Guinea Pig Liver Microsomes Research Articles

Novel amide and imidazole compounds as potent hematopoietic prostaglandin D2 synthase inhibitors

In seeking novel and potent small molecule hematopoietic prostaglandin D2 synthase (H-PGDS) inhibitors as potential therapies for PGD2-mediated diseases and conditions, we explored a series comprising multiple aryl/heteroaryl rings attached in a linear arrangement. Each compound incorporates an amide or imidazole “linker” between the pyrimidine or pyridine “core” ring and the “tail” ring system. We synthesized and screened twenty analogs by fluorescence polarization binding assay, thermal shift assay, glutathione S-transferase inhibition assay, and a cell-based assay measuring suppression of LPS-induced PGD2 stimulation. Amide analogs show ten-fold greater shift in the thermal shift assay in the presence of glutathione (GSH) versus the same assay run in the absence of GSH. The imidazole analogs did not produce a significant change in thermal shift between the two assay conditions, suggesting a possible stabilization effect of the amide linker in the synthase-GSH-inhibitor complex. Imidazole analog 23, (KMN-010034) demonstrates superior potency across the in vitro assays and good in vitro metabolic stability in both human and guinea pig liver microsomes.

Differences in xanthotoxin metabolites in seven mammalian liver microsomes.

Xanthotoxin, abundantly occurring in fruits, vegetables, grapefruit juice and oils, is widely used in medicine for the treatment of psoriasis and vitiligo. Xanthotoxin possesses the ability to inhibit mechanism-based cytochrome P450 (CYP450)-mediated activities in rats and mice. Furthermore, it time-dependently obstructs a number of CYP450-mediated functions in humans. CYP450 enzymes are most abundant in the liver and induce metabolic activation of numerous xenobiotic compounds. The present study aimed to identify the similarities and differences in xanthotoxin metabolism in liver microsomes of 7 mammalian species, including human liver microsomes (HLM), Rhesus monkey liver microsomes (RMLM), Cynomolgus monkey liver microsomes (CMLM), Sprague Dawley rat liver microsomes (RLM), mouse liver microsomes (MLM), Dunkin Hartley guinea pig liver microsomes (PLM) and Beagle dog liver microsomes (DLM). Ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometric analysis was used to determine the metabolites. A total of 3 metabolites were detected in RMLM, CMLM and RLM. Furthermore, two metabolites were observed in MLM, HLM, PLM and DLM. By analyzing the type and quantity of metabolites, the metabolism of xanthotoxin in MLM was indicated to be most similar to that in HLM. The metabolic transformations of xanthotoxin in the liver microsomes of the 7 species were analyzed in further detail. On the whole, the results of the present study provide a deeper understanding of the metabolic patterns of xanthotoxin in liver microsomes of different species, which may prove to be advantageous regarding the metabolic mechanisms of action of xanthotoxin. Further insight into drug metabolism with respect to different species will also aid in the selection of appropriate animal models for further research.

Distribution and excretion of 2,2′,3,4′,5,5′,6-heptachlorobiphenyl (CB187) and its metabolites in rats and guinea pigs

4-Hydroxy (OH)-2,2′,3,4′,5,5′,6-heptachlorobiphenyl (CB187) is a polychlorinated biphenyl (PCB) metabolite present in human serum at the highest concentration of the PCB metabolites. Our previous study demonstrated that CB187 was metabolized by rat and guinea pig liver microsomes to the major metabolite 4′-OH-2,2′,3,3′,5,5′,6-heptachlorobiphenyl (CB178), and the two minor metabolites 4-OH-CB187 and 4′-OH-2,2′,3,5,5′,6-hexachlorobiphenyl (CB151). In this study, the distribution of these metabolites in serum, liver and kidney, and their fecal excretion, were examined in rats and guinea pigs intraperitoneally dosed with CB187. Similarly to the in vitro study, 4′-OH-CB178 was a major metabolite in the liver, serum and feces of both animal species on day 4 after CB187 injection, and the level in the liver was about 20 times higher in untreated guinea pigs than in untreated rats. In addition, 4-OH-CB187, a minor metabolite, was detected in the serum and kidneys, but not in the feces, of both guinea pigs and rats. Another minor metabolite, 4′-OH-CB151, was detected at a lower level only in guinea pig feces; little was found in the serum or liver of either animals. Over the 30d following CB187 injection into guinea pigs, 4′-OH-CB178 and 4-OH-CB187 in the serum was observed at higher level on day 4 and day 16 after injection, respectively. The majority of the 4′-OH-CB178 was rapidly excreted to the feces following unmetabolized CB187, whereas 4-OH-CB187 was not found in guinea pig feces and liver during 30d. These results support previous reports that 4-OH-CB187 is retained persistently in animal blood.

