Acetylator phenotype and genotype in patients infected with HIV: discordance between methods for phenotype determination and genotype.

The acetylator phenotype and genotype of AIDS patients, with and without an acute illness, was compared with that of healthy control subjects (30 per group). Two probe drugs, caffeine and dapsone, were used to determine the phenotype in the acutely ill cohort. Polymerase chain reaction amplification and restriction fragment length polymorphism analysis served to distinguish between the 26 known NAT2 alleles and the 21 most common NAT1 alleles. The distribution (%) of slow:rapid acetylator phenotype seen among acutely ill AIDS patients differed with the probe substrate used: 70:30 with caffeine versus 53:47 with dapsone. Phenotype assignment differed considerably between the two methods and there were numerous discrepancies between phenotype and genotype. The NAT2 genotype distribution was 45:55 slow:rapid. Control subjects, phenotyped only with caffeine, were 67:33 slow:rapid versus 60:40 genotypically. Stable AIDS patients, phenotyped only with dapsone, were 55:45 slow:rapid versus 46:54 genotypically. Following resolution of their acute infections, 12 of the acutely ill subjects were rephenotyped with dapsone. Phenotype assignment remained unchanged in all cases. The distribution of NAT1 alleles was similar in all three groups. It is evident from the amount of discordance between caffeine phenotype and dapsone phenotype or genotype that caution should be exercised in the use of caffeine as a probe for NAT2 in acutely ill patients. It is also clear that meaningful study of the acetylation polymorphism requires both phenotypic and genotypic data.

Effect of streptolysin-O-on rat hepatic acetyl coenzyme-A: arylamine N-acetyltransferase and cytochrome P-450 2B 1/2 activities ex vivo.

Numerous immunostimulants have been found to increase N-acetylation in vivo but are not associated with a similar increase in vitro. Streptolysin-O (SLO), a thiol-activated (oxygen-labile) hemolytic and immune-stimulating exotoxin produced by group A streptococci, has been reported to increase the metabolic rate constant for sulfamethazine in vivo and arylamine N-acetyltransferase (NAT) activity toward procainamide (PA) ex vivo. The effect of SLO pretreatment of rats on cytochrome P-450-catalyzed tolbutamide hydroxylation and NAT activities toward PA (a substrate for NAT1), and p-aminobenzoic acid (a substrate for NAT2) was examined ex vivo. Subacute SLO (SIGMA Chemical Company, St. Louis, MO) pretreatment (100 Hemolytic Units/kg/day, intraperitoneal, for 4 days) did not alter body weight, liver weight or cytosolic protein content as compared with controls. SLO-pretreatment did not alter NAT activities measured ex vivo, nor was an alteration in tolbutamide hydroxylation observed. Pretreatment with an alternative SLO preparation (DIFCO Laboratories, Detroit, MI) also failed to alter the parameters of body weight, liver weight or cytosolic protein content as compared with controls. While treated animals had significantly reduced microsomal protein content, SLO pretreatment failed to alter the enzyme activities measured. We conclude that SLO does not serve as a useful model immunostimulant for mechanistic studies as it produces no consistent effect on drug metabolizing enzymes.

Amlodipine inhibits rat microsomal cytochrome P450-mediated drug biotransformation.

Calcium channel antagonists have been shown to inhibit cytochrome P-450-mediated metabolism both in vitro and in vivo. The purpose of the present study was to examine the effect of amlodipine on a suite of rat hepatic microsomal cytochrome P-450 activities to determine the potential for drug interactions. In this study, amlodipine (0.05 and 0.5 mM) decreased CYP1A-mediated ethoxyresorufin O-deethylase activity in microsomes prepared from noninduced (56 and 73% inhibition) and pyridine-induced (30 and 51% inhibition) rats. Amlodipine reduced pentoxyresorufin O-deethylase activity (a marker for CYP2B) to 15% of control in incubations utilizing microsomes from phenobarbital-treated rats, but had no effect on this enzyme reaction in noninduced microsomes. The para-nitrophenol hydroxylase, erythromycin N-demethylase, and lauric acid omega and omega-1 hydroxylase activities were significantly inhibited by 1 mM amlodipine in both noninduced and induced microsomes. These results suggest that amlodipine inhibits a number of different P450 forms and therefore has the potential to inhibit the metabolism of a large number of drugs.

