Human Microsomal Preparations Research Articles

Mechanistic Assessment of Extrahepatic Contributions to Glucuronidation of Integrase Strand Transfer Inhibitors.

Integrase strand transfer inhibitor (INSTI)-based regimens dominate initial human immunodeficiency virus treatment. Most INSTIs are metabolized predominantly via UDP-glucuronosyltransferases (UGTs). For drugs predominantly metabolized by UGTs, including INSTIs, in vitro data recovered from human liver microsomes (HLMs) alone often underpredict human oral clearance. While several factors may contribute, extrahepatic glucuronidation may contribute to this underprediction. Thus, we comprehensively characterized the kinetics for the glucuronidation of INSTIs (cabotegravir, dolutegravir, and raltegravir) using pooled human microsomal preparations from liver (HLMs), intestine (HIMs), and kidney (HKMs) tissues; human embryonic kidney 293 cells expressing individual UGTs; and recombinant UGTs. In vitro glucuronidation of cabotegravir (HLMs≈HKMs>>>HIMs), dolutegravir (HLMs>HIMs>>HKMs), and raltegravir (HLMs>HKMs>> HIMs) occurred in hepatic and extrahepatic tissues. The kinetic data from expression systems suggested the major enzymes in each tissue: hepatic UGT1A9 > UGT1A1 (dolutegravir and raltegravir) and UGT1A1 (cabotegravir), intestinal UGT1A3 > UGT1A8 > UGT1A1 (dolutegravir) and UGT1A8 > UGT1A1 (raltegravir), and renal UGT1A9 (dolutegravir and raltegravir). Enzymes catalyzing cabotegravir glucuronidation in the kidney and intestine could not be identified unequivocally. Using data from dolutegravir glucuronidation as a prototype, a "bottom-up" physiologically based pharmacokinetic model was developed in a stepwise approach and predicted dolutegravir oral clearance within 4.5-fold (hepatic data only), 2-fold (hepatic and intestinal data), and 32% (hepatic, intestinal, and renal data). These results suggest clinically meaningful glucuronidation of dolutegravir in tissues other than the liver. Incorporation of additional novel mechanistic and physiologic underpinnings of dolutegravir metabolism along with in silico approaches appears to be a powerful tool to accurately predict the clearance of dolutegravir from in vitro data.

Chromatographic separation of piracetam and its metabolite in a mixture of microsomal preparations, followed by an MS/MS analysis

A rapid bioanalytical method was evaluated for the simultaneous determination of piracetam and its metabolite (M1) in human microsomal preparations by fast ultra-performance liquid chromatography/tandem mass spectrometry (UPLC–MS/MS). In addition, a validated method of M1 in rat plasma was developed and successfully applied on pharmacokinetic studies. The present study was carried out to determine the metabolic pathways of piracetam for phase I metabolism and used cytochrome P450 isoforms responsible for the piracetam metabolism in human liver microsomes (HLMs). While additional potential metabolites of piracetam were suggested by computer-modeling. The resulting 2-(2-oxopyrrolidin-1-yl) acetic acid was the sole metabolite detected after the microsomal treatment. The amide hydrolysis mainly underwent to form a metabolite i.e., 2-(2-oxopyrrolidin-1-yl) acetic acid (M1).

In Vitro Metabolism of Phenstatin: Potential Pharmacological Consequences

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 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.

Abstract 3564: Design, synthesis and SAR studies leading to SB1518, a novel macrocyclic JAK2/FLT3 inhibitor in phase 2 clinical trials for myelofibrosis and lymphoma