Factors that Restrict the Cell Permeation of Cyclic Prodrugs of an Opioid Peptide, Part 3: Synthesis of Analogs Designed to have Improved Stability to Oxidative Metabolism

Previously, our laboratory reported that cyclic peptide prodrugs of the opioid peptide H-Tyr-D-Ala-Gly-Phe-D-Leu-OH (DADLE) are metabolized by cytochrome P450 (CYP450) enzymes, which limits their systemic exposure after oral dosing to animals. In an attempt to design more metabolically stable cyclic prodrugs of DADLE, we synthesized analogs of DADLE cyclized with a coumarinic acid linker (CA; CA-DADLE), which contained modifications in the amino acid residues known to be susceptible to CYP450 oxidation. Metabolic stability and metabolite identification studies of CA-DADLE and its analogs were then compared using rat liver microsomes (RLM), guinea pig liver microsomes (GPLM), and human liver microsomes (HLM), as well as recombinant human recombinant cytochrome P450 3A4 (hCYP3A4). Similar to the results observed for CA-DADLE, incubation of its analogs with RLM, GPLM, and HLM resulted in monohydroxylation of an amino acid side chain on these cyclic prodrugs. When CA-DADLE was incubated with hCYP3A4, similar oxidative metabolism of the peptide was observed. In contrast, incubation of the CA-DADLE analogs with hCYP3A4 showed that these amino-acid-modified analogs are not substrates for this CYP450 isozyme. These results suggest that the amino-acid-modified analogs of CA-DADLE prepared in this study could be stable to metabolic oxidation by CYP3A4 expressed in human intestinal mucosal cells.

Kinetics of N,N-Dimethyl Tyramine (Hordenine) Demethylation by Guinea-Pig Liver Microsomes

Kinetics of <u>N,N</u>-Dimethyl Tyramine (Hordenine) Demethylation by Guinea-Pig Liver Microsomes

Species and Gender Differences Affect the Metabolism of Emodin via Glucuronidation

The aim of the present study was to define the mechanisms responsible for poor bioavailability of emodin by determining its metabolism using in vitro and in situ disposition models of the intestine and liver. Liver microsomes of mice, rats, guinea pigs, dogs, and humans were used along with the rat intestinal perfusion model and the rat intestinal microsomes. In the rat intestine, excretion rates of emodin-3-O-glucuronide were significantly different (p < 0.05) in four regions of the intestine and were higher in males than in females (p < 0.01). Emodin glucuronidation in liver microsomes was species-dependent, and K (m) values varied 5.7-fold (3.2-18.2 microM) in males and 2.8-fold (4.6-13.0 microM) in females. The male intrinsic clearance (CL(int)) values differed by 5-fold (27.6-138.3 mL h(-1) mg(-1) protein), and female CL(int) values differed by 4.3-fold (24.3-103.5 mL h(-1) mg(-1) protein). Since CL(int) values of emodin glucuronidation were 10-fold higher than that of isoflavones, emodin was considered rapidly glucuronidated. In contrast to the large species-dependent effects on K (m) and CL(int) values, gender had a smaller effect on these kinetic parameters (2-fold, p < 0.05). Lastly, glucuronidation rates obtained using liver microsomes from various experimental animals of the same gender correlated well with those in human liver microsomes. In conclusion, Rapid metabolism by UDP-glucuronosyltransferase is the major reason why emodin has poor bioavailability. Species and gender affected emodin metabolism to a different degree, and experimental animals are expected to be useful in predicting emodin glucuronidation in humans.