Fluoroquinolone antibiotics inhibit cytochrome P450-mediated microsomal drug metabolism in rat and human.

The fluoroquinolone antibacterial agents have gained widespread use in the treatment of a broad range of bacterial infections. We recently described a possible interaction concerning the concomitant use of cyclosporine A and norfloxacin in pediatric renal transplant patients. We examined the effect of two common fluoroquinolone antibiotics on cytochrome P450-mediated drug biotransformations in human and rat liver microsomes. Rats were pretreated with inducers, which increased the levels of the P450 isozymes CYP3A2, CYP1A, CYP2E1, and CYP4A1. Ciprofloxacin and norfloxacin significantly depressed the N-demethylation of erythromycin by CYP3A4 in human microsomes and by CYP3A2 in rat microsomes. The inhibition was determined to be competitive in nature in rat microsomes, with ciprofloxacin and norfloxacin both exhibiting similar Ki values of 2.0 and 2.3 mM, respectively. Ciprofloxacin and norfloxacin also inhibited ethoxyresorufin-O-dealkylase (CYP1A). In contrast, ciprofloxacin and norfloxacin did not inhibit the metabolism of substrates that are specific for the P450 isozymes CYP2E1 and CYP4A1. Rats treated chronically with norfloxacin revealed no alterations in hepatic CYP3A2 protein levels or activity. These studies in hepatic microsomes demonstrate that fluoroquinolones can decrease CYP3A- and CYP1A-mediated biotransformation by competitive inhibition and that they have the potential to cause drug interactions with agents metabolized by these enzymes.

Norfloxacin interferes with cyclosporine disposition in pediatric patients undergoing renal transplantation.

Prophylactic treatment with norfloxacin was initiated in a group of pediatric patients undergoing renal transplantation who were receiving cyclosporine and were susceptible to recurrent urinary tract infections. At discharge from the hospital, the mean daily dose of cyclosporine needed to maintain trough cyclosporine blood levels of 150 to 400 ng/ml was 4.5 mg/kg/day for the patients who received norfloxacin compared with 7.4 mg/kg/day for patients who did not receive the antibiotic. This observation suggested that the clearance of cyclosporine was decreased by the concomitant use of norfloxacin. The effect of norfloxacin on specific drug-metabolizing cytochrome P450 isozymes in vitro was examined to determine if the metabolism and subsequent clearance of cyclosporine and possibly other drugs are altered through a metabolic interaction with norfloxacin. In human liver microsomes, the activity of cytochrome P4503A4, the isozyme that metabolizes cyclosporine in humans, was inhibited by norfloxacin. In rat liver microsomes, norfloxacin inhibited the activity of cytochrome P4503A2, the isozyme responsible for cyclosporine metabolism in this species, but did not alter the activity of the rat cytochrome P450 isozymes 1A, 2E1, and 4A1. The in vitro studies suggest that the lower cyclosporine dose required by pediatric patients who were given norfloxacin was caused by inhibition of the metabolism of cyclosporine.

Therapeutic drug monitoring in saliva. An update.

