Intrinsic Metabolic Clearance Research Articles

Prediction of hepatic clearance and availability by cryopreserved human hepatocytes: an application of serum incubation method.

A novel and convenient method was established for the prediction of in vivo metabolic clearance in human liver. The present method applied the in vitro-in vivo extrapolation paradigm previously established in rats to the in vitro data obtained from cryopreserved human hepatocytes. Predicted hepatic availability and clearance were compared with the reported oral bioavailability and plasma clearance in humans for 14 clinically used drugs (naloxone, buspirone, verapamil, lidocaine, imipramine, metoprolol, timolol, antipyrine, diazepam, quinidine, caffeine, propranolol, diclofenac, and phenacetin). A large interindividual variation was observed in the intrinsic metabolic clearance among separate cryopreserved preparations from different subjects. The prediction generally resulted in a marked underestimation when the biologically based scaling factor (3.1 x 10(9) cells/kg) was used for the extrapolation of in vitro data (milliliters per minutes per cells) to in vivo value (milliliters per minutes per kilograms). Reasonably good in vitro-in vivo correlations were obtained with empirically calculated scaling factors, 8.5 x 10(9) (cells/kg) from 10 individual preparations and 10.8 x 10(9) (cells/kg) from pooled preparation of two selected lots, which were 3- to 4-fold larger than the biologically based scaling factor. These data suggested that the calibration of inherent interindividual variation of metabolic activities among different cryopreserved preparations of human hepatocytes to obtain the empirical scaling factor, which is applicable only to the preparation used, was an essential step for more reliable and quantitative prediction of in vivo metabolic activity in humans.

Species Difference in Nisoldipine Oxidation Activity in the Small Intestine

Species difference in nisoldipine oxidation activities was investigated using small intestinal microsomes of rats, guinea pigs, dogs, monkeys and humans. The oxidation activities were estimated by measuring metabolites formation (BAY o 3199 and BAY r 9425) of nisoldipine. For the preparation of small intestinal microsomes of various animal species, the effect of protease inhibitors was preliminarily investigated. The formation of BAY o 3199 significantly increased in the rat small intestinal microsomes prepared with trypsin inhibitor. Using the trypsin inhibitor-treated small intestinal microsomes of various animals, metabolic intrinsic clearances (CL(int, in vitro)) for BAY o 3199 and BAY r 9425 formations were estimated based on an Eadie-Hofstee plot. The total CL(int,in vitro) estimated by the sum of CL(int, in vitro) for both formations in the small intestines of all species was much lower than that in the liver. There was a marked species difference in the nisoldipine oxidation activities in the small intestines, with the rank order being humans=monkeys>dogs>rats>guinea pigs, versus the following order in the liver: rats>monkeys=guinea pigs>humans>dogs. The formations of both BAY o 3199 and BAY r 9425 in the human intestinal microsomes were inhibited by pretreatment with troleandomycin (TAO) and antiserum against CYP3A4. Similar inhibition profile by TAO was obtained from the monkey intestinal microsomes. These results implied that monkeys would be a good predictor of human small intestinal metabolism for CYP3A4 substrates.

P450 interaction with farnesyl-protein transferase inhibitors metabolic stability, inhibitory potency, and P450 binding spectra in human liver microsomes

Methyl substitution at the 2-position of the imidazole ring greatly improved drug metabolism profiles, in human liver microsomes, of ras farnesyl-protein transferase inhibitor (FTI) candidates for drug development. Methyl substitution markedly reduced the P450 inhibitory potency of non-substituted FTIs for CYP3A4 (by a factor of 12-403) and 2C9 (by a factor of 4.2-28), while it had little effect on the CYP2D6 enzyme. An immunochemical inhibition study demonstrated that CYP3A4 plays a predominant role in the metabolism of both non-substituted and 2-methyl-substituted imidazole-containing FTI candidates. Very strong type II binding spectra with human liver microsomes were observed for all non-substituted FTIs, while methyl substitution markedly weakened type II spectra or shifted the type of spectra from II to I. This indicated that methyl substitution on the imidazole moiety interfered with the substrate-P450 heme interaction, likely due to a steric effect caused by the methyl group. A kinetics study revealed that the methyl substitution increased Vmax and Km values to the same extent. These studies suggested that the 2-methyl substitution on the imidazole ring improved its drug metabolism profile by reducing the potential to inhibit CYP3A4-mediated metabolism without affecting intrinsic metabolic clearance (Vmax/Km).