Abstract Introduction: Here we present the unique chemical structure of SB1518 and SAR studies leading to the discovery of this exciting novel JAK2/FLT3 inhibitor, currently in clinical trials for myelofibrosis and lymphoma. Methods/Results: As part of our innovative chemical strategy towards creating novel kinase scaffolds we discovered a series of unusual small molecular weight macrocycles which were optimized for selective JAK2 inhibitory potency culminating in the discovery of SB1518, which is selective for JAK2 (IC50 = 22nM) and its V617F mutant (IC50 = 19nM) over JAK1 (IC50 > 1µM) and JAK3 (IC50 > 0.5µM). FLT3 was identified as an additional kinase target (IC50 = 22nM and 6nM for its D835Y mutant) giving a broader potential in other hematological malignancies. The exciting and novel chemical structures of these compounds led us into a full lead optimization program to take full advantage of the unique nature of these molecules. SAR studies modifying the macrocyclic linker allowed unwanted protein kinase activity to be tuned out, whilst retaining a narrow spectrum of desired JAK2 kinase activity and selectivity over JAK1 and JAK3, a distinguishing feature. SB1518 and analogues were synthesized via a ring-closing metathesis (RCM) strategy: coupling of the two halves of the molecule provided a diene which was closed by RCM using either Grubbs or Zhan catalysts. SAR investigations, led by structure-based design, influenced the choice and position of a critical solubilising side-chain. Preferred compounds had improved aqueous solubility and high permeability. SB1518 is very stable both chemically and metabolically with a long half life in human microsomal preparations. With the desired balance of potency and oral pharmacokinetics, SB1518 was selected for further profiling. SB1518 demonstrated potent activity in cell lines driven by mutant JAK2 or FLT3 and was efficacious at very well tolerated oral doses in tumor models generated with these cell lines. Based on its attractive preclinical profile, SB1518 was selected for clinical development. Conclusions: Discovery of a new series of small molecule macrocycles of a unique and highly novel chemical structure are presented as inhibitors of JAK2 and FLT3 kinases. SAR studies directed us towards an optimal linker and solubilising side chain fine-tuning the molecular structure. SB1518 was selected as a development candidate with an attractive and distinguishing JAK2 selective inhibitory profile and additional activity against FLT3. SB1518 has shown encouraging clinical activity demonstrating that its attractive preclinical profile translates to the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3564. doi:10.1158/1538-7445.AM2011-3564

Malaria Causal Prophylactic Activity of Imidazolidinedione Derivatives

A series of acid-stable carboxamide derivatives of 2-guanidinoimidazolidinedione (5a-c and 6a-c) were prepared as potential malaria prophylactic and radical cure agents. The new compounds showed moderate to good causal prophylactic activity in mice infected with Plasmodium yoelii sporozoites. Three compounds were further tested for causal prophylactic activity in Rhesus monkeys infected with Plasmodium cynomolgi sporozoites, and all showed a delay in patency from 13 to 40 days at 30 mg/kg/day x 3 days by IM dosing. Two out of four compounds tested for radical curative activity in Rhesus showed cure at 30 mg/kg/day x 3 days. The other two compounds showed delay in relapse from 16 to 68 days. Conversion of new carboxamides (5 and 6) to s-triazine derivatives (7) was demonstrated in mouse and human microsomal preparations and in rat plasma. The results suggest the metabolites, s-triazine derivatives 7, may be the active species of the new carboxamides 5a-c and 6a-c prepared in this study.

Metabolism of Moexipril to Moexiprilat: Determination of In Vitro Metabolism Using HPLC-ES-MS

Moexipril is a long-acting, non-sulfhydryl angiotensine-converting enzyme inhibitor. It is used for treatment of arterial hypertension. Moexipril is the prodrug, yielding moexiprilat by hydrolysis of an ethyl ester group. Moexiprilat is the metabolite responsible for the pharmacological effect after moexipril administration. Samples of rat and human microsomal preparations exposed to moexipril treatment were analyzed by HPLC using octyl silica stationary phase and isocratic elution. To detect moexipril and moexiprilat the separation was monitored by both ultraviolet and mass specific detection. The rat liver microsomal preparation was more effective to in producing moexiprilat than the similar one derived from human liver cell lines. While additional potential metabolites of moexipril were suggested by computer-modeling, moexiprilat was the sole metabolite detected after microsomal treatment.

Metabolic activation of benzo[c]phenanthrene by cytochrome P450 enzymes in human liver and lung.

