Comparing coordination uranyl(vi) complexes with 2-(1H-imidazo[4,5-b]phenazin-2-yl)phenol and derivatives.

Four derivatives of 2-(1H-imidazo[4,5-b]phenazin-2-yl)phenol have been synthesized and characterized structurally using X-ray crystallography. Coordination complexes with uranyl (UO22+) and copper (Cu2+) were prepared and absorption/emission spectra detailed. We observed increased fluorescence upon uranyl binding, in stark contrast to rapid quenching observed with the addition of copper. These phenomena have been further examined by DFT computational methods.

The effects of age and liver disease on the disposition and elimination of diazepam in adult man.

This study investigates the separate effects of age and hepatocellular liver disease on the disposition and elimination of diazepam (Valium) in man. The drug was given either by rapid intravenous injection (0.1 mg/kg) or orally (10 mg) to 33 normal volunteers rnaging in age from 15 to 82 yr as well as to 9 individuals with alcoholic cirrhosis, 8 with acute viral hepatitis, and 4 with chronic active hepatitis. In the normal individuals, the terminal plasma half-life of diazepam, (t 1/2 (B)) exhibited a striking age-dependence; at 20 yr the t 1/2 (beta) was about 20 h, but it increased linearly with age to about 90 h at 80 yr. The plasma clearance of diazepam in the majority of the normal subjects was between 20 and 32 ml/min and showed no significant age-dependence. Cigarette smoking did not affect the half-life or the clearance. Additionally, neither the plasma binding (97.4 plus or minus 1.2%, mean plus or minus SD) nor the blood/plasma concentration ratio (0.58 plus or minus 0.16) of diazepam showed any age-related changes (P greater than 0.05). By contrast, analysis of the intravenous data according to a two-compartment open model indicated that both the initial distribution space (V1) and the volume of distribution at steady state [Vd(ss)] of diazepam increased linearly with age (P less than 0.005). The increase in Vd(ss) was secondary to the change in V1. It appears then that the prolongation of t 1/2 (beta) of diazepam with age is primarily dependent on an increase in the initial distribution volume of the drug. The plasma concentration/time course of the metabolite, desmethyldiazepam, was also affected by age. In older individuals, the initial presence and the peak values of desmethyldiazepam were observed later and the metabolite was present in lower concentrations. Despite the profound prolongation of t 1/2 (theta) with age, the constancy of diazepam clearance indicates that drug plasma concentrations will not accumulate any more in the old than the young, and chronic dosage more in the old than the young, and chronic dosage modifications based on pharmacokinetic considerations are unnecessary. Data obtained in patients with liver disease were compared with those found in age-matched control groups. Patients with cirrhosis showed a more than twofold prolongation in the half-life of diazepam (105.6 plus or minus 15.2 vs. 46.6 plus or minus 14.2 h, P less than 0.001).

Increased clearance of antipyrine and d-propranolol after phenobarbital treatment in the monkey. Relative contributions of enzyme induction and increased hepatic blood flow.

The effects of phenobarbital treatment for 12 days on the regional distribution of blood flow and on the disposition of two model drugs, antipyrine and d-propranolol, have been determined in six unanesthetized rhesus monkeys. Phenobarbital significantly increased total hepatic blood flow from 179+/-15 to 239+/-27 ml/min. Liver weight was increased to a similar degree (34%) in phenobarbital-treated animals as compared to control monkeys. The clearance of both antipyrine and d-propranolol was increased and the half-life decreased significantly by phenobarbital. Analysis of the data by a perfusion-limited pharmacokinetic model showed that the changes in antipyrine clearance were due almost entirely to enzyme induction. On the other hand, with d-propranolol, the increase in liver blood flow contributed as much to the enhanced clearance as did the stimulation of drug metabolism. The mechanism by which phenobarbital produces the frequently observed increase in drug clearance, therefore, depends upon the initial clearance value of the drug. For low clearance drugs like antipyrine, clearance changes occur largely as a result of enzyme induction. With higher clearance drugs, the effects of increased hepatic blood flow become progressively more important the greater the initial clearance value.

Plasma binding and transport of diazepam across the blood-brain barrier. No evidence for in vivo enhanced dissociation.