Stereospecific Toxicokinetics of Bromochloro- and Chlorofluoroacetate: Effect of GST-ζ Depletion

The chloro- and bromohaloacetates are drinking water disinfection by-products and rodent carcinogens. Chloro—bromo dihaloacetates are also mechanism-based inhibitors of glutathione S-transferase-ζ (GSTZ1–1). We studied the stereospecific toxicokinetics and in vitro metabolism of two chiral dihaloacetates in male F344 rats: (−),(+)-bromochloroacetate (BCA) and racemic chlorofluoroacetate (CFA), a non-GST-ζ-inhibiting dihaloacetate. These experiments were repeated in animals that had previously been treated with dichloroacetate (DCA) to deplete GST-ζ activity. Results indicated that the elimination half-life of (−)-BCA was 0.07 compared to 0.40 h for (+)-BCA in naive rats. A comparable difference in elimination half-life was also observed for the CFA stereoisomers (0.79 vs 0.11 h). In GST-ζ-depleted rats, stereospecific elimination of (−),(+)-BCA was absent, with both stereoisomers having an elimination half-life of approximately 0.4 h. This finding was in contrast to results for CFA, which still maintained the same relative difference in elimination rate between its stereoisomers, although overall elimination was diminished in GST-ζ-depleted rats. Results of in vitro metabolism experiments indicated (−)-BCA was affected by modulating GST-ζ activity, with the intrinsic metabolic clearance decreasing from 2.81 to 0.15 ml h−1 mg·protein−1 (naive, GST-ζ depleted) compared with values for (+)-BCA (0.30 and 0.31 ml h−1 mg·protein−1). Incubations with 350 μM diethyldithiocarbamate preferentially decreased (+)-BCA metabolism in naive and GST-ζ-depleted cytosol. These results indicate (+)-BCA is a poor substrate for GST-ζ and its metabolism is controlled by an additional GST isoenzyme.

Optimization of metabolic stability as a goal of modern drug design.

Metabolism and other pharmacokinetic (PK) studies have always played a critical role in helping to optimize the bioavailability and duration of action of new drugs thereby increasing their success rate. With the advent of automated combinatorial synthesis, high-throughput pharmacological testing, and the ability to create extensive databases in the past decade, drug discovery has undergone an amazing evolution. With the increased throughput of drug discovery, metabolism and other PK studies have evolved to keep pace. Often called "early ADME" studies, these studies are characterized by parallel processing and higher throughput than before. This article focuses on a particular class of early ADME (absorption, distribution mechanism, and excretion) studies known as "metabolic stability" studies. The theoretical basis for metabolic stability and its relationship to the concept of metabolic intrinsic clearance is briefly presented. Some key relationships between structure and metabolism are summarized. Several case studies from recent medicinal chemistry literature are reviewed to exemplify how metabolic stability studies influenced drug design and led to improvements in bioavailability and half-life. Finally, future trends in drug metabolism and analytical chemistry and how they may influence metabolic stability studies are reviewed.

Early ADME in support of drug discovery: the role of metabolic stability studies.

The area of Drug Discovery has undergone an amazing evolution in the past decade. This evolution is typified by the development of automated combinatorial synthesis and high throughput pharmacological testing. This, in turn, has lead to the ability to create and mine extensive databases and then model this new information. The overall result is a substantial increase in the rate of target identification, generation of new leads, and finally, optimization of those leads into clinical candidates. ADME studies have always played a critical role in helping to optimize the pharmacokinetic (PK) properties of new drugs thereby increasing their success rate. As a consequence of the increased throughput of drug discovery, ADME studies have evolved to keep pace. These so-called "early ADME" studies, are characterized by parallel processing and higher throughput than before. A primary concern of medicinal chemists is to design molecules that will have not only the desired activity, but also suitable potency and duration of action, which is influenced by pharmacokinetic properties such as bioavailability and half-life. This article focuses on a particular subset of eADME studies known as "metabolic stability", which can be an important contributor for a good pharmacokinetic profile. Metabolic stability studies represent the adaptation of more complex metabolism rate studies to a minimized system suitable for parallel processing of large numbers of compounds. The theoretical basis for metabolic stability lies in its relationship to the concept of metabolic intrinsic clearance. Typical metabolic stability protocols are discussed with respect to their relation to drug design. How metabolic stability studies have evolved to keep pace with advances in drug discovery is also discussed. Several case studies of the role of metabolic stability in drug design over the past few years are summarized to exemplify the utility of this kind of study. Finally, future trends in drug metabolism and analytical chemistry and how they may influence metabolic stability studies are reviewed.

