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Biomedical subjects

Andrew Parkinson

Publications and source records attributed to Andrew Parkinson.

14 recordsLinked to original sources
PubMed2006

CYP4F enzymes are the major enzymes in human liver microsomes that catalyze the O-demethylation of the antiparasitic prodrug DB289 [2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime].

DB289 [2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime] is biotransformed to the potent antiparasitic diamidine DB75 [2,5-bis(4-amidinophenyl) furan] by sequential oxidative O-demethylation and reductive N-dehydroxylation reactions. Previous work demonstrated that the N-dehydroxylation reactions are catalyzed by cytochrome b5/NADH-cytochrome b5 reductase. Enzymes responsible for catalyzing the DB289 O-demethylation pathway have not been identified. We report an in vitro metabolism study to characterize enzymes in human liver microsomes (HLMs) that catalyze the initial O-demethylation of DB289 (M1 formation). Potent inhibition by 1-aminobenzotriazole confirmed that M1 formation is catalyzed by P450 enzymes. M1 formation by HLMs was NADPH-dependent, with a Km and Vmax of 0.5 microM and 3.8 nmol/min/mg protein, respectively. Initial screening showed that recombinant CYP1A1, CYP1A2, and CYP1B1 were efficient catalysts of M1 formation. However, none of these three enzymes was responsible for M1 formation by HLMs. Further screening showed that recombinant CYP2J2, CYP4F2, and CYP4F3B could also catalyze M1 formation. An antibody against CYP4F2, which inhibited both CYP4F2 and CYP4F3B, inhibited 91% of M1 formation by HLMs. Two inhibitors of P450-mediated arachidonic acid metabolism, HET0016 (N-hydroxy-N'-(4-n-butyl-2-methylphenyl)formamidine) and 17-octadecynoic acid, effectively inhibited M1 formation by HLMs. Inhibition studies with ebastine and antibodies against CYP2J2 suggested that CYP2J2 was not involved in M1 formation by HLMs. Additionally, ketoconazole preferentially inhibited CYP4F2, but not CYP4F3B, and partially inhibited M1 formation by HLMs. We conclude that CYP4F enzymes (e.g., CYP4F2, CYP4F3B) are the major enzymes responsible for M1 formation by HLMs. These findings indicate that, in human liver, members of the CYP4F subfamily biotransform not only endogenous compounds but also xenobiotics.

Antiparasitic Agents↗
PubMed2006

The effect of fenbuconazole on cell proliferation and enzyme induction in the liver of female CD1 mice.

Fenbuconazole, a triazole fungicide, has been associated with an increase in the incidence of liver adenomas in female mice following long-term dietary exposure. The aim of this study was to evaluate whether the mode of action for liver tumor formation by fenbuconazole is similar to that of phenobarbital. Treatment of CD1 mice with 0, 20, 60, 180 or 1300 ppm fenbuconazole for up to 4 weeks caused a dose-dependent increase in liver weight that was associated with centrilobular hepatocellular hypertrophy, cytoplasmic eosinophilia and panlobular hepatocellular vacuolation, as well as an initial increase in the cell proliferation labeling index. Fenbuconazole also caused a dose-dependent increase in liver microsomal cytochromes b(5) and P450 and the levels of immunoreactive CYP2B10 and its associated activity 7-pentoxyresorufin O-dealkylation (PROD). Treatment of mice with 1000 ppm phenobarbital elicited the same effects as treatment of mice with 1300 ppm fenbuconazole, except that phenobarbital was more effective than fenbuconazole at inducing PROD activity, even though fenbuconazole induced CYP2B10 to the same extent as did phenobarbital. This difference was attributed to the ability of fenbuconazole to bind tightly to CYP2B10 and partially mask its catalytic activity in liver microsomes, which is characteristic of several azole-containing drugs. All hepatocellular changes and induced enzyme activity returned to control levels within 4 weeks of discontinuing treatment with fenbuconazole or phenobarbital, indicating that the observed changes were fully reversible. We conclude that fenbuconazole is a phenobarbital-type inducer of mouse liver cytochrome P450, and the mode of action by which fenbuconazole induces liver adenomas in mice is similar to that of phenobarbital.