The tissue uptake of extensively plasma-bound compounds is reportedly inconsistent with the conventional free-drug hypothesis limiting transport to unbound moiety in rapid intracapillary equilibrium with bound complex. Instead, protein-mediated/cell surface enhancement of dissociation has been postulated to occur in the microvasculature. This possibility was investigated by studying the passive transport of diazepam across the blood-brain barrier. Microdialysis probes placed within the vena cava and brain cortex were used to directly compare steady-state, interstitial unbound diazepam levels in both Wistar and genetically analbuminemic rats. The absence of albumin in the latter increased the unbound fraction of diazepam by almost fivefold; however, in both groups, the ratio of unbound concentrations in brain and blood at equilibrium was equal to unity. If enhanced dissociation occurred in the microvasculature, then the unbound brain level should have been greater than that in the systemic circulation. It is probable that earlier findings suggestive of protein-mediated transport reflect a nonequilibrium phenomenon. Comparison of the extent of diazepam's in vivo binding in blood by microdialysis to that estimated in vitro using conventional equilibrium dialysis with microcells showed good agreement, thus validating a widely accepted assumption of equivalency of these two values.

The drug transporter P-glycoprotein limits oral absorption and brain entry of HIV-1 protease inhibitors.

Currently available HIV-1 protease inhibitors are potent agents in the therapy of HIV-1 infection. However, limited oral absorption and variable tissue distribution, both of which are largely unexplained, complicate their use. We tested the hypothesis that P-glycoprotein is an important transporter for these agents. We studied the vectorial transport characteristics of indinavir, nelfinavir, and saquinavir in vitro using the model P-glycoprotein expressing cell lines L-MDR1 and Caco-2 cells, and in vivo after intravenous and oral administration of these agents to mice with a disrupted mdr1a gene. All three compounds were found to be transported by P-glycoprotein in vitro. After oral administration, plasma concentrations were elevated 2-5-fold in mdr1a (-/-) mice and with intravenous administration, brain concentrations were elevated 7-36-fold. These data demonstrate that P-glycoprotein limits the oral bioavailability and penetration of these agents into the brain. This raises the possibility that higher HIV-1 protease inhibitor concentrations may be obtained by targeted pharmacologic inhibition of P-glycoprotein transport activity.

Encainide and its metabolites. Comparative effects in man on ventricular arrhythmia and electrocardiographic intervals.

To assess the relative contributions of encainide and its putatively active metabolites, O-demethyl encainide (ODE) and 3 methoxy-O-demethyl encainide (3MODE), to the drug's pharmacologic effects, we compared intravenous infusions and sustained oral therapy in two phenotypically distinct groups of patients, extensive and poor metabolizers of encainide. Unlike poor metabolizers, extensive metabolizers had appreciable quantities of both metabolites detectable in plasma and had fourfold shorter elimination half-lives for encainide. By quantitating electrocardiogram intervals, arrhythmia frequency, and plasma concentrations, we found that, in poor metabolizers, arrhythmia suppression and ventricular complex (QRS) prolongation were correlated positively with encainide concentrations (r greater than or equal to 0.570, P less than 0.014). In these two subjects, antiarrhythmic concentrations of encainide (greater than 265 ng/ml) were at least fivefold higher than those sustained in the six extensive metabolizers during steady state oral therapy. In extensive metabolizers, encainide concentrations were uncorrelated with effects. Arrhythmia suppression and QRS prolongation in extensive metabolizers correlated best with ODE (r greater than or equal to 0.816, P less than 0.001); QTc change correlated positively with both 3MODE and ODE. Arrhythmia suppression paralleled QRS prolongation; the relationship between them appeared similar in both phenotypic groups. In most patients, extensive metabolizers, encainide effects during oral therapy are mediated by metabolites, probably ODE.

P-glycoprotein and cytochrome P-450 3A inhibition: dissociation of inhibitory potencies.

