters isoflavone response in the MCF-7 human breast cancer cell line

The soyabean isoflavones genistein and daidzein, which may protect against some cancers, cardiovascular disease and bone mineral loss, undergo substantial Phase 2 metabolism, predominantly glucuronidation. We observed a correlation between rates of metabolism of marker substrates of specific UGTs and rates of glucuronidation of genistein and daidzein in vitro by a panel of human liver microsomes, demonstrating that UGT1A1 and UGT1A9, but not UGT1A4, make a major contribution to the metabolism of these isoflavones by human liver. These findings were substantiated by observations that recombinant human UGT1A1 and UGT1A9, but not UGT1A4, catalysed the production of the major glucuronides of both genistein and daidzein in vitro. Recombinant human UGT1A8 also metabolised both genistein and daidzein, whereas UGT1A6 was specific to genistein and UGTs 2B7 and 2B15 were inactive, or only marginally active, with either isoflavone as substrate. The intestinal isoform UGT1A10 metabolised either both isoflavones or genistein only, depending on the commercial supplier of the recombinant enzyme, possibly as a result of a difference in amino acid sequence, which we were unable to confirm. Daidzein (16 μM) increased cell death in the MCF-7 human breast cancer cell line and this effect was reversed by glucuronidation. In view of a well-characterised functional polymorphism in UGT1A1, these observations may have implications for inter-individual variability in the potential health-beneficial effects of isoflavone consumption.

Effect of Breed upon Cytochromes P450 and Phase II Enzyme Expression in Cattle Liver

Cattle represent an important source of animal-derived food-products; nonetheless, our knowledge about the expression of drug-metabolizing enzymes (DMEs) in present and other food-producing animals still remains superficial, despite the obvious toxicological consequences. Breed represents an internal factor that modulates DME expression and catalytic activity. In the present work, the effect of breed upon relevant phase I and phase II DMEs was investigated at the pretranscriptional and post-translational levels in male Charolais (CH), Piedmontese (PM) and Blonde d'Aquitaine (BA) cattle. Because specific substrates for cattle have not yet been identified, the breed effect upon specific cytochrome P450 (P450), UDP-glucuronosyltransferase (UGT), or glutathione S-transferase (GST) DMEs, in terms of catalytic activity, was determined by using human marker substrates. Among P450s, benzphetamine N-demethylase, 16beta-, 6beta-, and 2beta-testosterone hydroxylase, aniline and p-nitrophenol hydroxylase, and alpha-naphthol and p-nitrophenol UGT activities were significantly higher in CH; in contrast, lower levels of CYP1A1-, CYP1A2-, CYP2B6-, CYP2C9-, CYP2C18-, CYP3A4-, and UGT1A1-like mRNAs were noticed, with CH < PM < or = BA as a trend. CYP2B and CYP3A mRNA results were confirmed with immunoblotting, too. As regards conjugative DMEs, UGT1A6-like mRNA levels were consistent with respective catalytic activities. Both 1-chloro-2,4-dinitrobenzene and 3,4-dichloronitrobenzene GST activities were higher in BA, and these results agreed with GSTA1-, GSTM1-, and GSTP1-like mRNA amounts. Correlation analysis between catalytic activities and mRNAs showed either significant or uneven results, depending on the substrate. These findings confirm previous data obtained in laboratory species; however, further studies are required to ascribe this behavior to pretranscriptional or post-translational phenomena.

Approach to predict the contribution of cytochrome P450 enzymes to drug metabolism in the early drug-discovery stage: The effect of the expression of cytochrome b5 with recombinant P450 enzymes

In order to evaluate the potential adverse effects due to genetic polymorphism and/or inter-individual variation, it is necessary to calculate the cytochrome P450 (CYP) contribution to the metabolism of new drugs. In the current study, the in vitro intrinsic clearance (CLint) values of marker substrates and drugs were determined by measuring metabolite formation and substrate depletion, respectively. Recombinant CYP microsomes expressing CYP2C9, CYP2C19 and CYP3A4 with co-expressed cytochrome b5 were used, but those expressing CYP1A2 and CYP2D6 did not have co-expressed cytochrome b5. The following prediction methods were compared to determine the CLint value using data from recombinant CYP enzymes: (1) relative CYP enzyme content in human liver microsomes; (2) relative activity factor (RAF) estimated from the Vmax value; and (3) RAF estimated from the CLint value. Estimating RAF from CLint proved the most accurate prediction method among the three tested, and differences in the CYP3A4 marker reactions did not affect its accuracy. The substrate depletion method will be useful in the early drug-discovery stage when the main metabolite and/or metabolic pathway has not been identified. In addition, recombinant CYP microsomes co-expressed with cytochrome b5 might be suitable for the prediction of the CLint value.