Determination of microsomal CYP2A6 activity by high performance liquid chromatography

Coumarin has been reported to be a reliable probe for the determination of human microsomal CYP2A6 activity. Coumarin is converted to 7-hydroxycoumarin by CYP2A6. A high pressure liquid chromatographic assay for the estimation of CYP2A6 activity in microsomes was evaluated. A RP C-18 Novapak Waters (15 cm x 3.9 mm, 5 µm) column was used for the assay. The mobile phase composition was methanol : 1% glacial acetic acid (35:65 v/v) (pH~3.1), with a flow rate of 0.6 ml/min, injection volume of 100 µl and detection at 320 nm. The retention times for coumarin and 7-hydroxycoumarin were 8.7 min and 5.3 min, respectively. The limit of detection (LOD) was 0.05 µM, and the limit of quantitation (LOQ) was 0.1 µM, for 7-hydroxycoumarin. The percent coefficient of variation associated with 7-hydroxycoumarin determination after duplicate estimation was found to be in the range 0.03 to 3.6% for buffer matrix and 0.1 to 6.5% for microsomal matrix. The mean rate of 7-hydroxycoumarin formation in guinea pig liver microsomes was 0.084 nmol/min/nmol P450. Coumarin 7-hydroxylase activity was absent in rat liver microsomes. No interference was observed from incubation mixture components.

Comparative Study on Nobiletin Metabolism with Liver Microsomes from Rats, Guinea Pigs and Hamsters and Rat Cytochrome P450

In vitro metabolism of nobiletin, a polymethoxy-flavonoid abundantly present in citrus peels, was studied using liver microsomes of rats, hamsters and guinea pigs and ten cDNA-expressed rat cytochrome P450 (P450). The effects of P450 inducers on nobiletin metabolism were also investigated. Aerobical incubation with NADPH and animal liver microsomes transformed nobiletin to five metabolites, M-1, M-2, M-3, M-4 and M-5. From LC-MS and (1)H-NMR data and a time-course study, these were assumed to be 4'-hydroxy (OH)-, 7-OH-, 6-OH-, 3',4'-diOH- and 6,7-diOH-metabolites, respectively. Pretreatment of animals with phenobarbital increased M-2 and M-3 to about 2-fold that in untreated animals. Pretreatment with 3-methylcholanthrene (MC) resulted in remarkable increases of both M-1 and M-4 (3 to 9-fold that of untreated). Males had 2-3 times higher M-2 and M-3 formation activities in rats, and for M-2 in hamsters than did females. Immunoinhibition study using antiserum against P450 revealed the involvement of hamster CYP1A2 in the formation of M-1 and M-4 in hamster liver. Of ten rat P450s, CYP2C11, CYP3A1, CYP3A2 and CYP2D1 had high activities for the formation of M-1, M-2 and M-3. Another P450s (CYP1A1, CYP2C12 and CYP1A2) also showed activity for the formation of M-1. Only CYP1A1 produced 3',4'-diOH-metabolites (M-4). However, CYP2A1, CYP2B1 and CYP2E1 had no activity for nobiletin. These results suggested that constitutive P450s such as CYP2C11, CYP2D1, CYP3A1, CYP3A2 and CYP2C12 are responsible for the demethylation at the 6-, 7-, 3'- and 4'-positions; whereas, MC-inducible P450s, CYP1A1 and CYP1A2, preferentially catalyzed demethylation at the 3'-and 4'-positions.