Many P-glycoprotein (P-gp) inhibitors studied in vitro and in vivo are also known or suspected to be substrates and/or inhibitors of cytochrome P-450 3A (CYP3A). Such overlap raises the question of whether CYP3A inhibition is an intrinsic characteristic of P-gp inhibitors, a matter of concern in the development and rational use of such agents. Thus, the purpose of the present study was to determine whether the ability to inhibit P-gp and CYP3A is, in fact, linked and whether specific P-gp inhibitors with limited ability to inhibit CYP3A can be identified. Therefore, the potency of a series of 14 P-gp inhibitors was assessed by measuring their inhibition of the transepithelial flux across Caco-2 cells of digoxin, a prototypical P-gp substrate. CYP3A inhibition was determined from the impairment of nifedipine oxidation by human liver microsomes. Determination of the apparent Ki values for CYP3A inhibition and the IC50s for P-gp and CYP3A inhibition allowed comparison of the relative inhibitory potency of the compounds on the two proteins' function. The IC50s for P-gp inhibition ranged from 0.04 to 3.8 microM. All compounds inhibited CYP3A with apparent Ki values of between 0.3 and 76 microM and IC50s between 1.5 and 50 microM. However, no correlation was found between the extent of P-gp inhibition and CYP3A inhibition, and the ratio of the IC50 for CYP3A inhibition to the IC50 for P-gp inhibition varied from 1.1 to 125. These results demonstrate that, although many P-gp inhibitors are potent inhibitors of CYP3A, a varying degree of selectivity is present. The development and use of P-gp inhibitors with minimal or absent CYP3A inhibitory effects should decrease the impact of drug interactions on the therapeutic use of such compounds.

Genetic predisposition to bladder cancer: ability to hydroxylate debrisoquine and mephenytoin as risk factors.

The hypothesis that the frequency distribution of indices of oxidative drug-metabolizing activity is different between patients with bladder cancer (n = 98) and age, sex-matched control subjects (n = 110) has been investigated. Urinary recovery ratios of debrisoquine and R/S ratios of mephenytoin have been measured in an 8-h urine sample after simultaneous administration of debrisoquine (10 mg) and racemic mephenytoin (100 mg). In addition, alcohol consumption, smoking habit, and acetylation phenotype (using 100 mg dapsone as a substrate) have been measured. Patients with bladder cancer were classified on histological criteria as having aggressive (Stage III) (34%) or nonaggressive (Stages I and II) (66%) disease. The median of the frequency distribution of the debrisoquine urinary recovery ratio in patients with aggressive bladder cancer was greater than in control subjects, and only four patients had recovery ratios lower than the mean of the control group. Using logistic regression analysis, efficient debrisoquine metabolism and a synergistic interaction between smoking and ethanol consumption were significant, independent risk factors, while S-mephenytoin hydroxylation and acetylation phenotype were not significant risk factors. In contrast, patients with non-aggressive bladder cancer had a significant, but weaker, association with rapid hydroxylation of S-mephenytoin, which was independent of a significant synergistic interaction between smoking and alcohol consumption. Acetylation phenotype and debrisoquine urinary recovery ratio were not associated with increased risk of nonaggressive cancer. These results are consistent with the concept that oxidative isozymes might be responsible for conversion of environmental agents to proximate bladder carcinogens in nonindustrial-related bladder cancer. They also suggest that different etiological factors are involved in the pathogenesis of aggressive and nonaggressive bladder cancer.

Pharmacokinetics of high-dose etoposide (VP-16-213) administered to cancer patients.

Plasma urine, and cerebrospinal fluid etoposide concentrations have been measured in 12 adult patients after administration of high-dose (400 to 800 mg/sq m) etoposide in order to determine the pharmacokinetics of this drug at these elevated dosages. Increasing the drug dosage produced proportionally higher peak plasma etoposide concentrations (27 to 114 micrograms/ml) and total areas under the concentration-time curve (9,200 to 48,000 micrograms/ml X min). The etoposide mean (+/- S.D.) terminal half-life of 8.05 +/- 4.3 hr and plasma clearance of 28.0 +/- 9.7 ml/min/sq m, however, were independent of the dosage given. The mean etoposide renal clearance in 5 patients was 10.0 +/- 4.3 ml/min/sq m, representing from 35 to 40% of the total clearance of this drug from plasma. Cerebrospinal fluid etoposide concentrations ranged from 0.1 to 1.4 micrograms/ml, as measured in 6 patients at 1 to 8 hr after high-dose etoposide therapy, and were 1.8 +/- 1.7% of the simultaneously measured plasma levels. Pleural fluid removed from one patient at 18 hr posttherapy contained etoposide at 1.8 micrograms/ml. Our data, combined with data published previously, indicate that the pharmacokinetics of high-dose etoposide is linear within the dosage range tested and similar to that seen with lower drug doses. They also suggest that etoposide penetrates poorly into the cerebrospinal fluid.