Quantitative prediction of in vivo drug-drug interactions from in vitro data based on physiological pharmacokinetics: use of maximum unbound concentration of inhibitor at the inlet to the liver.

To assess the degree to which the maximum unbound concentration of inhibitor at the inlet to the liver (I(inlet,u,max), used in the prediction of drug-drug interactions, overestimates the unbound concentration in the liver. The estimated value of I(inlet,u,max) was compared with the unbound concentrations in systemic blood, liver, and inlet to the liver, obtained in a simulation study based on a physiological flow model. As an example, a tolbutamide/sulfaphenazole interaction was predicted taking the plasma concentration profile of the inhibitor into consideration. The value of I(inlet,u,max) differed from the concentration in each compartment, depending on the intrinsic metabolic clearance in the liver, first-order absorption rate constant, non-hepatic clearance and liver-to-blood concentration ratio (Kp) of the inhibitor. The AUC of tolbutamide was predicted to increase 4-fold when co-administered with sulfaphenazole, which agreed well with in vivo observations and was comparable with the predictions based on a fixed value of I(inlet,u,max). The blood concentration of tolbutamide was predicted to increase when it was co-administered with as little as 1/100 of the clinical dose of sulfaphenazole. Although I(inlet,u,max) overestimated the unbound concentration in the liver, the tolbutamide/sulfaphenazole interaction could be successfully predicted by using a fixed value of I(inlet,u,max) indicating that the unbound concentration of sulfaphenazole in the liver after its clinical dose is by far larger than the concentration to inhibit CYP2C9-mediated metabolism and that care should be taken when it is co-administered with drugs that are substrates of CYP2C9.

Pharmacokinetics of prilocaine after intravenous administration in volunteers: enantioselectivity.

Prilocaine exists in two stereoisomeric configurations, the enantiomers S(+)- and R(-)-prilocaine. The drug is clinically used as the racemate. This study examined the pharmacokinetics of the enantiomers after intravenous administration of the racemate. Ten healthy male volunteers received 200 mg racemic prilocaine as a 10-min intravenous infusion. Blood samples were collected for 8 h after the start of the infusion. Plasma concentrations were measured by stereoselective high-performance liquid chromatography (HPLC). Unbound fractions of the enantiomers in blank blood samples, spiked with racemic prilocaine, were determined using equilibrium dialysis. The unbound fraction of R(-)-prilocaine (mean +/- SD, 70%+/-8%) was smaller (P < 0.05) than that of S(+)-prilocaine (73%+/-5%). The total plasma clearance of R(-)-prilocaine (2.57+/-0.46 l/min) was larger (P < 0.0001) than that of S(+)-prilocaine (1.91+/-0.30 l/min). The steady-state volume of distribution of R(-)-prilocaine (279+/-94 l) did not differ from that of S(+)-prilocaine (291+/-93 l). The terminal half-life of R(-)-prilocaine (87+/-27 min) was shorter (P < 0.05) than that of S(+)-prilocaine (124+/-64 min), as was the mean residence time of R(-)-prilocaine (108+/-30 min) compared with S(+)-prilocaine (155+/-59 min; P < 0.005). The pharmacokinetics of prilocaine are enantioselective. The difference in clearance is most likely a result of a difference in intrinsic metabolic clearance. The difference in the pharmacokinetics of the enantiomers of prilocaine does not seem to be clinically relevant.

Determination of the rate-limiting step in the hepatic elimination of YM796 by isolated rat hepatocytes.

