Thomas K. H. Chang

Identification of the polymorphically expressed CYP2C19 and the wild-type CYP2C9-ILE359 allele as low-Km catalysts of cyclophosphamide and ifosfamide activation

Cyclophosphamide and ifosfamide are alkylating agent prodrugs that require activation by cytochrome P450 (CYP) to manifest their cancer chemotherapeutic activity. The present study investigates the activity of four individual human CYP2C enzymes and their allelic variants in cyclophosphamide and ifosfamide activation as an initial attempt to gain insight into the underlying basis for the large interpatient differences in the clinical pharmacokinetics and metabolism of these anticancer drugs. Recombinant CYP2C8, CYP2C19, two allelic variants of CYP2C18, and six variants of CYP2C9 expressed in a yeast cDNA expression system were each enzymatically active, as judged by the ability of the isolated microsomes to catalyse 7-ethoxycoumarin O-deethylation after reconstitution with purified NADPH-cytochrome P450 reductase and cytochrome b5. With cyclophosphamide as substrate, CYP2C19 had the lowest apparent Km, followed by CYP2C9, CYP2C18 and CYP2C8, whereas in the case of ifosfamide, the rank order was: Km CYP2C19 < CYP2C18 < CYP2C9 < CYP2C8. CYP2C18 had the highest in vitro intrinsic clearance/catalytic efficiency (apparent Vmax/Km) in cyclophosphamide and ifosfamide activation, followed by 2C19 > 2C9 approximately 2C8. Examination of a panel of CYP2C allelic variants revealed that CYP2C18-Thr385 had both a higher Vmax and a higher apparent Km toward cyclophosphamide than CYP2C18-Met385 with no difference in catalytic efficiency, whereas with ifosfamide the Thr385 allele exhibited a strikingly lower apparent Km resulting in a six-fold higher catalytic efficiency. In the case of CYP2C9, a Ile359 to Leu mutation associated with poor metabolism of the hypoglycemic drug tolbutamide decreased catalytic efficiency toward cyclophosphamide by increasing the apparent Km, whereas the same mutation reduced the efficiency of this P450 toward ifosfamide by decreasing the Vmax. Substitution of CYP2C9-Gly417 by Asp resulted in a two-fold lower catalytic efficiency for cyclophosphamide metabolism but a three-fold higher efficiency for ifosfamide metabolism. A His276 to Gly substitution resulted in an increase in both Vmax and apparent Km with no net change in catalytic efficiency for either oxazaphosphorine. Mutations at CYP2C9 residues 144 and 358 had little or no effect. Thus (a) wild type CYP2C19 and CYP2C9 are relatively low Km catalysts of cyclophosphamide and ifosfamide activation, and (b) all four human CYP2C enzymes activate these two anticancer prodrugs with varying efficiencies and with striking differences among naturally occurring allelic variants in the case of CYP2C9 and CYP2C18.

Pharmacogenetics: The therapeutic drug monitoring of the future?

Genetic variability in drug response occurs as a result of molecular alterations at the level of drug-metabolising enzymes, drug targets/receptors, and drug transport proteins. In this paper, we discuss the possibility that therapeutic drug monitoring (TDM) in the future will involve not the mere measurement and interpretation of drug concentrations but will include both traditional TDM and pharmacogenetics-oriented TDM. In contrast to traditional TDM, which cannot be performed until after a drug is administered to the patient. pharmacogenetics-oriented TDM can be conducted even before treatment begins. Other advantages of genotyping over traditional TDM include, but are not limited to, the following: (i) it does not require the assumption of steady-state conditions (or patient compliance) for the interpretation of results; (ii) it can often be performed less invasively (with saliva, hair root or buccal swab samples); (iii) it can provide predictive value for multiple drugs [e.g. a number of cytochrome P450 (CYP) 2D6, CYP2C 19 or CYP2C9 substrates] rather than a single drug; (iv) it provides mechanistic, instead of merely descriptive, information; and (v) it is constant over an individuals lifetime (and not influenced by concurrent drug administration, alteration in hormonal levels or disease states). Pharmacogenetic information can be applied a priori for initial dose stratification and identification of cases where certain drugs are simply not effective. However, traditional TDM will still be required for all of the reasons that we use it now. In current clinical practice, pharmacogenetic testing is performed for only a few drugs (e.g. mercaptopurine, thioguanine, azathioprine, trastuzumab and tacrine) and in a limited number of teaching hospitals and specialist academic centres. We propose that other drugs (e.g. warfarin, phenytoin, codeine, oral hypoglycaemics, tricyclic antidepressants, aminoglycosides, digoxin, cyclosporin, cyclophosphamide, ifosfamide, theophylline and clozapine) are potential candidates for pharmacogenetics-oriented TDM. However, prospective studies of phaymacogenetics-oriented TDM must be performed to determine its efficacy and cost effectiveness in optimising therapeutic effects while minimising toxicity. In the future, in addition to targeting a patients drug concentrations within a therapeutic range, pharmacists are likely to be making dosage recommendations for individual drugs on the basis of the individual patients genotype. As we enter the era of personalised drug therapy, we will be able to identify not only the best drug to be administered to a particular patient, but also the most effective and safest dosage from the outset of therapy.