Point:Counterpoint: There is/is not capillary recruitment in active skeletal muscle during exercise

#### Focus. The key issue here is whether resting skeletal muscle is fully perfused. We will make the case that it is not, and thus there exists a reserve of unperfused capillaries (capillary reserve) that are recruited to carry flow not only by muscle contraction, but also by insulin. #### Our

In vitro evaluation of human cytochrome P450 and P-glycoprotein-mediated metabolism of some phytochemicals in extracts and formulations of African potato

African potato (Hypoxis hemerocallidea, AP) is a traditional herbal medicine widely used as an immune booster and also for the treatment of various ailments such as urinary diseases, prostrate hypertrophy and cancer. Amongst the chemical components contained in AP, the norlignan glycoside, hypoxoside (HYP) is purported to be the most important phytochemical in terms of AP's medicinal value. Additional constituents in AP include the sterols, beta-sitosterol (BSS), stigmasterol (STG), and the stanol, stigmastanol (STN). The potential of extracts of AP, AP formulations as well as HYP, its aglycone rooperol (ROP) and the sterols to inhibit in vitro metabolism of drug marker substrates by human cytochrome P450 (CYP) enzymes such as CYP3A4, 3A5 and CYP19 were investigated. Samples were also assessed for their effect on drug transport proteins such as P-glycoprotein (P-gp). The effects on CYP-mediated metabolism were studied by fluorometric microtitre plate assay. The potential interaction with P-gp was investigated by measuring the efflux of the fluorescent dye rhodamine 123 (Rh 123) in the CaCo-2 (colon carcinoma) cell line. Various extracts of AP, AP formulations, only STG and the norlignans, in particular the aglycone ROP, exhibited inhibitory effects on CYP3A4-, 3A5- and 19-mediated metabolism. The extracts and the formulations that contained a significant amount of HYP showed high induction of P-gp compared to the positive control, ritonavir. Whilst extrapolation of the current in vitro findings to clinical effects may well be considered speculative, these in vitro data should be heeded as a signal of possible in vivo interactions. Appropriate measures are therefore necessary to explore the possibility of such in vitro-in vivo correlations.

Rifampin Induces the in Vitro Oxidative Metabolism, but Not the in Vivo Clearance of Diclofenac in Rhesus Monkeys

Effects of rifampin on in vitro oxidative metabolism and in vivo pharmacokinetics of diclofenac (DF), a prototypic CYP2C9 marker substrate, were investigated in rhesus monkeys. In monkey hepatocytes, rifampin markedly induced DF 4'-hydroxylase activity, with values for EC(50) of 0.2 to 0.4 microM and E(max) of 2- to 5-fold over control. However, pretreatment with rifampin did not alter the pharmacokinetics of DF obtained after either i.v. or intrahepatic portal vein (i.pv.) administration of DF to monkeys. At the dose studied, plasma concentrations of rifampin reached 10 microM, far exceeding the in vitro EC(50) values. Under similar treatment conditions, rifampin was previously shown to induce midazolam (MDZ) 1'-hydroxylation in rhesus monkey hepatocytes (EC(50) and E(max) values approximately 0.2 microM and approximately 2- to 3-fold, respectively), and markedly affected the in vivo pharmacokinetics of MDZ (>10-fold decreases in the i.pv. MDZ systemic exposure and its hepatic availability, F(h)) in this animal species. In monkey liver microsomes, DF underwent, predominantly, glucuronidation, and, modestly, oxidation; the intrinsic clearance (CL(int) = V(max)/K(m)) value for the glucuronidation pathway accounted for >95% (versus about 75% in human liver microsomes) of the total (glucuronidation + hydroxylation) intrinsic clearance value. In monkey hepatocytes, the hydroxylation also was a minor component (< or =10%) relative to the glucuronidation, supporting the liver microsomal finding. Collectively, our results suggest that the oxidative metabolism is not the major in vivo clearance mechanism of DF in either untreated or rifampin-treated monkeys and, conceivably, also in humans, raising a question about the utility of DF as an in vivo CYP2C9 probe.

EFFECTS OF INDIVIDUAL GINSENOSIDES, GINKGOLIDES AND FLAVONOIDS ON CYP2C19 AND CYP2D6 ACTIVITY IN HUMAN LIVER MICROSOMES

1. The effects of four individual ginsenosides (Rb1, Rb2, Rc and Rd), two ginkgolides (A and B) and one flavonoid (quercetin) on CYP2C19-dependent S-mephenytoin 4 cent-hydroxylation and CYP2D6-mediated bufuralol 1 cent-hydroxylation were evaluated in human liver microsomes. 2. Increasing concentrations of each test compound were added to microsomal incubation mixtures containing a well-characterized marker substrate (S-mephenytoin for CYP2C19 or bufuralol for CYP2D6) to determine their IC(50) values (compound concentration yielding 50% inhibition of a marker enzyme activity), which were estimated by graphical inspection. 3. For CYP2C19, the IC(50) values were 46, 46 and 62 micromol/L for ginsenoside Rd, quercetin and ginsenoside Rb2, respectively, whereas only ginsenoside Rd had an IC(50) value of 57 micromol/L for CYP2D6. 4. The data suggest that the tested compounds are not likely to inhibit the metabolism of the concurrent use of a given drug whose primary route of elimination is through CYP2C19 or CYP2D6.