High affinity uptake by isolated rat hepatocytes of a linear pseudo-hexapeptide, ditekiren.

The hepatic elimination of many oligopeptides is both rapid and extensive, and often limits their potential as therapeutic agents. The linear, hydrophobic pseudo-hexapeptide ditekiren, a renin inhibitor, is one such example. The mechanism(s) involved in its hepatic clearance are largely unknown; accordingly, the characteristics of ditekiren's transport into isolated rat hepatocytes was investigated. In addition to a concentration-independent, linear process, uptake also involved a carrier-mediated component (Km = 0.2 +/- 0.05 microM; Vmax = 11.6 +/- 0.6 pmol (mg protein)[-1] min[-1]). Phenobarbital pretreatment in vivo resulted in marked induction of such transport. Negative results from cis-inhibition studies with substrates and/or inhibitors of well-established hepatic transport systems, e.g., sodium-dependent bile acid, sodium-independent multispecific bile acid and cation carriers, ruled out their involvement in ditekiren's uptake. By contrast, a number of cyclic and linear oligopeptides inhibited the uptake process to varying extents and in the case of EMD-59121, the most inhibitory compound, the interaction was competitive in nature. Collectively, these data suggest the presence of a novel high affinity, low capacity transporter in rat hepatocytes with specific affinity for ditekiren and possibly other oligopeptides.

Human MRP3 transporter: identification of the 5'-flanking region, genomic organization and alternative splice variants.

In humans, at least six members of the multidrug resistance-associated protein (MRP) family are thought to exist. Here we report the molecular cloning of two splice variants of MRP3 from human liver. In addition, MRP3 genomic organization including the 5'-flanking region and a major portion of the MRP3 intron-exon organization are identified and characterized.

Stereoselective disposition of hexobarbital and its metabolites: relationship to the S-mephenytoin polymorphism in Caucasian and Chinese subjects.

In vitro studies with human liver preparations suggest that the metabolism of hexobarbital involves CYP2CMP--the determinant of the S-mephenytoin 4'-hydroxylation polymorphism, but no in vivo evidence of interphenotypic differences exist. The pharmacokinetics and urinary excretion of hexobarbital and its metabolites were, therefore, investigated following oral administration of a differentially labelled pseudoracemate that allowed determination of the fate of the individual enantiomers. Studies were undertaken in 10 Caucasian and nine Chinese healthy subjects known to be either extensive (EM) or poor (PM), metabolizers of mephenytoin. No inter-racial differences were observed in any of the measured parameters within a given phenotype. However, pronounced stereoselectivity in disposition was noted in EMs with R-(-)-hexobarbital's oral clearance being five- to six-fold greater than that for the S-(+)-enantiomer. By contrast, the S-(+)-isomer was eliminated twice as fast as R-(-) hexobarbital in PMs and, in addition, the oral clearances of both enantiomers were significantly reduced compared with their values in EMs. Formation of 3'-hydroxy- and 3'-ketohexobarbital and 1,5-dimethylbarbituric acid were the major identified routes of metabolism for each enantiomer in both phenotypes. Furthermore, these pathways were found to co-segregate with the mephenytoin polymorphism and in EMs they were primarily responsible for the observed stereoselectivity in disposition. These findings, therefore, confirm the stereoselectivity in hexobarbital's disposition in humans and identify the major pathways of metabolism involved. Additionally, the results indicate that CYP2CMP is a major determinant of the in vivo metabolism of both of hexobarbital's enantiomers but especially that of the R-(-)-enantiomer.

The ability to 4-hydroxylate debrisoquine is related to recurrence of bladder cancer.

