Biomedical subjects

Michael B Fisher

Publications and source records attributed to Michael B Fisher.

9 recordsLinked to original sources

PubMed2006

In vivo use of the P450 inactivator 1-aminobenzotriazole in the rat: varied dosing route to elucidate gut and liver contributions to first-pass and systemic clearance.

The small intestine is regarded as an absorptive organ in the uptake of orally administered drugs, but also has the ability to metabolize drugs by both phase 1 and phase 2 reactions. The amount of drug that reaches the systemic circulation can be reduced by both intestinal and hepatic metabolism. 1-Aminobenzotriazole (ABT) is an irreversible inhibitor of cytochrome P450s. Through in vivo and in vitro studies, ABT has been evaluated for its utility in studying intestinal metabolism in rats. Rats have been exposed to ABT through varied routes of administration followed by p.o. and i.v. administration of midazolam (MDZ), a CYP3A substrate. The MDZ bioavailablity in rats dosed orally and in rats dosed intravenously with ABT is 58.5% and 0.7%, respectively (%F = 2.3% w/o ABT). The approximately 80-fold difference between the two groups suggests the majority of the extraction occurs in the intestine following an oral dose. To further study the utility of ABT, the antihistamine fexofenadine (Fex), which is not significantly metabolized and is a substrate for the uptake and efflux transporters, OATP and P-gp, was tested in rat. There was no change in oral or systemic exposure of Fex when animals were predosed with ABT, suggesting that ABT does not affect these transporters. These findings may lead to a better understanding of the interdependent role of absorption and metabolism and the specificity of ABT. This method should have utility in drug discovery for the identification of factors limiting oral bioavailability.

Administration, Oral↗

PubMed2006

The complexities inherent in attempts to decrease drug clearance by blocking sites of CYP-mediated metabolism.

Oxidative metabolism by the cytochromes P450 (CYPs) is the most common metabolic pathway of drug clearance. Medicinal chemists in drug discovery often synthesize analogs of lead molecules to reduce clearance due to metabolism. One method generally used when attempting to reduce CYP metabolism is to identify the site of modification to 'block' it. Substituting fluorine in the place of hydrogen at metabolically labile positions, for example, is a common approach, although deuterium can also be considered here for simplicity. In this case, the rate of metabolism via a specific pathway is attenuated, but the rate of overall substrate consumption or overall clearance is not significantly altered, due to a compensatory increase in the rate of formation of an alternate metabolite. The concepts and evidence behind this phenomenon as it relates to complexities in blocking metabolic clearance are presented herein.

Animals↗

PubMed2005

A streamlined method to predict hepatic clearance using human liver microsomes in the presence of human plasma.

INTRODUCTION: Human liver microsomal incubations are often used to predict the metabolic lability of new chemical entities. The clearance values are scaled-up from in vitro data and mathematically corrected for plasma protein binding, or in some cases the free fraction ratio of plasma to microsomes, using well-established scaling methods such as the well-stirred model. This can be time consuming for multiple compounds since it requires separate experiments to determine in vitro lability, and free fraction. METHODS: We attempted to streamline clearance predictions by combining experiments into one. Firstly, we combined the free fraction experiments into one free fraction ratio by measuring the partitioning of compound between plasma and microsomes, and by applying this experimental ratio to clearance predictions found that it performed at least as well as free fractions determined separately. We also incubated compounds with plasma added to the incubation mixture and compared the predicted clearances to values determined using traditional mathematical protein binding corrections. RESULTS: Consistently, incubations with added plasma resulted in CL predictions closer to literature values than incubations only mathematically corrected for protein binding. For example, incorporating plasma into a ketamine incubation resulted in a CL value of 15.1 mL/min/kg, compared with a value of 10.2 using mathematical binding corrections. The literature value is 16.4 mL/min/kg. DISCUSSION: This work characterizes this new method and compares it to the traditional microsomal incubation method using several literature compounds, and suggests that streamlining the methods may generate quality data faster and with less resource investment.

Adrenergic beta-Antagonists↗

PubMed2005

Altered AZT (3'-azido-3'-deoxythymidine) glucuronidation kinetics in liver microsomes as an explanation for underprediction of in vivo clearance: comparison to hepatocytes and effect of incubation environment.