Hormonal Regulation of Liver Cytochrome P450 Enzymes

Sex differences characterize hepatic expression of specific cytochrome P450 (CYP) enzymes and their associated CYP genes, and underlie the widespread sex differences in drug and xenobiotic metabolism and toxicity seen in animal models and in humans. Sex differences in P450 expression first emerge around puberty, as exemplified by the postnatal developmental expression patterns characterizing the prototypic sex-specific rat P450 enzymes CYP2C11 (male-specific) and CYP2C12 (female-specific). The sex-dependent expression of liver CYPs is primarily determined by sex differences in the temporal patterns of pituitary growth hormone secretion, which confer widespread sex differences in chromatin accessibility, epigenetic marks, and chromatin states. Mechanistic studies have identified an intracellular signaling protein and transcription factor known as signal transducer and activator of transcription 5b (STAT5b) as a major molecular mediator of the action of growth hormone on the sex-dependent transcription of liver CYP genes. Gonadal hormones (testosterone and estrogen) regulate hepatic expression of sex-dependent P450 enzymes primarily by indirect mechanisms, via gonadal hormone effects on the hypothalamic–pituitary axis, which in turn dictate the sex-dependent temporal pattern of pituitary growth hormone secretion. The expression of sex-dependent liver P450 enzymes can also be altered by hormonal perturbation induced by drugs and other xenobiotics, disease states, including diabetes mellitus, liver cirrhosis, and kidney failure, and dietary factors such as vitamin A.

Activation of Pregnane X Receptor (PXR) and Constitutive Androstane Receptor (CAR) by Herbal Medicines

Pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are transcription factors that control the expression of a broad array of genes involved not only in transcellular transport and biotransformation of many drugs, other xenochemicals, and endogenous substances, such as bile acid, bilirubin, and certain vitamins, but also in various physiological/pathophysiological processes such as lipid metabolism, glucose homeostasis, and inflammation. Ligands of PXR and CAR are chemicals of diverse structures, including naturally occurring compounds present in herbal medicines. The overall aim of this article is to provide an overview of our current understanding of the role of herbal medicines as modulators of PXR and CAR.

Influence of CYP2C9 genotypes on the formation of a hepatotoxic metabolite of valproic acid in human liver microsomes

ABSTRACTThe present study investigated the effect of cytochrome P450 2C9 (CYP2C9) genetic polymorphism on the biotransformation of valproic acid (VPA) to its hepatotoxic metabolite, 4-ene-VPA, and compared that to the formation of the inactive 4-OH-VPA and 5-OH-VPA. cDNA-expressed CYP2C9*2 and CYP2C9*3 variants were less efficient than the CYP2C9*1 wild type in catalyzing the formation of these metabolites, as assessed by the ratio of Vmax and apparent Km (in vitro intrinsic clearance). The reduced efficiency by CYP2C9*2 was due to a reduced Vmax, whereas, in the case of CYP2C9*3, it was the result of increased apparent Km. The formation rates of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA in human liver microsomes were reduced by 29, 28, and 31%, respectively, in samples with one mutated CYP2C9 allele, and by 61, 73, and 58%, respectively, in samples with two mutated CYP2C9 alleles. Overall, the homozygote and heterozygote CYP2C9*2 and CYP2C9*3 genotypes may compromise hepatic VPA biotransformation.

Identification of Ginkgo biloba as a Novel Activator of Pregnane X Receptor

Pregnane X receptor (PXR; NR1I2) is a ligand-activated transcription factor that plays a role not only in drug metabolism and transport but also in various other biological processes. Ginkgo biloba is a herbal medicine commonly used to manage memory impairment. Treatment of primary cultures of rat hepatocytes with G. biloba extract increases the mRNA expression of CYP3A23, which is a target gene for rat PXR. The present study was conducted to test the hypothesis that G. biloba extract activates PXR. Treatment of mouse PXR (mPXR) or human PXR (hPXR)-transfected HepG2 cells with G. biloba extract at 200 μg/ml increased mPXR and hPXR activation by 3.2- and 9.5-fold, respectively. Dose-response analysis showed a log-linear increase in hPXR activation by the extract over the range of 200 to 800 μg/ml. To determine whether G. biloba extract induces hPXR target gene expression, cultured LS180 human colon adenocarcinoma cells were treated for 72 h with the extract. G. biloba extract at 200, 400, and 800 μg/ml increased CYP3A4 mRNA expression by 1.7-, 2.4-, and 2.5-fold, respectively. The same concentrations of the extract increased CYP3A5 (1.3-3.6-fold) and P-glycoprotein (ABCB) 1 (2.7-6.4-fold) mRNA expression. At concentrations (5 and 10 μM) that did not down-regulate PXR gene expression and were not cytotoxic, l-sulforaphane (an hPXR antagonist) decreased CYP3A4, CYP3A5, and ABCB1 gene expression in cells treated with G. biloba extract. In summary, G. biloba extract activated mPXR and hPXR in a cell-based reporter gene assay and induced CYP3A4, CYP3A5, and ABCB1 gene expression in hPXR-expressing LS180 cells.