The membrane permeability clearance and intrinsic metabolic clearance of a drug in the liver were estimated using isolated rat hepatocytes, and the rate-limiting step in the overall intrinsic clearance of the drug in vivo was investigated. For this purpose, an anti-dementia drug, (S)-(-)-2,8-dimethyl-3-methylene-1-oxa-8-azaspiro [4,5] decane-L-tartarate monohydrate (YM796) was used as a model drug. The parent drug and its metabolites in both medium and cells were separated by thin-layer chromatography (TLC). The total amount of drug taken up by hepatocytes and the total amount of metabolites were plotted against the AUC of YM796 in the medium or cells to obtain the kinetic parameters. While the influx clearance (PS(inf)) through the sinusoidal membrane defined in terms of YM796 concentration in the medium was almost constant, irrespective of the concentration of YM796 in the medium, the intrinsic metabolic clearance (CL(int)) and the efflux clearance (PS(eff)) defined in terms of the total concentration of YM796 in the cells markedly decreased and increased, respectively, as the concentration of YM796 increased. The overall intrinsic metabolic clearance (CL(int,all), defined in terms of the YM796 concentration in the medium, corresponding to the hepatic intrinsic clearance obtained from the in vivo pharmacokinetic data on the drug, was comparable with PS(inf) at low concentrations of YM796. As the YM796 concentration increased, however, CL(int,all) fell markedly approaching CL(int). While, at low concentrations of YM796, CL(int,all) was predominantly affected by membrane permeability clearance, at high concentrations it was completely rate-determined by the intrinsic metabolic clearance because of the marked reduction in CL(int) resulting from the saturation of YM796 metabolism.

PREDICTION OF THE &lt;I&gt;IN VIVO&lt;/I&gt; DRUG METABOLIZING ACTIVITY IN HUMAN LIVER FROM &lt;I&gt;IN VITRO&lt;/I&gt; DATA ON METABOLISM : THE USE OF HUMAN LIVER MICROSOMES AND RECOMBINANT HUMAN CYP ISOZYMES

We have successfully predicted in vivo drug metabolic clearances in human liver from in vitro data obtained using human liver samples. The magnitude of differences between in vivo intrinsic hepatic clearances (CLint, in vivo) and in vitro intrinsic metabolic clearances (CLint, in vitro) calculated from the in vitro metabolic data with human liver microsomes was less than 3-fold for about 60 % of the 50 metabolic reactions catalyzed by cytochrome P450 (CYP). In particular, the predictability was good for those metabolic pathways mediated mainly by CYP3A4. Furthermore, as far as all the 9 metabolic pathways we studied were concerned, the values of CLint, in vitro obtained using recombinant human CYP isozymes were in good agreement with those obtained using human liver microsomes, irrespective of the CYP isozymes involved. This finding suggests that recombinant human CYP isozymes are a good substitute for human liver microsomes as far as predicting in vivo metabolic clearance is concerned.

Metabolism of an anionic fluorescent dye, 1-anilino-8-naphthalene sulfonate (ANS) by rat liver microsomes.

The present study was designed to examine the metabolism of 1-anilino-8-naphthalene sulfonate (ANS), an anionic compound which is transported into liver via "multispecific organic anion transporter", with rat hepatic microsomes. TLC analysis indicated that the fluorescent metabolites were not produced to a measurable extent, which made it possible to assess the ANS metabolism by measuring the fluorescence disappearance. The metabolism of ANS was remarkably inhibited by the presence of SKF-525A as well as by the substitution of O2 by CO gas. ANS metabolism by microsomes also required NADPH as a cofactor. These results indicated that the microsomal monooxygenase system might be mainly responsible for the ANS metabolism. The maximum velocity (Vmax) and Michaelis constant (K(m)) were calculated to be 4.3 +/- 0.2 nmol/min/mg protein and 42.1 +/- 2.0 microM, respectively. Assuming that 1 g of liver contains 32 mg of microsomal protein, the Vmax value was extrapolated to that per g of liver (Vmax'). The intrinsic metabolic clearance (CLint) under linear conditions calculated from this in vitro metabolic study was 3.3 ml/min/g liver, being comparable with that (3.0 ml/min/g liver) calculated by analyzing the in vivo plasma disappearance curve in a previous study. Furthermore, the effects of other organic anions on the metabolism of ANS were examined. Bromophenolblue (BPB) and rose bengal (RB) competitively inhibited the metabolism of ANS, while BSP inhibited it only slightly. The inhibition constant (Ki) of BPB (6 microM) was much smaller than that of RB (200 microM). In conclusion, the microsomal monooxygenase system plays a major role in the metabolism of ANS, and other unmetabolizable organic anions (BPB and RB) compete for this metabolism.