Oxidative metabolism by cytochrome P450 enzymes is often involved in the activation of environmental procarcinogens. Debrisoquine, mephenytoin, and dapsone were used as in vivo probes for the activities of P4502D6, 2CMP, and 3A4, respectively, as well as dapsone for N-acetyltransferase, in order to assess the relationship between such activities and the relative risk of recurrence of bladder cancer. Urinary recovery ratios of debrisoquine and dapsone and the R/S ratio of mephenytoin were measured in an 0-8 h urine sample after simultaneous administration of debrisoquine (10 mg) and racemic mephenytoin (100 mg), and the administration of dapsone (100 mg) one week later, to patients undergoing local surgical resection of transitional cell bladder cancer of G-I, G-II, or G-III histopathology. In addition, plasma levels of dapsone and mono-acetyldapsone were determined in an 8 h plasma sample to determine the N-acetylation phenotype. Patients were followed for 3 years, to the time of tumour recurrence, or death. Three patients were lost to follow-up; of the remaining 95 patients, 55 had tumour recurrence. The debrisoquine recovery ratio was significantly greater in patients with recurrence than in individuals who remained disease-free. Among the 65 patients with non-aggressive (G-I and G-II) histopathology, two patients were lost to follow-up and 32 had tumour recurrence. In this subgroup, the debrisoquine recovery ratio was again found to be significantly greater in those individuals with tumour recurrence (p < 0.003).(ABSTRACT TRUNCATED AT 250 WORDS)

Disposition of debrisoquine in Caucasians with different CYP2D6-genotypes including those with multiple genes.

Debrisoquine is a major prototypic in-vivo probe used to assess polymorphic CYP2D6 activity in humans, based on the 0-8 h urinary excretion of unchanged drug and its 4-hydroxy metabolite (the so-called metabolic ratio). The primary purpose of the study was to investigate further the relationship between genotype and phenotype by determining the overall disposition characteristics of the drug in selected groups of healthy Swedish Caucasian individuals. Debrisoquine (20 mg) was orally administered to five poor metabolizers with no functional CYP2D6 gene, five heterozygous extensive metabolizers, five homozygous extensive metabolizers, five ultrarapid metabolizers with duplicated/triplicated CYP2D6*2 genes and one individual with 13 copies of CYP2D6*2. Peak plasma levels of debrisoquine and 4-hydroxydebrisoquine were attained within 2-4 h and then declined in a multi-exponential fashion over 96 h. However, the post 8-h period of the elimination process was characterized by irregular fluctuations that prevented formal pharmacokinetic analysis. Nevertheless, marked differences were apparent in the compounds' plasma level-time profiles between the CYP2D6 genotypes. For example, in the case of debrisoquine, the mean ratio of the AUC(0-8) values was 22:22:7:6:1, corresponding to 0, 1, 2, 3/4 and 13 genes and, for 4-hydroxydebrisoquine, the respective values were 1:7:19:28:17. The 0-96 h urinary recovery of debrisoquine differed 100-fold between the genotypes, being essentially complete in poor metabolizers and zero in the individual with 13 CYP2D6*2 genes. 4-hydroxydebrisoquine excretion increased according to the number of functional CYP2D6 genes. A highly significant correlation (r(s) = 0.95, P < 0.001) was observed between the plasma AUC(0-8) ratio for debrisoquine to 4-hydroxydebrisoquine and the 0-8 h urinary metabolic ratio. This study demonstrates that the number of functional CYP2D6 alleles is critically important in the plasma concentration-time curves of debrisoquine and its CYP2D6-mediated 4-hydroxy metabolite. Concentration-related pharmacologic effects would be expected to be similarly affected by gene dosage and it is likely that the same situation also applies to other drugs whose elimination is importantly determined by this enzyme; for example, many antidepressants and neuroleptics, antiarrhythmic agents, beta-adrenoceptor antagonists and opiates.

Low activity of dapsone N-hydroxylation as a susceptibility risk factor in aggressive bladder cancer.