Spectrophotometric Analysis of Human CYP2E1-Catalyzed p-Nitrophenol Hydroxylation

The cytochrome P450 enzyme CYP2E1 catalyzes the oxidative metabolism of many solvents and other small organic molecules. A spectrophotometric method is described for determination of CYP2E1 activity by monitoring the formation of p-nitrocatechol from p-nitrophenol by cDNA-expressed CYP2E1 or isolated liver microsomes. The enzymatic product, p-nitrocatechol, is assayed at 535 nm after acidification of the reaction mixture with trichloroacetic acid followed by neutralization using 2 M NaOH. This method is applicable to enzymatic studies for determination of P450-catalyzed p-nitrophenol hydroxylation activity.

Enzymatic Analysis of cDNA-Expressed Human CYP1A1, CYP1A2, and CYP1B1 with 7-Ethoxyresorufin as Substrate

Cytochrome P450 (P450) enzymes belonging to the CYP1 family are highly inducible by polycyclic aromatic hydrocarbons and other environmental chemicals and play a major role in the metabolism of many foreign chemicals and endogenous substances. We describe a spectrofluorometric method for determining 7-ethoxyresorufin O-dealkylation catalyzed by CYP1A1, CYP1A2, and CYPB1. The formation of the enzymatic product, resorufin, is monitored continuously by fluorescence using an excitation wavelength of 530 nm and an emission wavelength of 580 nm. This method can be applied to assay P450-catalyzed formation of resorufin from other alkoxyresorufins, such as 7-methoxyresorufin, 7-benzyloxyresorufin, and 7-pentoxyresorufin. It can also be used to assay 7-ethoxyresorufin O-dealkylation activity in isolated hepatocytes and cultured cells that express this P450 activity.

The effect of valproic acid on hepatic and plasma levels of 15-F2t-isoprostane in rats

The mechanism by which valproic acid (VPA) induces liver injury remains unknown, but it is hypothesized to involve the generation of toxic metabolites and/or reactive oxygen species. This studys objectives were to determine the effect of VPA on plasma and hepatic levels of the F(2)-isoprostane, 15-F(2t)-IsoP, a marker for oxidative stress, and to investigate the influence of cytochrome P450- (P450-) mediated VPA biotransformation on 15-F(2t)-IsoP levels in rats. In rats treated with VPA (500 mg/kg), plasma 15-F(2t)-IsoP was increased 2.5-fold at t(max) = 0.5 h. Phenobarbital pretreatment (80 mg/kg/d for 4 d) in VPA-treated rats increased plasma and liver levels of free 15-F(2t)-IsoP by 5-fold and 3-fold, respectively, when compared to control groups. This was accompanied by an elevation in plasma and liver levels of P450-mediated VPA metabolites. Pretreatment with SKF-525A (80 mg/kg) or 1-aminobenzotriazole (100 mg/kg), which inhibited P450-mediated VPA metabolism, did not attenuate the increased levels of plasma 15-F(2t)-IsoP in VPA-treated groups. Plasma and hepatic levels of 15-F(2t)-IsoP were further elevated after 14 d of VPA treatment compared to single-dose treatment. Our data indicate that VPA increases plasma and hepatic levels of 15-F(2t)-IsoP and this effect can be enhanced by phenobarbital by a mechanism not involving P450-catalyzed VPA biotransformation.

Growth hormone regulation and developmental expression of rat hepatic CYP3A18, CYP3A9, and CYP3A2

The present study investigated the role of growth hormone (GH) in hepatic CYP3A18 and CYP3A9 expression in prepubertal and adult male rats. For comparison, the effects of GH on CYP3A2 expression were also measured. Initial experiments demonstrated that CYP3A18 mRNA levels were greater during puberty and adulthood than during the prepubertal period, CYP3A9 mRNA was not expressed until puberty and its expression increased in adulthood, and CYP3A2 mRNA levels were relatively constant from prepuberty to adult life. Hypophysectomy, which results in the loss of multiple pituitary factors including GH, increased CYP3A2 and CYP3A18 mRNA expression 3- to 4-fold, but it did not affect CYP3A9 mRNA levels or CYP3A-mediated testosterone 2beta- or 6beta-hydroxylase activity in adult rats. GH administered as twice daily s.c. injections (0.12 microg/g body weight) to hypophysectomized or intact adult rats did not affect CYP3A18 or CYP3A9 mRNA expression. The same treatment decreased CYP3A2 mRNA and protein and testosterone 2beta- and 6beta-hydroxylase activity levels in intact but not hypophysectomized rats. However, in intact prepubertal rats, intermittent GH administration decreased CYP3A18 and CYP3A2 mRNA levels, but a higher dosage (3.6 microg/g) was required to suppress CYP3A2. Overall, the present study demonstrated that: (a) the constitutive expression of CYP3A18, CYP3A9, and CYP3A2 does not require the presence of GH, (b) CYP3A18 is more sensitive than CYP3A9 to GH modulation in adult rats; and (c) CYP3A2 is less sensitive to the suppressive influence of GH during the prepubertal period than during adult life.