Animals↗
PubMed2006

Distribution, metabolism, and excretion of the anti-angiogenic compound SU5416.

SU5416, 3-(3,5-dimethyl-1H-pyrrol-2-ylmethylene)-1,3-dihydro-indol-2-one, is a potent inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinase, Flk-1/KDR (fetal liver kinase 1/kinase insert domain-containing receptor), also known as VEGF receptor 2 (VEGFR2). It was the first VEGFR2 inhibitor to enter clinical trials for the treatment of colorectal and non-small cell lung cancers. Pre-clinical evaluation of SU5416 included studies related to the distribution, metabolism and excretion of this compound. These studies have provided information useful in understanding the disposition and metabolism of the indolinone class of chemicals, which has not been studied previously with therapeutic intent. The lessons we learned from SU5416 have been successfully applied in developing next generation indolinone compounds targeting tumor angiogenesis.

Angiogenesis Inhibitors↗
PubMed2006

On the mechanism of hepatocarcinogenesis of benzodiazepines: evidence that diazepam and oxazepam are CYP2B inducers in rats, and both CYP2B and CYP4A inducers in mice.

The aim of this study was to evaluate diazepam and oxazepam as cytochrome P450 inducers at doses previously shown to cause liver tumors in mice but not rats. In rats, diazepam and oxazepam induced CYP2B, and were as effective as phenobarbital despite lacking phenobarbital's tumor-promoting effect in rats. In mice, diazepam and oxazepam induced both CYP2B and CYP4A at dietary doses associated with liver tumor formation. It remains to be determined why diazepam and oxazepam induce CYP4A in mice but not rats and whether this difference accounts for the apparent species difference in the tumor-promoting activity of diazepam and oxazepam.

Animals↗
PubMed2005

Glucuronidation converts gemfibrozil to a potent, metabolism-dependent inhibitor of CYP2C8: implications for drug-drug interactions.

Gemfibrozil more potently inhibits CYP2C9 than CYP2C8 in vitro, and yet the opposite inhibitory potency is observed in the clinic. To investigate this apparent paradox, we evaluated both gemfibrozil and its major metabolite, an acyl-glucuronide (gemfibrozil 1-O-beta-glucuronide) as direct-acting and metabolism-dependent inhibitors of the major drug-metabolizing cytochrome P450 enzymes (CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4) in human liver microsomes. Gemfibrozil most potently inhibited CYP2C9 (IC50 of 30 microM), whereas gemfibrozil glucuronide most potently inhibited CYP2C8 (IC50 of 24 microM). Unexpectedly, gemfibrozil glucuronide, but not gemfibrozil, was found to be a metabolism-dependent inhibitor of CYP2C8 only. The IC50 for inhibition of CYP2C8 by gemfibrozil glucuronide decreased from 24 microM to 1.8 microM after a 30-min incubation with human liver microsomes and NADPH. Inactivation of CYP2C8 by gemfibrozil glucuronide required NADPH, and proceeded with a K(I) (inhibitor concentration that supports half the maximal rate of enzyme inactivation) of 20 to 52 microM and a k(inact) (maximal rate of inactivation) of 0.21 min(-1). Potent inhibition of CYP2C8 was also achieved by first incubating gemfibrozil with alamethicin-activated human liver microsomes and UDP-glucuronic acid (to form gemfibrozil glucuronide), followed by a second incubation with NADPH. Liquid chromatography-tandem mass spectrometry analysis established that human liver microsomes and recombinant CYP2C8 both convert gemfibrozil glucuronide to a hydroxylated metabolite, with oxidative metabolism occurring on the dimethylphenoxy moiety (the group furthest from the glucuronide moiety). The results described have important implications for the mechanism of the clinical interaction reported between gemfibrozil and CYP2C8 substrates such as cerivastatin, repaglinide, rosiglitazone, and pioglitazone.