The environmentally occurring polycyclic aromatic hydrocarbon (PAH) benzo[c]phenanthrene (B[c]PH) is a weak carcinogen in rodents. In contrast, the dihydrodiol-epoxides of B[c]PH are among the most carcinogenic PAH metabolites tested so far. In rodents, B[c]PH is predominantly metabolized to B[c]PH-5,6-dihydrodiol (B[c]PH-5,6-DH) and only to a minor extent to B[c]PH-3,4-DH, the proximate precursor of the highly potent ultimate carcinogen, B[c]PH-3,4-DH-1,2-epoxide. This might explain why in rodents B[c]PH is a weak carcinogen. However, little is known about human metabolism of B[c]PH. Using microsomal preparations from human liver and lung, we investigated the metabolic activation of B[c]PH. In contrast to the findings in experimental animals, human liver microsomes predominantly generated B[c]PH-3,4-DH and only to a minor extent B[c]PH-5,6-DH. Only one lung tissue sample was found to be metabolically active, producing B[c]PH-5,6-DH together with small amounts of B[c]PH-3,4-DH. Catalytic activities known to be associated with specific cytochrome P450 (P450) enzyme activities were determined and correlated with the spectrum of B[c]PH metabolites. The results indicate that B[c]PH-DH formation in human liver is mainly mediated by P450 1A2. Studies with P450 enzyme selective inhibitors confirmed these findings. Further support was obtained using preparations of the respective human recombinant P450 enzymes expressed in Escherichia coli and yeast. In addition to P450 1A2, P450 1B1 effectively mediated B[c]PH-metabolism. The umu-assay for induction of SOS repair response in Salmonella typhimurium TA 1535 pSK 1002 containing a umuC-lacZ reporter gene was used to study metabolic generation of genotoxic metabolites from B[c]PH-DHs in human microsomal preparations. B[c]PH-3,4-DH was activated by human liver microsomes to a potent genotoxic agent. Taken together, the results clearly demonstrate that human liver microsomes can effectively catalyze the biotransformation of B[c]PH into highly genotoxic metabolites. The results provide evidence that B[c]PH should be considered a potentially potent carcinogen in humans, and that rodent models may underestimate the risk.

Assessment of [ 18F]fluoroethylflumazenil metabolites using high-performance liquid chromatography and tandem mass spectrometry

A simple procedure using HPLC and tandem mass spectrometry has been developed for the determination of fluoroethylflumazenil metabolites. Samples were precipitated with acetonitrile, evaporated to dryness followed by reconstitution with methanol. As mobile phase, 50 m M ammonium formate–methanol (58:42, v/v) was used. The method is valid both for cold and radiolabelled metabolites. Various cold metabolites (hydroxylated and/or dealkylated) were identified in rat and human microsome preparations. Radiolabelled metabolites arise from two or more transformations including hydroxylation. The methodology developed can be applied for further characterisation of metabolites, and for the determination of non metabolised [ 18F]fluoroethylflumazenil in routine clinical analysis.

Metabolic activation of pyrrolizidine alkaloids by human, rat and avocado microsomes

Two pyrrolizidine alkaloids and one pyrrolizidine alkaloid-N-oxide were incubated with microsomal preparations from humans, rat and avocado and the product profiles examined. The alkaloids were converted to dehydroretronecine, the putative toxic metabolite, by both rat and human microsomal preparations. In addition, alkaloid-N-oxides, the major detoxication products from pyrrolizidine alkaloids, were also formed. The pyrrolizidine alkaloid-N-oxide was converted to both dehydroretronecine and the parent alkaloid. This suggests that the toxicity of pyrrolizidine alkaloid-N-oxides could be greater than suggested hitherto as a result of conversion to the toxic metabolite via the parent alkaloid. Quantitative differences in the proportions of products formed by the different microsomal preparations may be of significance in the extrapolation of toxicological data from animal models such as the rat to humans.

Oxidation of 1,2-epoxy-3-butene to 1,2:3,4-diepoxybutane by cDNA-expressed human cytochromes P450 2E1 and 3A4 and human, mouse and rat liver microsomes

1,3-Butadiene is carcinogenic in B6C3F1 mice and Sprague-Dawley rats, and has been classified as a probable human carcinogen. The genetic basis for butadiene carcinogenicity is likely mediated by its metabolite, 1,2:3,4-diepoxybutane (BDE). Oxidation of butadiene to 1,2-epoxy-3-butene (BMO) and further activation to BDE is catalysed by cytochrome P450 (CYP) isozymes. The production of BMO from butadiene is mediated by CYP2E1 and, at high butadiene concentrations, by CYP2A6. The purpose of the present study was to identify which human CYP isozymes have the ability to oxidize BMO to BDE, and to determine the extent to which this reaction occurs in B6C3F1 mouse, Sprague-Dawley rat, and human liver microsomes. Of the human cDNA-expressed CYP isozymes tested, only CYP2E1 formed detectable concentrations of BDE at 80 microM BMO. CYP2E1 and CYP3A4 were active at 5.0 mM BMO. Interindividual and interspecies variation in the initial rate of oxidation of 80 microM BMO to BDE was determined using 10 samples of human liver microsomes and single pooled samples from rats and mice. Those experiments revealed a 60-fold variation in activity among 10 human liver samples (range: 0.005-0.324 nmol/mg protein/min). Rates of BMO oxidation for mouse and rat liver microsomes were 0.473 and 0.166 nmol/mg protein/min, respectively. Apparent kinetic constants for the oxidation of BMO to BDE by four human microsomal preparations, and pooled samples from mice and rats were estimated from detailed investigations of BMO oxidation at various BMO substrate concentrations. Apparent Km for the human liver samples ranged from 0.304-0.880 mM, and Vmax values ranged from 0.38 to 1.2 nmol/mg protein/min. The apparent values of Km and Vmax for mouse liver microsomes were 0.141 +/- 0.007 mM (mean +/- SE) and 1.303 +/- 0.141 nmol/mg protein/min, respectively. For rat liver microsomes, apparent Km and Vmax were 0.145 +/- 0.036 mM and 0.408 +/- 0.031 nmol/mg protein/min, respectively. Measured rates of BDE formation correlated well with CYP2E1 protein concentrations in the human microsome samples. These results implicate human CYP2E1 as a hepatic isoform responsible for the oxidation of BMO to BDE at low concentrations of BMO. Moreover, our in vitro results reveal that microsomes prepared from human, rat and mouse liver possess the ability to form BDE from BMO. Previous in vitro results suggest that following exposure to butadiene more BMO would probably be present in mice than in rats or humans. Thus, in mice more BMO would be available for activation to BDE.(ABSTRACT TRUNCATED AT 400 WORDS)

