Drug metabolism in human liver in vitro: Establishment of a human liver bank

Bahr, Christer von; Groth, Carl‐Gustav; Jansson, Helena; Lundgren, G; Lind, Margarete; Glaumann, Hans

doi:10.1038/clpt.1980.102

Cited by 164 publications

(55 citation statements)

References 8 publications

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“…18888) were purchased from BD Gentest (Woburn, MA, USA). Rat liver microsomes were prepared by differential centrifugation [11], and the protein concentration of the microsomes was determined using the Lowry method.…”

Section: Chemicalsmentioning

confidence: 99%

PQ-69, a novel and selective adenosine A1 receptor antagonist with inverse agonist activity

Lu¹,

Wang²,

Zhang³

et al. 2014

Purinergic Signalling

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Potent and selective adenosine A 1 receptor (A 1 AR) antagonists with favourable pharmacokinetic properties used as novel diuretics and antihypertensives are desirable. Thus, we designed and synthesized a series of novel 4-alkylamino substitution-2-arylpyrazolo[4,3-c]quinolin-3-one derivatives. The aim of the present study is to characterize the biological profiles of the optimized compound, PQ-69. In vitro binding assay revealed a K i value of 0.96 nM for PQ-69 in cloned hA 1 receptor, which was 217-fold more selective compared with hA 2A receptors and >1,000-fold selectivity for hA 1 over hA 3 receptor. The results obtained from [ 35 S]-GTPγS binding and cAMP concentration assays indicated that PQ-69 might be an A 1 AR antagonist with inverse agonist activity. In addition, PQ-69 displayed highly inhibitory activities on isolated guinea pig contraction (pA2 value of 8.99) induced by an A 1 AR agonist, 2-chloro-N6-cyclopentyl adenosine. Systemic administration of PQ-69 (0.03, 0.3, 3 mg/kg) increased urine flow and sodium excretion in normal rats. Furthermore, PQ-69 displayed better metabolic stability in vitro and longer terminal elimination half-life (t 1/2 ) in vivo compared with 1,3-dipropyl-8-cyclopentylxanthine. These findings suggest that PQ-69 exhibits potent antagonist effects on A 1 AR in vitro, ex vivo and in vivo, it might be a useful research tool for investigating A 1 AR function, and it could be developed as a potential therapeutic agent.

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Section: Chemicalsmentioning

confidence: 99%

PQ-69, a novel and selective adenosine A1 receptor antagonist with inverse agonist activity

Lu¹,

Wang²,

Zhang³

et al. 2014

Purinergic Signalling

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“…30,31 QIAamp s Tissue DNA Preparation Kit (Qiagen, Hilden, Germany) was used to isolate the genomic DNA. Identification of the CYP2B6 C1459T mutation (CYP2B6*5) was made by allelic discrimination with a 5 0 -nuclease assay.…”

Section: Genotyping Of Cyp2b6mentioning

confidence: 99%

“…30,31 Except three Japanese donor livers, the samples were from Swedish origin. The microsomes were stored in potassium phosphate buffer (50 mM, pH 7.4) at -801C until use.…”

Section: Preparation Of Human Liver Microsomesmentioning

confidence: 99%

Role of polymorphic human CYP2B6 in cyclophosphamide bioactivation

et al. 2003

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The role of polymorphic CYP2B6 in cyclophosphamide (CPA) bioactivation was investigated in human liver microsomes. A total of 67 human liver specimens were first genotyped with respect to the CYP2B6*5 and CYP2B6*6 variant alleles. CYP2B6 apoprotein levels in 55 liver microsomal preparations were assessed by immunoblotting. 4-Hydroxy-CPA and hydroxy-bupropion were quantified by using HPLC and LC-MS, respectively. 7-Ethoxy-4-trifluoromethyl coumarin O-deethylase activity was measured fluorometrically. The frequencies of CYP2B6*5 and CYP2B6*6 mutant alleles were 9.0 and 16.4%, respectively. CYP2B6 protein expression was detected in 80% of the samples, with a large variation (0.003-2.234, arbitrary units). There was a high correlation between CYP2B6 apoprotein content and CPA 4-hydroxylation (n ¼ 55, r ¼ 0.81, Po0.0001). When based on the CYP2B6 apoprotein levels, the *6 carriers had significantly higher CPA 4-hydroxylation (Po0.05). CPA 4-hydroxylation also correlated significantly with other CYP2B6-specific reactions (n ¼ 20, Po0.0001). V max and K m for CPA 4-hydroxylation in recombinant CYP2B6 enzyme were 338 nmol/min/nmol enzyme and 1.4 mM, respectively. CYP2B6 showed much higher in vitro intrinsic clearance than previously observed in recombinant CYP2C19 and CYP2C9 variants in yeast expression system. Our results demonstrate that the polymorphic CYP2B6 is a major enzyme in the bioactivation of CPA. Moreover, we identified a strong impact of CYP2B6*6 on CPA 4-hydroxylation.