Alamethicin↗
PubMed2004

The effects of gender, age, ethnicity, and liver cirrhosis on cytochrome P450 enzyme activity in human liver microsomes and inducibility in cultured human hepatocytes.

We have measured cytochrome P450 (CYP) activity in nearly 150 samples of human liver microsomes and 64 samples of cryopreserved human hepatocytes, and we have performed induction studies in over 90 preparations of cultured human hepatocytes. We have analyzed these data to examine whether the expression of CYP enzyme activity in liver microsomes and isolated hepatocytes or the inducibility of CYP enzymes in cultured hepatocytes is influenced by the gender, age, or ethnicity of the donor (the latter being limited to Caucasians, African Americans, and Hispanics due to a paucity of livers from Asian donors). In human liver microsomes, there were no statistically significant differences (P > 0.05) in CYP activity as a function of age, gender, or ethnicity with one exception. 7-Ethoxyresorufin O-dealkylase (CYP1A2) activity was greater in males than females, which is consistent with clinical observation. Liver microsomal testosterone 6beta-hydroxylase (CYP3A4) activity was slightly greater in females than males, but the difference was not significant. However, in cryopreserved human hepatocytes, the gender difference in CYP3A4 activity (females = twice males) did reach statistical significance, which supports the clinical observation that females metabolize certain CYP3A4 substrates faster than do males. Compared with those from Caucasians and African Americans, liver microsomes from Hispanics had about twice the average activity of CYP2A6, CYP2B6, and CYP2C8 and half the activity of CYP1A2, although this apparent ethnic difference may be a consequence of the relatively low number of Hispanic donors. Primary cultures of hepatocytes were treated with beta-naphthoflavone, an inducer of CYP1A2, phenobarbital or rifampin, both of which induce CYP2B6, CYP2C9, CYP2C19, and CYP3A4, albeit it to different extents. Induction of these CYP enzymes in freshly cultured hepatocytes did not appear to be influenced by the gender or age of the donor. Furthermore, CYP3A4 induction in hepatocytes isolated from cirrhotic liver was comparable to that in normal hepatocytes, which supports the "healthy hepatocyte, sick environment" hypothesis of liver cirrhosis. This review summarizes these findings and discusses their implications for the use of human liver microsomes and hepatocytes for in vitro studies of drug metabolism and enzyme induction, which play a key role in drug development.

Aging↗
PubMed2003

Decreased coumarin 7-hydroxylase activities and CYP2A6 expression levels in humans caused by genetic polymorphism in CYP2A6 promoter region (CYP2A6*9).

One of seven poor metabolizers of coumarin found in Thai subjects was previously genotyped as heterozygote for the CYP2A6*4 (whole deletion) and CYP2A6*9. Thus, we aimed to investigate the relationship between the genetic polymorphism in the TATA box of the CYP2A6 gene (CYP2A6*9), expression levels of CYP2A6 mRNA and coumarin 7-hydroxylase activities in human livers. Levels of CYP2A6 mRNA were quantified by real-time quantitative reverse transcriptase-polymerase chain reaction. The mean expression levels of CYP2A6 mRNA in individuals with CYP2A6*1/*4, CYP2A6*1/*9 and CYP2A6*4/*9 were 58%, 71% and 21% of the individuals genotyped as CYP2A6*1/*1, respectively. The mean in-vitro coumarin 7-hydroxylase activities in subjects carrying CYP2A6*1/*4, CYP2A6*1/*9 and CYP2A6*4/*9 were 41%, 71% and 12%, respectively, compared to those of the subjects judged as wild-type. Vmax values for coumarin 7-hydroxylation in the liver microsomes from human subjects with genotypes of CYP2A6*1/*1, CYP2A6*1/*4, CYP2A6*1/*9 and CYP2A6*4/*9 were 0.58, 0.26, 0.44 and 0.13 nmol/min/nmol total P450, respectively. CYP2A6 protein levels in human liver microsomes with the CYP2A6*4 and the CYP2A6*9 alleles were markedly decreased. These results suggest that the genetic polymorphism in the promoter region of the CYP2A6 gene (CYP2A6*9) reduced the expression levels of CYP2A6 mRNA and protein in human livers, resulting in the decrease of coumarin 7-hydroxylase activities. Individuals judged as CYP2A6*4/*9 were expected to be poor metabolizers, having extremely low activity of CYP2A6.