Human liver microsomes are a reagent commonly used to predict human hepatic clearance of new chemical entities via phase 1 metabolism. Another common metabolic pathway, glucuronidation, can also be observed in human liver microsomes, although the scalability of this process has not been validated. In fact, several groups have demonstrated that clearance estimated from liver microsomes with UDP-glucuronic acid typically underpredicts the actual in vivo clearance more than 10-fold for compounds that are predominantly glucuronidated. In contrast, clearance predicted using human hepatocytes, for these same compounds, provides a more accurate assessment of in vivo clearance. We sought to characterize the kinetics of glucuronidation of the selective UGT2B7 substrate AZT (3'-azido-3'-deoxythymidine), a selective UGT2B7 substrate, in human liver microsomes (HLMs), recombinant UGT2B7, and human hepatocytes. Apparent Km values in these three preparations were 760, 490, and 87 microM with apparent Vmax values highest in hepatocytes. The IC50 for ibuprofen against AZT glucuronidation, when run at its Km concentration in HLMs and hepatocytes, was 975 and 170 microM respectively. Since incubation conditions have been shown to modulate glucuronidation rates, AZT glucuronidation was performed in various physiological and nonphysiological buffer systems, namely Tris, phosphate, sulfate, carbonate, acetate, human plasma, deproteinized human liver cytosol, and Williams E medium. The data showed that carbonate and Williams E medium, more physiologically relevant buffers, yielded the highest rates of AZT glucuronidation. Km observed in HLM/carbonate was 240 microM closer to that found in hepatocytes, suggesting that matrix differences might cause the kinetic differences observed between liver preparations. Caution should be exercised when extrapolating metabolic lability via glucuronidation or inhibition of UGT enzymes from human liver microsomes, since this system appears to underpredict the degree of lability or inhibition, respectively, due in part to an apparent decrease in substrate affinity.

Buffers↗

PubMed2005

Assessment of UDP-glucuronosyltransferase catalyzed formation of ethyl glucuronide in human liver microsomes and recombinant UGTs.

While ethanol is primarily metabolized to acetaldehyde and acetic acid via alcohol dehydrogenase, a minor but increasingly important pathway in the field of forensic science involves the conjugation of glucuronic acid to form an ethyl glucuronide (EtG) metabolite. The kinetics of ethyl glucuronide formation were examined in human liver microsomes (HLM) and recombinant UDP-glucuronosyltransferases (UGTs). The metabolite exhibited a relatively slow rate of formation in a human liver microsome mix of 75.4 pmol/(min/mg). Further investigation identified multiple UGT isoforms to be responsible for catalyzing the addition of glucuronic acid to ethanol, with UGT1A1 and 2B7 being the two most prevalent isoforms. Co-incubation with bilirubin or 3'-azido-3'-deoxythymidine (UGT1A1 and 2B7 inhibitors, respectively) inhibited the greatest amount of ethyl glucuronide formation, though other UGT inhibitors also showed some effect. Enzyme kinetics were performed in human liver microsomes and recombinant UGT enzymes. The apparent Km (Km app) and Vmax values were determined to be 0.17+/-0.08 mM and 75.98+/-5.63 pmol/(min/mg) (human liver microsomes), 0.03+/-0.01 mM and 25.22+/-3.45 pmol/(min/mg) (UGT1A1), and 0.11+/-0.04 mM and 52.03+/-9.8 pmol/(min/mg) (UGT2B7). Thus, it appears that multiple UGTs are responsible for the formation of ethyl glucuronide and that any functional differences in the enzymology underlying ethyl glucuronide formation would most likely be masked by a combination of other enzymatic pathways.

Alcohol Drinking↗

PubMed2004

Impact of incubation conditions on bufuralol human clearance predictions: enzyme lability and nonspecific binding.

Human liver microsomes (HLMs) are frequently utilized in drug discovery to predict the human clearance of a compound. The extent to which the incubation conditions affect the accuracy of a human clearance prediction was determined for bufuralol. HLMs were preincubated at 37 degrees C for varying times (5-120 min) with and without NADPH, and the remaining enzyme activity was determined by incubating compounds that have been characterized to be selective for individual cytochromes P450 or flavin-containing monooxygenase 3. CYP2D6, the high-affinity component of bufuralol metabolism, was shown to be the least stable of the isoforms studied. The loss of CYP2D6 activity was further examined by determining the kinetics of 1'-hydroxybufuralol formation after different preincubation time periods, by using reactive oxygen species (ROS) scavengers, and by utilizing Western blotting techniques. A 3-fold decrease in Vmax was observed over 2 h, whereas the Km remained constant. ROS scavengers were able to block enzyme lability, and Western blots revealed no apparent loss of immunoreactive enzyme. The protein binding of bufuralol was determined in HLMs, recombinant CYP2D6, and human plasma. A prediction of theoretical bufuralol concentrations over a 120-min incubation that incorporated enzyme lability was performed and shown to be closer to actual data than if enzyme lability were ignored. Finally, a similar prediction using literature bufuralol data, coupled with the observed protein binding data, was used to illustrate that the most accurate predictions of bufuralol clearance are obtained when the amount of protein in the incubation is kept to a minimum and the overall incubation time is less than 20 min.