Affinity labeling of vertebrate oxidosqualene cyclases with a tritiated suicide substrate

Pig and rat liver oxidosqualene cyclase (OSC) enzymes were purified to homogeneity and showed single bands on SDS-polyacrylamide gel electrophoresis with molecular masses of 75 kDa (pig) and 78 kDa (rat). Pig liver OSC was purified for the first time (441-fold with a yield of 39%). Chemical affinity labeling of pure or crude preparations of the liver cyclases using the mechanism-based irreversible inhibitor of OSC, [3H]29-methylidene-2,3-oxidosqualene ([3H]29-MOS), showed a single radioactive band at 75 kDa (pig) and 78 kDa (rat). Affinity labeling experiments were also performed with dog and human microsomal preparations and with yeast and plant cyclases. All of the vertebrate OSC enzymes were specifically labeled with [3H]29-MOS and gave a single band with molecular masses ranging from 70 to 80 kDa (rat, 78 kDa; dog, 73 kDa; pig, 75 kDa; and human, 73 kDa). In contrast, yeast lanosterol cyclase and plant cycloartenol cyclase were not labeled, demonstrating subtle differences in the active sites of animal, plant, and fungal enzymes.

Isolation of an immunosuppressive metabolite of FK506 generated by human microsome preparations

Metabolism of FK506, a 23 member macrolide under clinical investigation as immunosuppressant after transplantation, was studied using human liver microsomes. Two fractions isolated by semi-preparative HPLC were identified by negative fast atom bombardment mass spectrometry as FK506 metabolites with mass peaks at m/z = 790 indicating demethylation of the mother compound. The immunosuppressive activity of one metabolite was evaluated in a ConA-stimulated peripheral rat lymphocyte assay. FK506 had an IC50 of 0.186 nmol/L and the metabolite tested of 1.89 nmol/L.

Stereochemical studies on the cytochrome P-450 catalyzed oxidation of (S)-nicotine to the (S)-nicotine delta 1'(5')-iminium species.

Mammals metabolize the tobacco alkaloid (S)-nicotine primarily to the lactam (S)-cotinine by a pathway involving an initial cytochrome P-450 catalyzed two-electron oxidation at the prochiral 5'-carbon atom. The stereochemical course of this oxidation was examined with human microsomal preparations and the E and Z diastereomers of (S)-nicotine-5'd1. The metabolically generated delta 1'(5')-iminium ion intermediate was trapped and analyzed as the corresponding diastereomeric 5'-cyano derivatives by a capillary column GC-EIMS selected ion monitoring assay. The results of these studies established that this biotransformation proceeds with the stereoselective abstraction of the 5'-pro-E proton, that is, the C-5' proton trans to the bulky pyridyl group. The observed stereoselectivity was independent of proton vs. deuteron abstraction. Additionally, the extent of (S)-cotinine formation was minor and did not influence the stereochemical composition of the metabolically derived alpha-cyano amines. Studies with male Dutch rabbit liver microsomal preparations gave similar results. These findings suggest that the structure of the complex formed between (S)-nicotine and the active site of cytochrome P-450 is highly ordered and dictates the stereochemical course of the reaction pathway.