Assessment of the effect of malaria infection on hepatic clearance of dihydroartemisinin using rat liver perfusions and microsomes.

1. The clearance of dihydroartemisinin (DHA) in control and malaria-infected (MI) rats was investigated using the isolated perfused rat liver (IPRL) model and hepatic microsomal studies. 2. In the recirculating IPRL, clearance of DHA was reduced from a mean (s.d.) of 8.2+/-1.8 ml min(-1) in controls (n=8) to 6.0+/-1.0 ml min(-1) in MI (n=8; P<0.01). Clearance in control livers was similar to the perfusion flow rate, suggesting a high hepatic extraction ratio for DHA. 3. Single-pass IPRL studies in controls (n=8) showed that DHA bioavailability at 1.3, 8 and 38 microm was 0.026+/-0.020, 0.043+/-0.025 and 0.14+/-0.06, respectively (P<0.001 for 8 microM vs 38 microM). In MI livers (n=5), DHA bioavailability at 8 and 38 microM was 0.18+/-0.07 and 0.40+/-0.08, respectively (P=0.002). Bioavailability was higher in the MI group than in controls (P=0.01 at 8 microM and P<0.001 at 38 microM). DHA-glucuronide was the sole biliary metabolite. 4. Hepatic microsomal studies of DHA-glucuronide formation showed a significantly lower Vmax but no significant change in Km, in MI compared to control livers (n=6). Intrinsic metabolic clearance (Vmax/Km) was higher in control than in MI livers (5.2+/-1.3 and 2.5+/-1.4 microl min(-1) mg(-1), respectively; P=0.006). 5. These studies demonstrate that DHA has a high, concentration-dependent hepatic extraction ratio that is reduced by 20-30% in the P. berghei rodent malaria model. The impaired hepatic clearance of DHA in MI is attributable to a reduction in intrinsic metabolic clearance.

Pharmacokinetics and Metabolism of Bisoprolol Enantiomers in Humans

□ The plasma concentrations and urinary excretions of bisoprolol enantiomers in four Japanese male healthy volunteers after a single oral administration of 20 mg of racemic bisoprolol were evaluated. The AUCǴ∞ and elimination half-life of (S)-(–)-bisoprolol were slightly larger than those of (R)-(+)-bisoprolol in all subjects. The metabolic clearance of (R)-(+)-bisoprolol was significantly (P<0.05) larger than that of (S)-(–)-bisoprolol (S/R ratio: 0.79±0.03), although the difference was small. In contrast, no stereoselective in vitro protein binding of bisoprolol in human plasma was found. An in vitro metabolic study using recombinant human cytochrome P450 (CYP) isoforms indicated that oxidation of both bisoprolol enantiomers was catalyzed by the two isoforms, CYP2D6 and CYP3A4. CYP2D6 metabolized bisoprolol stereoselectively (R>S), whereas the metabolism of bisoprolol by CYP3A4 was not stereoselective. The S/R ratio of the mean clearance due to renal tubular secretion was 0.68, indicating a moderate degree of stereoselective renal tubular secretion. These findings taken together suggest that the small differences in the pharmacokinetics between (S)-(–)- and (R)-(+)-bisoprolol are mainly due to the stereoselectivity in the intrinsic metabolic clearance by CYP2D6 and renal tubular secretion.

Venlafaxine oxidation in vitro is catalysed by CYP2D6.

1. Several selective 5-HT reuptake inhibitors (SSRIs) are inhibitors of the genetically polymorphic drug metabolizing enzyme, CYP2D6. We studied the interaction of venlafaxine, a new SSRI, with CYP2D6 in human liver microsomes. 2. Venlafaxine was a less potent inhibitor of this enzyme activity in vitro than other SSRIs tested. The average apparent Ki values determined using CYP2D6-dependent dextromethorphan O-demethylation were: 33, 52 and 22 microM for rac-venlafaxine, R(+)-venlafaxine and S(-)-venlafaxine, respectively, vs 0.065 to 1.8 microM for paroxetine, fluoxetine, norfluoxetine, fluvoxamine and sertraline. 3. Microsomes from human livers (n = 3) and from yeast transformed with an expression plasmid containing human CYP2D6 cDNA catalyzed the O-demethylation of venlafaxine, which is the major metabolic pathway in vivo. Intrinsic metabolic clearance values (Vmax/Km) indicated that S(-)-venlafaxine was cleared preferentially via this pathway. 4. In microsomes from CYP2D6-deficient livers (n = 2), Vmax/Km of O-demethylation of venlafaxine was one to two orders of magnitude lower and was similar to the rate of N-demethylation. 5. Studies with chemical probes which preferentially inhibit P450 isoforms suggested that CYP3A3/4 is involved in venlafaxine N-demethylation. 6. These in vitro findings predict phenotypic differences in the kinetics of venlafaxine in vivo, although the clinical importance of this is unclear as O-demethylvenlafaxine is pharmacologically similar to the parent drug. The findings also predict relatively limited pharmacokinetic interaction between venlafaxine and other CYP2D6 substrates.