Adrenergic beta-Antagonists↗

PubMed2003

Prevention of organ allograft rejection by a specific Janus kinase 3 inhibitor.

Because of its requirement for signaling by multiple cytokines, Janus kinase 3 (JAK3) is an excellent target for clinical immunosuppression. We report the development of a specific, orally active inhibitor of JAK3, CP-690,550, that significantly prolonged survival in a murine model of heart transplantation and in cynomolgus monkeys receiving kidney transplants. CP-690,550 treatment was not associated with hypertension, hyperlipidemia, or lymphoproliferative disease. On the basis of these preclinical results, we believe JAK3 blockade by CP-690,550 has potential for therapeutically desirable immunosuppression in human organ transplantation and in other clinical settings.

Animals↗

PubMed2002

Flavin-containing monooxygenase activity in hepatocytes and microsomes: in vitro characterization and in vivo scaling of benzydamine clearance.

Liver microsomes, and more recently cryopreserved hepatocytes, are commonly used in the in vitro characterization of the metabolism of new xenobiotics. The flavin-containing monooxygenases (FMO) are a major non p450 oxidase present in liver microsomes and hepatocytes. Since FMO is known to be thermally labile, and this enzyme may be involved in the metabolic clearance of some drugs, we sought to more completely characterize the metabolic competency of this enzyme in cryopreserved hepatocytes and in liver microsomes preincubated under various conditions using benzydamine as an in vitro and in vivo probe. The metabolism of benzydamine to its major metabolite, the N-oxide, is mediated by FMO3 in humans. We found that the in vitro microsomal t(1/2) was 70% longer when incubations were prewarmed at 37 degrees C in the absence of NADPH compared with prewarming in the presence of an NADPH-regenerating system, and N-oxide formation was inhibited >99%. Interestingly, the in vivo clearance predicted from these incubations and from human hepatocytes overpredicted the observed clearance of benzydamine in humans (>10.5 versus 2.4 ml/min/kg). In contrast, rat hepatocytes successfully predicted rat in vivo benzydamine clearance to within approximately 30% (>68 versus 48 ml/min/kg). Benzydamine N-oxidation in liver microsomes from all common preclinical species demonstrated heat sensitivity. This information should be considered when extrapolating metabolism data of xenobiotics from these in vitro systems.

Animals↗

PubMed2002

Characterization by liquid chromatography-nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry of two coupled oxidative-conjugative metabolic pathways for 7-ethoxycoumarin in human liver microsomes treated with alamethicin.

The microsomal metabolism of 7-ethoxycoumarin (7-EC) was investigated using liquid chromatography (LC)-NMR and liquid chromatography-mass spectrometry (LC-MS) to characterize the coupling of oxidative-conjugative metabolism events. Within microsomes, cytochromes P450 (P450s) and UDP-glucuronosyltransferases (UGTs) are spatially disparate, each having surface and luminal localization, respectively. To optimize cofactor and substrate transit to UGT without compromising P450 activity, the pore-forming peptide alamethicin was used for microsomal perforation. Aqueous extracts of microsomal incubations containing NADPH and UDP-glucuronic acid were injected for LC-NMR and LC-MS analysis. The analytical complementarity of LC-NMR and LC-MS permitted the identification of four metabolites (M1 to M4). The metabolites M1 and M2 are novel microsomal metabolites for 7-EC, consistent with 3-hydroxylation and subsequent glucuronidation, respectively. Metabolites M3 and M4 were 7-hydroxycoumarin (7-HC) and 7-HC glucuronide, respectively. Viewed collectively, these results illustrate the utility of alamethicin in the examination of coupled oxidative-conjugative metabolism and the synergy of LC-NMR and LC-MS in metabolite identification.

7-Alkoxycoumarin O-Dealkylase↗