Isolation and catalytic competence of different animal liver microsomal fractions prepared by calcium-aggregation method

Rat, mouse, rabbit, and guinea pig liver microsomes were prepared, using the calcium-aggregation method. The mean specific spectral cytochrome P450 content obtained for rat, mouse, rabbit, and guinea pig liver microsomes were 0.547, 0.394, 0.677, and 0.378 nmol cytochrome P450/mg protein, respectively. All the microsomal samples showed the ability to biotransform p-nitrophenol to p-nitrocatechol. The mean rate of formation of p-nitrocatechol by rat, mouse, rabbit, and guinea pig liver microsomes was 0.272, 0.247, 0.497 and 0.424 nmole/min/nmole cytochrome 450, respectively. This method circumvents the need of an ultracentrifuge for isolation of microsomes, by the conventional differential centrifugation method.

Estimation of microsomal CYP1A2 activity by high performance liquid chromatography

Phenacetin has been reported to be a reliable probe for the estimation of microsomal CYP1A2 activity. Phenacetin is converted to paracetamol by CYP1A2. A high pressure liquid chromatographic assay for the estimation of CYP1A2 activity in microsomes, was evaluated with caffeine as the internal standard. An RP C-18 supelcosil (150 × 4.6 mm, 5 µm) column was used for the assay. The mobile phase composition was acetonitrile:water (15:85), with a flow rate of 0.7 ml/min, injection volume of 100 µl, and detection at 240 nm. The retention times for paracetamol, caffeine and phenacetin were found to be 4.9, 8.6 and 15.5 min, respectively. The limit of detection was 0.1 µM for phenacetin, and the limit of quantitation was 0.2 µM for paracetamol. The percent coefficient of variation associated with paracetamol determination after duplicate estimation, was found to be in the range 0.02 to 7.7% for buffer matrix, and 0.8 to 9.7% for microsomal matrix. The mean rate of paracetamol formation in rat liver and guinea pig liver microsomes was 0.027 and 0.161 nmoles/min/nmole of P450, respectively. No interference was observed from incubation mixture components.

Significant differences in the disposition of cyclic prodrugs of opioid peptides in rats and guinea pigs following IV administration

The stabilities of DADLE ([D-Ala2,D-Leu5]-Enk, H-Tyr-D-Ala-Gly-Phe-D-Leu-OH), the capped derivative Ac-DADLE-NH2, and the oxymethyl-coumarinic acid (OMCA)-based cyclic prodrug of DADLE and [D-Ala2,Leu5]-Enk (H-Tyr-D-Ala-Gly-Phe-Leu-OH) were determined at 37 degrees C in rat and guinea pig liver microsomes in the presence and absence of paraoxon, an esterase B inhibitor, and ketoconazole, a CYP3A4 inhibitor. These studies showed that the order of stability in microsomes was: DADLE >> Ac-DADLE-NH2 > OMCA-DADLE = OMCA-[D-Ala2,Leu5]-Enk. While paraoxon produced no significant effect on the stability of the studied compounds in liver microsomes, ketoconazole inhibited the metabolism, suggesting that the capped peptide and the cyclic prodrugs are substrates for cytochrome P450 enzymes. For pharmacokinetic studies, the cyclic prodrugs of DADLE and [D-Ala2,Leu5]-Enk were administered i.v. to rats and guinea pigs. Various biological fluids and tissue (brain, bile, and blood) were collected and analyzed for the free peptide and the prodrugs by high performance liquid chromatography with tandem mass spectrometric detection (LC-MS-MS). These studies showed that the conversion of the cyclic prodrugs to the respective linear peptides (i.e., DADLE and [D-Ala2,Leu5]-Enk) was rapid in rat and guinea pig. In terms of drug elimination, only trace amounts of OMCA-DADLE and OMCA-[D-Ala2,Leu5]-Enk were recovered in guinea pig bile (3.3% and 0.82%, respectively), while significant amounts were recovered in rat bile (38.1% and 51.7%, respectively). Brain uptake of the cyclic prodrugs in guinea pigs compared to previously determined brain uptake of OMCA-DADLE in rats was also significantly different. For OMCA-DADLE, the brain levels of the cyclic prodrug and DADLE in guinea pigs were approximately 80 and 8.5 times greater, respectively, than the levels observed in rat brain. The brain-to-plasma prodrug concentration ratios in guinea pigs (>or= 0.6) were significantly higher than the ratio observed in rats (0.01). These species differences are most likely due to the different substrate specificities of the efflux transporters that facilitate liver clearance of these prodrugs and limit their permeation into the brain.