This article re-examines the issue of salivary therapeutic drug monitoring (STDM). The anatomy and physiology of saliva and the salivary glands, as well as the effects of disease and drugs on salivary secretion and composition, are discussed briefly. Drugs for which therapeutic drug monitoring (TDM) has been shown useful are individually considered to determine if salivary drug concentrations (Csal) are reflective of plasma free drug concentrations (C(up)). That is, is the Csal/C(up) ratio time- and concentration-independent, as supported by a review of literature data? The primary determinant which appears to govern the potential utility of STDM for many of the drugs is the pKa of the drug. Drugs which are not ionisable or are un-ionised within the salivary pH range (phenytoin, carbamazepine, theophylline) are candidates for STDM based on current literature data. Digoxin and cyclosporin are potential candidates for STDM; however, further studies are necessary to confirm these preliminary findings. On the basis of current literature data, STDM does not appear to be useful for other drugs therapeutically monitored in serum/plasma.

Immunomodulation and drug acetylation: influence of the immunomodulator tilorone on hepatic, renal and blood N-acetyltransferase activity and on hepatic cytosolic acetyl coenzyme A content.

The biochemical alteration responsible for immunomodulator enhancement of drug acetylation in vivo was probed ex vivo and in vitro in the rat. Rat liver or kidney cytosol, obtained by differential centrifugation, or whole blood served as the source of N-acetyltransferase (NAT). Addition of tilorone (0.5-8.0 mM) to incubation mixtures containing procainamide (PA, 0.6 mM) and acetyl coenzyme A (AcCoA, 0.42 mM) resulted in the inhibition of N-acetylprocainamide formation, while lower concentrations of tilorone had no effect. Pretreatment of rats with tilorone (50 mg/kg) administered orally 48 hr prior to sacrifice did not alter hepatic apparent Km and Vmax for NAT toward PA compared to control animals. Utilization of an AcCoA regenerating system in the incubation mixtures also resulted in no significant differences in the apparent Michaelis-Menten parameters obtained. Acetylation activity in kidney and whole blood also was not altered by immunomodulator pretreatment. Hepatic cytosolic AcCoA content was reduced significantly 48 hr after tilorone pretreatment (5.10 +/- 2.1 vs 11.97 +/- 2.2 nmol/mg protein) (P less than 0.05). These data indicate that an increase in NAT content or activity is not the biochemical alteration responsible for immunomodulator enhancement of drug acetylation, and that the required cofactor, cytosolic AcCoA, is decreased by immunomodulator pretreatment.

Effects of chloroquine and primaquine on rat liver cytosolic N-acetyltransferase activity.

Chloroquine caused only slight reductions in NAT activity when added in vitro, and had no detectable influence when animals were pretreated with it for 4 days. This would suggest that the previously reported reduced excretion of acetylated metabolites of INH and SDD following chloroquine pretreatment is not the result of inhibition of NAT. In contrast, we found that primaquine significantly (P less than 0.05) reduced NAT activity when added in vitro, suggesting the need for further study with this agent.

Effect of glutathione depletion on the in vivo inhibition of drug metabolism by agents forming an inactive cytochrome P-450 Fe(II):metabolite complex. Studies with amiodarone and troleandomycin.

The relative contribution of competitive inhibition versus formation of a P-450:metabolite complex to the in vivo inhibition of drug metabolism for several agents is unclear. The present investigation examined the contribution of these two mechanisms to the in vivo inhibition of drug metabolism by amiodarone through manipulation of glutathione turnover. In vivo P-450-dependent metabolism in rats was assessed by determining antipyrine clearance. Pretreatment with amiodarone (50 mg/kg, iv) decreased antipyrine clearance with or without prior glutathione depletion. Depletion of glutathione by buthionine sulfoximine (1.6 g/kg, ip) did not enhance the magnitude of inhibition of antipyrine clearance by amiodarone. Moreover, administration of a normally subinhibitory dose of amiodarone after buthionine sulfoximine pretreatment did not influence antipyrine clearance. Similarly, depletion of glutathione via buthionine sulfoximine or diethylmaleate (1 mL/kg, po) did not influence the magnitude of inhibition caused by a single po dose of troleandomycin (500 or 350 mg/kg, respectively). These data indicate that glutathione content may not be a critical determinant for the in vivo inhibition of drug metabolism by agents which form a P-450:metabolite complex.