Adult↗
PubMed2003

Effects of prototypical microsomal enzyme inducers on cytochrome P450 expression in cultured human hepatocytes.

Cultured human hepatocytes are a valuable in vitro system for evaluating new molecular entities as inducers of cytochrome P450 (P450) enzymes. The present study summarizes data obtained from 62 preparations of cultured human hepatocytes that were treated with vehicles (saline or dimethylsulfoxide, 0.1%), beta-naphthoflavone (33 microM), phenobarbital (100 or 250 microM), isoniazid (100 microM) and/or rifampin (20 or 50 microM), and examined for the expression of P450 enzymes based on microsomal activity toward marker substrates, or in the case of CYP2C8, the level of immunoreactive protein. The results show that CYP1A2 activity was markedly induced by beta-naphthoflavone (on average 13-fold, n = 28 preparations), and weakly induced by phenobarbital (1.9-fold, n = 25) and rifampin (2.3-fold, n = 22); CYP2A6 activity tended to be increased with phenobarbital (n = 7) and rifampin (n = 3) treatments, but the effects were not statistically significant; CYP2B6 was induced by phenobarbital (6.5-fold, n = 13) and rifampin (13-fold, n = 14); CYP2C8 was induced by phenobarbital (4.0-fold, n = 4) and rifampin (5.2-fold, n = 4); CYP2C9 was induced by phenobarbital (1.8-fold, n = 14) and rifampin (3.5-fold, n = 10); CYP2C19 was markedly induced by rifampin (37-fold, n = 10), but relatively modestly by phenobarbital (7-fold, n = 9); CYP2D6 was not significantly induced by phenobarbital (n = 5) or rifampin (n = 5); CYP2E1 was induced by phenobarbital (1.7-fold, n = 5), rifampin (2.2-fold, n = 5), and isoniazid (2.3-fold, n = 5); and, CYP3A4 was induced by phenobarbital (3.3-fold, n = 42) and rifampin (10-fold, n = 61), but not by beta-naphthoflavone. Based on these observations, we generalize that beta-naphthoflavone induces CYP1A2 and isoniazid induces CYP2E1, whereas rifampin and, to a lesser extent phenobarbital, tend to significantly and consistently induce enzymes of the CYP2A, CYP2B, CYP2C, CYP2E, and CYP3A subfamilies but not the 2D subfamily.

Aryl Hydrocarbon Hydroxylases↗
PubMed2003

Regulation of drug transporter gene expression by nuclear receptors.

Pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are key regulators of xenobiotic-inducible cytochrome P450 gene expression. Whereas much is known about their role in regulating drug metabolism, little is known regarding their role in regulating drug transport in vivo. Wild-type mice and mice lacking PXR (PXR-KO) were used to examine the inducible expression of two drug transporter genes, Oatp2 (Slc21a5) and Mrp3 (Abcc3), in liver following treatment with selective PXR and CAR activators. Selective activation of PXR or CAR induced Oatp2 and Mrp3 expression in wild-type mice but not in PXR-KO mice. Basal expression levels of Oatp2 and Mrp3 gene were significantly higher in PXR-KO mice when compared with wild-type mice. Additionally, phenobarbital (PB)-inducible Oatp2 and Mrp3 gene expression was significantly increased in the PXR-KO mice when compared with wild-type PB-treated mice. We also examined the effect of PXR ablation on PB-inducible hepatic CYP3A activity in vivo. Microsomes isolated from PB-treated PXR-KO mice exhibited a significantly elevated rate of testosterone 6 beta-hydroxylation when compared with microsomes isolated from wild-type PB-treated mice. PB treatment produced significantly increased levels of hepatomegaly in PXR-KO mice when compared with wild-type PB-treated mice. Taken together, these results suggest that nonliganded PXR plays a net negative role in coregulating shared PXR/CAR-target gene expression in vivo and extend the hypothesis that PXR and CAR coregulate not only drug metabolism but also drug transport.