In vitro metabolism of 2,2′,3,4′,5,5′,6-heptachlorobiphenyl (CB187) by liver microsomes from rats, hamsters and guinea pigs

The metabolism of 2,2′,3,4′,5,5′,6-heptachlorobiphenyl (heptaCB) (CB187) was studied using liver microsomes of rats, hamsters and guinea pigs, and the effect of cytochrome P450 (CYP) inducers, phenobarbital (PB) and 3-methylcholanthrene (MC), was also investigated. In untreated animals, guinea pig liver microsomes formed three metabolites which were deduced to be 4′-hydroxy-2,2′,3,5,5′,6-hexachlorobiphenyl (M-1), 4′-hydroxy-2,2′,3,3′,5,5′,6-heptaCB (M-2) and 4-OH-CB187 (M-3) from the comparison of GC/MS data with some synthetic authentic samples. The formation rate of M-1, M-2 and M-3 was 18.1, 36.6, 14.7 pmol h−1 mg protein−1, respectively. Liver microsomes of untreated rats and hamsters did not form CB187 metabolites. In guinea pigs, PB-treatment increased M-1 and M-2 significantly to 1.9- and 3.4-fold of untreated animals but did not affect the formation of M-3. In rats, PB-treatment resulted in the appearance of M-2 and M-3 with formation rates of 87.1 and 13.7 pmol h−1 mg protein−1, respectively, but M-1 was not observed. In hamsters, PB-treatment formed only M-2 at a rate of 29.4 pmol h−1 mg protein−1. On the other hand, MC-treatment of guinea pigs decreased the formation of M-1 and M-2 to less than 50% of untreated animals. MC-microsomes of rats and hamsters produced no metabolites. Preincubation of antiserum (300 µl) against guinea pig CYP2B18 with liver microsomes of PB-treated guinea pigs produced 80% inhibition of M-1 and the complete inhibition of M-2 and M-3. These results suggest that PB-inducible CYP forms, especially guinea pig CYP2B18, rat CYP2B1 and hamster CYP2B, are important in CB187 metabolism and that CB187 metabolism in guinea pigs may proceed via the formation of 3,4- or 3′,4′-oxide and subsequent NIH-shift or dechlorination.

Hepatic Metabolism of Methyl Anthranilate and Methyl N-Methylanthranilate as Food Flavoring Agents in Relation to Allergenicity in the Guinea Pig

To assess the safety of the food flavoring agents, methyl anthranilate (MA) and methyl N-methylanthranilate (MNMA), their relationships with metabolism and allergenicity were examined in guinea pigs. Both MA and MNMA were hydrolyzed to anthranilic acid (AA) and N-methylanthranilic acid (N-methylAA), respectively, by guinea pig liver microsomes. These hydrolytic activities at 1000 µM were 320 and 35 nmol/min/mg protein, respectively. Moreover, MNMA was N-demethylated to MA by the liver microsomes; the oxidative activity at 1000 µM of MNMA was 2.8 nmol/min/mg protein. The N-demethylase activity for N-methylAA at 1000 µM in the liver microsomes was 3.9 nmol/min/mg protein. Kinetic analysis indicated that the Vmax/Km values of MA and MNMA hydrolyses were 15and 7.4-fold greater in guinea pig liver microsomes than in the cytosol, respectively, suggesting that these hydrolytic activities were predominantly localized in the microsomes. Liver microsomal activities for the hydrolysis of these flavoring esters were markedly inhibited by diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, and bis(pnitrophenyl)phosphate but not by physostigmine. These hydrolytic activities were suppressed by aspirin, a substrate of carboxylesterase, in a concentration-dependent manner. The N-demethylations of MNMA and N-methylAA were inhibited by SKF 525-A, a nonselective inhibitor of cytochrome P450. At the same time as the metabolic study described above, skin reactions in guinea pigs were investigated using MA, MNMA, N-methylAA, and AA. All compounds examined elicited positive skin reactions, although MNMA and AA exhibited relatively greater sensitizing properties. These results may provide useful information about metabolism in the toxicologic evaluations of MA and MNMA.