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“…Liver microsomes were prepared essentially as described [13] from 35 different human livers belonging to a donor liver biobank established at the Division of Clinical Pharmacology, Huddinge University Hospital, after approval from the hospital Ethics Committee. Each liver was genotyped for the CYP2C9 * 1, CYP2C9 * 2 and CYP2C9 * 3 alleles [7].…”

Section: Preparation Of Human Liver Microsomesmentioning

confidence: 99%

Oxidation of celecoxib by polymorphic cytochrome P450 2C9 and alcohol dehydrogenase

Sandberg

Yaşar

Strömberg

et al. 2002

Brit J Clinical Pharma

102

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Aims Celecoxib is a novel selective cyclooxygenase-2 inhibitor, which is subject to extensive hepatic metabolism. The aims of the present in vitro investigation were 1) to compare the rate of celecoxib hydroxylation by different genetic variants of cytochrome P450 2C9 (CYP2C9), and 2) to identify the enzyme(s) involved in the formation of the major metabolite carboxycelecoxib. Methods Hydroxycelecoxib formation was studied in human liver microsomes from 35 genotyped livers, as well as in yeast microsomes with recombinant expression of different P450 variants. Carboxycelecoxib formation was studied in liver microsomes incubated in the absence or presence of liver cytosol. The metabolites were identified and quantified by h.p.l.c. In addition, hydroxycelecoxib oxidation by different variants of recombinant human alcohol dehydrogenase (ADH1-3) was analysed by spectrophotometric monitoring of NADH generation from NAD + . Results The intrinsic clearance of celecoxib hydroxylation was significantly lower for yeast-expressed CYP2C9.3 (0.14 ml min − 1 nmol − 1 enzyme) compared with CYP2C9.1 (0.44 ml min − 1 nmol − 1 enzyme). In human liver microsomes, a significant 2-fold decrease in the rate of hydroxycelecoxib formation was evident in CYP2C9 * 1/ * 3 samples compared with CYP2C9 * 1/ * 1 samples. There was also a marked reduction (up to 5.3 times) of hydroxycelecoxib formation in a liver sample genotyped as CYP2C9 * 3/ * 3 . However, the CYP2C9 * 2 samples did not differ significantly from CYP2C9 * 1 in any of the systems studied. Inhibition experiments with sulphaphenazole (SPZ) or triacetyloleandomycin indicated that celecoxib hydroxylation in human liver microsomes was mainly dependent on CYP2C9 and not CYP3A4. The further oxidation of hydroxycelecoxib to carboxycelecoxib was completely dependent on liver cytosol and NAD + . Additional experiments showed that ADH1 and ADH2 catalysed this reaction in vitro with apparent K m values of 42 µ M and 10 µ M , respectively, whereas ADH3 showed no activity. Conclusions The results confirm that CYP2C9 is the major enzyme for celecoxib hydroxylation in vitro and further indicate that the CYP2C9 * 3 allelic variant is associated with markedly slower metabolism. Furthermore, it was shown for the first time that carboxycelecoxib formation is dependent on cytosolic alcohol dehydrogenase, presumably ADH1 and/or ADH2.

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Drug metabolism in human liver in vitro: Establishment of a human liver bank

Cited by 164 publications

References 8 publications

PQ-69, a novel and selective adenosine A1 receptor antagonist with inverse agonist activity

PQ-69, a novel and selective adenosine A1 receptor antagonist with inverse agonist activity

Role of polymorphic human CYP2B6 in cyclophosphamide bioactivation

Oxidation of celecoxib by polymorphic cytochrome P450 2C9 and alcohol dehydrogenase

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