Animals↗
PubMed2002

In vivo and in vitro induction of cytochrome P450 enzymes in beagle dogs.

The aim of this study was to determine the in vitro and in vivo effects of several prototypical inducers, namely beta-naphthoflavone, 3-methylcholanthrene, phenobarbital, isoniazid, rifampin, and clofibric acid, on the expression of cytochrome P450 (P450) enzymes in beagle dogs. For the in vitro induction study, primary cultures of dog hepatocytes were treated with enzyme inducers for 3 days, after which microsomes were prepared and analyzed for P450 activities. For the in vivo induction study, male and female beagle dogs were treated with enzyme inducers for 4 days (with the exception of phenobarbital, which was given for 14 days), after which the livers were removed and microsomal P450 activities were determined ex vivo. Treatment of male beagle dog hepatocyte cultures (n = 3) with beta-naphthoflavone or 3-methlychloranthrene resulted in up to a 75-fold increase in microsomal 7-ethoxyresorufin O-dealkylase (CYP1A1/2) activity, whereas in vivo treatment of male and female beagle dogs with beta-naphthoflavone followed by ex vivo analysis resulted in up to a 24-fold increase. Phenobarbital caused a 13-fold increase in 7-benzyloxyresorufin O-dealkylase (CYP2B11) activity in vitro and up to a 9.9-fold increase in vivo. Isoniazid had little or no effect on 4-nitrophenol hydroxylase activity in vitro. Rifampin caused a 13-fold induction of testosterone 6beta-hydroxylase (CYP3A12) activity in vitro and up to a 4.5-fold increase in vivo. Treatment of dogs in vivo or dog hepatocytes in vitro with clofibric acid appeared to have no effect on CYP4A activity as determined by the 12-hydroxylation of lauric acid. In general, the absolute rates (picomoles per minute per milligram of microsomal protein) of P450 reactions catalyzed by microsomes from cultured hepatocytes (i.e., in vitro rates) were considerably lower than those catalyzed by microsomes from dog liver (i.e., ex vivo rates). These results suggest that beagle dogs have CYP1A, CYP2B, CYP2E, and CYP3A enzymes and that the induction profile resembles the profile observed in humans more than in rats.

Animals↗
PubMed2002

Hepatocyte nuclear factor-1alpha is a causal factor responsible for interindividual differences in the expression of UDP-glucuronosyltransferase 2B7 mRNA in human livers.

UDP-glucuronosyltransferase (UGT) 2B7 is one of the most important UGT isozymes expressed in human livers. This enzyme is reported to show more than 10-fold interindividual differences in its enzyme activities. Thus, the amounts of UGT2B7 mRNA in 12 human livers were quantified by quantitative reverse transcription-polymerase chain reaction. The amounts of UGT2B7 mRNA in the subjects ranged from 0.22 to 2.63 copies/10(3) copies of beta-actin. A novel point mutation (-253G to A) found in this study did not affect the level of UGT2B7 mRNA in the subjects. To clarify a causal factor(s) determining the expression level of UGT2B7 mRNA, we examined the correlation between the amounts of mRNAs for UGT2B7 and hepatocyte nuclear factor (HNF)-1alpha, which regulates the expression of UGT2B7 gene. HNF-1alpha mRNA was expressed at a level ranging from 2.99 to 24.76 copies/10(6) copies of beta-actin in the subjects. The amounts of mRNAs for UGT2B7 expressed in these individual liver samples were highly associated with the amount of mRNA for HNF-1alpha (r = 0.786, p = 0.002), suggesting that HNF-1alpha is a factor limiting the expression of UGT2B7 mRNA and a causal factor responsible for an interindividual difference in human livers.