Species Differences in the Metabolism of (+)- and (−)-Limonenes and their Metabolites, Carveols and Carvones, by Cytochrome P450 Enzymes in Liver Microsomes of Mice, Rats, Guinea Pigs, Rabbits, Dogs, Monkeys, and Humans

(+)-Limonene is shown to cause renal toxicity in male rats, but not in female rats and other species of animals including mice, guinea pigs, rabbits, and dogs. We have previously shown that male-specific rat CYP2C11 (but not female-specific CYP2C12) is able to convert limonenes to carveols and perillyl alcohols (M. Miyazawa, M. Shindo, and T. Shimada: Chem. Res. Toxicol., 15, 15-20, 2002). Here, we investigated whether (+)- and (-)-limonene enantiomers are differentially metabolized by P450 enzymes in liver microsomes of mice, rats, guinea pigs, rabbits, dogs, monkeys, and humans. Limonene enantiomers were converted to respective carveols, perillyl alcohols, and carvones (oxidative metabolites of carveols) by liver microsomes of dogs, rabbits, and guinea pigs. Mice, rats, monkeys, and humans produced carveols and perilly alcohols, but not carvones. Reconstituted monooxygenase systems containing purified rabbit CYP1A2 and 2B4 and NADPH-P450 reductase were found to catalyze (+)-limonene to (+)-carveol, (+)-carvone, and (+)-perillyl alcohol, being more active with CYP2B4. When (+)-carveol and (+)-carvone were used as substrates, dogs, rabbits, and guinea pigs metabolized them to (+)-carvone and (+)-carveol, respectively. Again humans, monkeys, rats, and mice did not convert (+)-carveol to (+)-carvone, but metabolized (+)-carvone to (+)-carveol, with male rats having the highest rates. CYP2C enzymes were suggested to play major roles in metabolizing (+)-carveol to (+)-carvone and (+)-carvone to (+)-carveol by liver microsomes, since the activities were inhibited significantly by anti-human CYP2C9 antibodies in these animal species. Studies with recombinant P450 enzymes suggested that CYP2C9 and 2C19 in humans and CYP2C11 in untreated male rats were the major enzymes in metabolizing (+)-carvone. These results suggest that there are species-related differences in the metabolism of limonenes by P450 enzymes, particularly in the way from (+)-carveol to (+)-carvone. However, it remains unclear whether these differences in limonene metabolism by these animal species explain species-related differences in limonene-induced renal toxicity.

Development and Characterization of Cholinephosphotransferase Antibody

In the present study, we generated antibodies in rabbits against two synthetic peptides, one based on peptide sequence from yeast CPT cDNA (position 86 to 98 of the amino acid sequence) and the other from our guinea pig CPT cDNA (it corresponds to amino acid positions 119 to 130 according to yeast CPT gene). The antibody titers were measured by both dot blot analysis and ELISA using Keyhole limpets hemocyanin coupled CPT peptides. The CPT antibody recognized a single band by Western blot analysis of proteins from guinea pig liver mitochondria and microsomes. The molecular weight of the protein recognized by Western blot analysis is close to the predicted molecular weight (46 kDa) of yeast CPT. Further analysis revealed that the antibody inhibited CPT activity in both subcellular fractions in a dose dependent manner, thus confirming the specificity of the antibody against both subcellular CPT.