N-arylamines involved in the pathogenesis of bladder cancer, require metabolic activation via N-hydroxylation. The efficiency of in vivo N-hydroxylation of dapsone, a non-carcinogenic arylamine, may, therefore, provide a host susceptibility measure of risk of developing bladder cancer. To investigate this possibility, the dapsone recovery ratio, a phenotypic measure of the efficiency of dapsone hydroxylation, has been measured in a case control study in an urban UK population, comparing patients with aggressive bladder cancer (n = 33), non-aggressive bladder cancer (n = 60) and controls (n = 108). Dapsone recovery ratio in controls exhibited a unimodal distribution. Patients with aggressive bladder cancer had a similar distribution but significantly lower mean value (p < 0.005). Logistic regression analysis, controlling for sex, age, smoking habit and alcohol consumption confirmed a significant (p < 0.05) association between the dapsone recovery ratio and aggressive bladder cancer. Subjects in the lowest tertile of dapsone recovery ratio had a relative risk to 5.4-fold greater than subjects in the upper tertile (p < 0.009), and a trends test was significant (p < 0.001). There was no significant association between dapsone recovery ratio and non-aggressive bladder cancer. These results do not support the hypothesis that the drug metabolizing enzymes involved in dapsone N-hydroxylation are involved in causing bladder cancer. Instead, they suggest the opposite, the observation that low enzyme activity was associated with increased risk is consistent with this enzyme providing a detoxification mechanism for environmental procarcinogens.

An additional defective allele, CYP2C19*5, contributes to the S-mephenytoin poor metabolizer phenotype in Caucasians.

The metabolism of the anticonvulsant drug mephenytoin exhibits a genetic polymorphism in humans. This polymorphism exhibits marked racial heterogeneity, with the poor metabolizer PM phenotype representing 13-23% of oriental populations, but only 2-5% of Caucasian populations. Two defective CYP2C19 alleles (CYP2C19*2 and CYP2C19*3) have been described, which account for more than 99% of Oriental poor metabolizer alleles but only approximately 87% of Caucasian poor metabolizer alleles. Therefore, additional defects presumably contribute to the poor metabolizer in Caucasians. Recent studies have found a third mutation CYP2C19*4, which accounts for approximately 3% of Caucasian poor metabolizer alleles. A fourth rare mutation (CYP2C19*5A) (C99,A991,Ile331;C1297T,Arg433-->Trp) resulting in an Arg433 to Trp substitution in the heme-binding region has been reported in a single Chinese poor metaboliser outlier belonging to the Bai ethnic group. The present study identifies a second variant allele CYP2C19*5B (C99-->T; A991-->G, Ile331-->Val; C1297-T, Arg433-->Trp in one of 37 Caucasian poor metabolizers. The frequency of the CYP2C19*5 alleles is low in Chinese (approximately 0.25% in the Bai ethnic group) and Caucasians (< 0.9%). However, these alleles contribute to the poor metabolizer phenotype in both ethnic groups and increases the sensitivity of the genetic tests for identifying defective alleles to approximately 100% in Chinese poor metabolizers and 92% in Caucasian poor metabolizers genotyped in our laboratory. The Arg433 to Trp mutation in the heme-binding region essentially abolishes activity of recombinant CYP2C19*5A toward S-mephenytoin and tolbutamide, which is consistent with the conclusion that CYP2C19*5 represents poor metabolizer alleles.

Allelic, genotypic and phenotypic distributions of S-mephenytoin 4'-hydroxylase (CYP2C19) in healthy Caucasian populations of European descent throughout the world.

Impaired S-mephenytoin 4'-hydroxylation is a well-described genetic polymorphism affecting drug metabolism in humans. The reported population prevalence of the CYP2C19 poor metabolizer phenotype in Caucasians of European descent has been described as ranging from 0.9% to 7.7%. To address the question of whether the difference in the frequency of poor metabolizers represents an ethnic genetic microheterogeneity in the structure and expression of the CYP2C19 gene in Caucasian individuals, we performed a pooled analysis of available studies. Combined data from the 22 homogeneous studies showed that the frequency of poor metabolizers in healthy unrelated Caucasians determined by phenotyping was 2.8% (110 of 3990; 95% confidence interval 2.3-3.3). Data obtained from eight homogeneous studies that determined the frequency of poor metabolizers by genotyping showed that the genotypic frequency of poor metabolizers was 2.1% (28 of 1356; 95% confidence interval 1.3-2.8), consistent with the poor metabolizer frequency determined by phenotyping. In the extensive metabolizers, 26% (471 of 1786; 95% confidence interval 24.4-28.4) were heterozygotes. The observed frequencies of the three Mendelian genotypes were 73% for wt/wt, 26% for wt/m, and 2.1% for m/m. Based on the overall phenotypic poor metabolizer frequency of 2.8%, the expected genotypic frequencies were 69% for wt/wt, 28% for wt/m and 2.8% for m/m, which are in good agreement to the observed values. However, in the 84 Caucasian phenotyped and genotyped poor metabolizers, approximately 10% of the putative poor metabolizer alleles (17 of 168) were unknown. This study provides a systematic overview of the population distribution of the CYP2C19 poor metabolizer phenotype and CYP2C19 alleles and genotypes in healthy Caucasians living in different geographical areas, and shows a similar polymorphic pattern in the structure and expression of the CYP2C19 gene in the worldwide Caucasian populations.