DNA-Binding Proteins↗
PubMed2002

CYP3A4 induction by drugs: correlation between a pregnane X receptor reporter gene assay and CYP3A4 expression in human hepatocytes.

Induction of cytochrome P450 3A4 (CYP3A4) is determined typically by employing primary culture of human hepatocytes and measuring CYP3A4 mRNA, protein and microsomal activity. Recently a pregnane X receptor (PXR) reporter gene assay was established to screen CYP3A4 inducers. To evaluate results from the PXR reporter gene assay with those from the aforementioned conventional assays, 14 drugs were evaluated for their ability to induce CYP3A4 and activate PXR. Sandwiched primary cultures of human hepatocytes from six donors were used and CYP3A4 activity was assessed by measuring microsomal testosterone 6beta-hydroxylase activity. Hepatic CYP3A4 mRNA and protein levels were also analyzed using branched DNA technology/Northern blotting and Western blotting, respectively. In general, PXR activation correlated with the induction potential observed in human hepatocyte cultures. Clotrimazole, phenobarbital, rifampin, and sulfinpyrazone highly activated PXR and increased CYP3A4 activity; carbamazepine, dexamethasone, dexamethasone-t-butylacetate, phenytoin, sulfadimidine, and taxol weakly activated PXR and induced CYP3A4 activity, and methotrexate and probenecid showed no marked activation in either system. Ritonavir and troleandomycin showed marked PXR activation but no increase (in the case of troleandomycin) or a significant decrease (in the case of ritonavir) in microsomal CYP3A4 activity. It is concluded that the PXR reporter gene assay is a reliable and complementary method to assess the CYP3A4 induction potential of drugs and other xenobiotics.

Adult↗
PubMed2002

Twenty one novel single nucleotide polymorphisms (SNPs) of the CYP2A6 gene in Japanese and Caucasians.

We sequenced all nine exons and exon-intron junctions of the CYP2A6 gene from 33 Japanese and 28 Caucasians. We found twenty one single nucleotide polymorphisms (SNPs) including four SNPs causing amino acid substitutions, one silent SNP in exon 5, one SNP in a 5'-flanking region, four SNPs in a 3'-untranslated region, and eleven SNPs in introns. The four mutations (13G>A and 86G>A in exon 1, and 2134A>G and 2161C>T in exon 4) causing amino acid substitutions (Gly(5)Arg, Ser(29)Asn, Lys(194)Glu, and Arg(203)Ser), respectively, were as follows: SNP, 020719Kiyotani004; GENE NAME, CYP2A6; ACCESSION NUMBER, NG_000008.4; LENGTH, 25 base; 5'-ATGCTGGCCTCAG/AGGATGCTTCTGG-3'. SNP, 020719Kiyotani005; GENE NAME, CYP2A6; ACCESSION NUMBER, NG_000008.4; LENGTH, 25 base; 5'-AGCAGAGGAAGAG/ACAAGGGGAAGCT-3'. SNP, 020719Kiyotani011; GENE NAME, CYP2A6; ACCESSION NUMBER, NG_000008.4; LENGTH, 25 base; 5'-CGCTTTGACTATA/GAGGACAAAGAGT-3'. SNP, 020719Kiyotani012; GENE NAME, CYP2A6; ACCESSION NUMBER, NG_000008.4; LENGTH, 25 base; 5'-CTGTCACTGTTGC/TGCATGATGCTAG-3'. New alleles having these SNPs were designated as CYP2A6( *)13-CYP2A6( *)16.

Journal Article↗