P-644 - Stimulation of Carbon Tetrachloride Metabolism by Alcohols in Guinea-Pig Liver Microsomes

P-644 - Stimulation of Carbon Tetrachloride Metabolism by Alcohols in Guinea-Pig Liver Microsomes

Total synthesis of an antioxidant isolated from yeast via palladium-catalyzed coupling and its application for related compounds

A total synthesis of an antioxidant ( 1) having a benzofuran skeleton was achieved in four steps via the palladium(0)-catalyzed cross-coupling reaction. We also prepared several related compounds bearing a variety of aromatic or heterocyclic rings. Some of these compounds demonstrate more potent than 1 for antioxidative activity using guinea pig liver microsomes.

P–158 - Suppression of reductive dehalogenation of halothane in guinea-pig liver microsomes by nitric oxide.

P–158 - Suppression of reductive dehalogenation of halothane in guinea-pig liver microsomes by nitric oxide.

Studies on the Metabolic Fate of NK-104, a New Inhibitor of HMG-CoA Reductase. (5). In Vitro Metabolism and Plasma Protein Binding in Animals and Human.

NK-104 is a new and very potent competitive inhibitor of HMG-CoA reductase. NK-104 bound to plasma protein of mouse, rat, rabbit, dog and monkey, at a binding ratio of more than 96%. NK104 also highly bound to human serum albumin and a1 acid glycoprotein. However, no interaction in protein binding was found between NK-104 and selected highly protein binding drugs at their therapeutic concentrations in human plasma. The in vitro metabolism of 14C-NK 104 was investigated using the liver microsomes of rat, rabbit, guinea pig, dog, monkey and human. The radioactivity was mostly due to unchanged NK-104 except for monkey. A relatively large amount of M-13 (8-hydroxy NK-104) was observed in monkey, but not in other animal species. The kinetic study of NK 104 metabolism suggested that M-13 was formed with relatively low intrinsic clearance. Based on three different in vitro approaches, namely 1) chemical inhibition, 2) immunoinhibition and 3) metabolism by recombinant human P450, it is concluded that CYP2C9 and CYP2C8 are the enzymes responsible for the metabolism of NK-104. In addition, no inhibitory effect on CYP mediated 4-hydroxylation of tolbutamide (CYP2C9) in the presence of NK-104 was detected. In conclusion, NK-104 was highly bound to plasma protein whereas no mutual interaction in protein binding was found between NK-104 and various commonly used drugs. Furthermore, NK-104 was scarcely metabolized in the liver microsomes, and no drug/drug interaction between NK-104 and tolbutamide was noted in in vitro metabolism.

Triple marker screening, Japanese experience

In order to provide evidence that a cytochrome P450 belonging to the IIB subfamily is expressed as a constitutive form in the guinea pig, we tried to purify an isozyme from liver microsomes of untreated guinea pigs by assessing its reactivity with anti-P450b antibody in the present study. One form of cytochrome P450, named P450GP-1, was obtained. The minimum molecular weight of this isozyme was estimated to be 52,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino terminal sequence up to the 33rd amino acid of P450GP-1 was determined. As expected, comparison of the amino acid sequence with those of cytochrome P450 isozymes from other species reported so far indicated that P450GP-1 was highly homologous to P450s categorized in the IIB subfamily; that is, 67% similarity to rat P450b, 82% to rabbit LM2, 76% to dog PBD-2, 70% to mouse pf 346, and 73% to human IIB1. On the other hand, P450GP-1 showed only low similarity, less than 41%, to other cytochrome P450s of the II subfamily and those of the I, III, and IV families. Affinity of P450GP-1 to anti-P450b immunoglobulin G was confirmed to be comparable with that of a principal antigen, P450b. Immunoblot analysis revealed that P450GP-1 in the guinea pig liver microsomes was induced by phenobarbital treatment, but the increase was not as large as in the rat. P450GP-1 efficiently catalyzed benzphetamine N-demethylation, strychnine 2-hydroxylation, and testosterone 16β-hydroxylation, all of which are also catalyzed by P450b. Based on these results, it was strongly suggested that the IIB-type of cytochrome P450 in guinea pigs, at least one of them, is a constitutive form which is moderately induced by phenobarbital.