The procarcinogen hypothesis for bladder cancer: activities of individual drug metabolizing enzymes as risk factors.

Bladder cancer provides the most definitive example for an association between environmental agents and cancer. However, in the absence of industrial occupational exposure, the primary carcinogen is rarely identified, and the mechanisms involved in cancer formation are poorly understood. The environmental procarcinogen hypothesis of tumour pathogenesis proposes that many carcinogens require metabolic activation by drug metabolizing enzymes to form the proximate carcinogen. A balance of exposure to the carcinogen, the activity of the enzymes involved in either formation of proximate carcinogen, or production of non-toxic metabolites, will determine tumour risk. We have used mephenytoin, debrisoquine and dapsone as selective probes for the phenotypic measures of activity of CYP2C19, CYP2D6, and CYP3A4, respectively. Within subject reproducibility of phenotypic measures, and the lack of cross-inhibition when the three drugs are given in a concurrent cocktail, have been confirmed. We have applied the cocktail drug approach in two, non-overlapping series of cases with bladder cancer and matched controls. In both series, patients with aggressive bladder cancer (GIII histopathology) had a history of excess alcohol intake, an under-representation of poor metabolizers of debrisoquine, a significant mean reduction in dapsone recovery ratio, but no difference in mephenytoin phenotype. Collectively, these observations involving multiple routes of drug metabolism support the procarcinogen environmental hypothesis for bladder cancer and suggest that measurement of activity of selected individual drug metabolizing enzymes involved in the pathogenesis of this tumour can be used to identify subjects at high risk of developing bladder cancer.

In-vivo phenotyping for CYP3A by a single-point determination of midazolam plasma concentration.

We investigated whether a single plasma midazolam concentration could serve as an accurate predictor of total midazolam clearance, an established in-vivo probe measure of cytochrome P450 3A (CYP3A) activity. In a retrospective analysis of data from 224 healthy volunteers, non-compartmental pharmacokinetic parameters were estimated from plasma concentration-time curves following intravenous (IV) and/or oral administration. Based on statistical moment theory, the concentration at the mean residence time (MRT) should be the best predictor of the total area under the curve (AUC). Following IV or oral midazolam administration, the average MRT was found to be approximately 3.5 h, suggesting that the optimal single sampling time to predict AUC was between 3 and 4 h. Since a 4-h data point was common to all studies incorporated into this analysis, we selected this time point for further investigation. The concentrations of midazolam measured 4 h after an IV or oral dose explained 80 and 91% of the constitutive interindividual variability in midazolam AUC, respectively. The 4-h midazolam measurement was also an excellent predictor of drug-drug interactions involving CYP3A induction and inhibition. Compared with baseline values, the direction and magnitude of change in midazolam AUC and the 4-h concentration were completely concordant for all study subjects. We conclude that a single 4-h midazolam concentration following IV or oral administration represents an accurate marker of CYP3A phenotype under constitutive and modified states. Moreover, the single-point approach offers an efficient means to phenotype and identify individuals with important genetic polymorphisms that affect CYP3A activity.

Commentary: a physiological approach to hepatic drug clearance.

A physiological approach has been developed recognizing that hepatic blood flow, the activity of the overall elimination process (intrinsic clearance), drug binding in the blood, and the anatomical arrangement of the hepatic circulation are the major biological determinants of hepatic drug clearance. This approach permits quantitative prediction of both the unbound and total drug concentration/time relationships in the blood after intravenous and oral administration, and any changes that may occur as a result of alterations in the above biological parameters. These considerations have led to a classification of drug metabolism based on the hepatic extraction ratio. The proposed classification allows prediction and interpretation of the effects of individual variations in drug-metabolizing activity, route of administration, pharmacokinetic interactions, and disease states on hepatic drug elimination.