Xenobiotic acyl-CoA formation: evidence of kinetically distinct hepatic microsomal long-chain fatty acid and nafenopin-CoA ligases

Multiplicity of hepatic microsomal coenzyme A ligases catalyzing acyl-CoA thioester formation is an important factor for consideration in relation to the metabolism of xenobiotic carboxylic acids. In this study the kinetic characteristics of rat hepatic microsomal nafenopin-CoA ligase were studied and compared with those of long-chain fatty acid (palmitoyl) CoA ligase. The high affinity component of palmitoyl-CoA formation was inhibited by nafenopin ( K i 53 μM) and ciprofibrate ( K i 1000 μM). Analagous to palmitoyl-CoA, nafenopin-CoA formation was catalyzed by an apparent high affinity low capacity isoform ( K m 6 ± 2.5 μM, (V max 0.33 ± 0.12 nmol/mg per min) which was inhibited competitively by palmitic acid (mean K i 1.7 μM, n = 5) and R-ibuprofen (mean K i 10.8 μM, n = 5) whilst ciprofibrate and clofibric acid were ineffective as inhibitors. The intrinsic metabolic clearance of nafenopin to nafenopin-CoA (V max/ K m 0.057 ± 0.011 nmol/mg/min ± M) was similar to that reported recently for the formation of ibuprofenyl-CoA by rat liver microsomes. Evidence of both a substantial difference between the K m and K i for nafenopin and lack of commonality with regard to xenobiotic inhibitors suggests that the high affinity microsomal nafenopin-CoA and long-chain fatty acid-CoA ligases are kinetically distinct. Thus until the current ‘long-chain like’ xenobiotic-CoA ligases are fully characterised in terms of substrate specificity, inhibitor profile, etc, it will be impossible to rationalize (and possibly predict) the metabolism and hence toxicity of xenobiotic carboxylic acids forming acyl-CoA thioester intermediates.

Evaluation of the effect of fluoxetine on the formation of carbamazepine epoxide.

Fluoxetine has been reported to increase carbamazepine (CBZ) plasma concentrations and cause adverse effects. CBZ-10, 11 epoxide (CBZE), the major metabolite of CBZ, contributes to the clinical effect and toxicity of CBZ. The objective of the present study was to investigate the effect of fluoxetine and its major metabolite, norfluoxetine, on CBZE formation in isolated perfused rat liver, in vitro human liver (n = 5) microsomes, and patients (n = 14), after either CBZ monotherapy or polytherapy with fluoxetine. In isolated perfused rat liver, there was no effect of fluoxetine (n = 8) or norfluoxetine (n = 6) on the formation clearance of CBZE (12.8 +/- 5.3 and 11.7 +/- 3.8 ml/min, respectively) or the intrinsic metabolic clearance of CBZ (6.6 +/- 2.7 and 6.3 +/- 1.8 ml/min, respectively). Studies on human liver microsomes confirmed that neither fluoxetine or norfluoxetine inhibited formation of CBZE until concentrations were > 20 times those found clinically. In support of this, there was no difference in the ratio of CBZE to CBZ plasma concentrations in patients also receiving fluoxetine when compared to patients on CBZ monotherapy; however, there was a trend toward a decrease in the apparent plasma clearance of CBZ. In conclusion, increased plasma concentrations of CBZ found when fluoxetine is added are not due to decreased formation of CBZE. Clinically, if fluoxetine causes an increase in CBZ levels, CBZE plasma concentrations will increase proportionately and contribute to the toxicity.

PLASMA CONCENTRATIONS OF THE STEREOISOMERS OF PRILOCAINE AFTER ADMINISTRATION OF THE RACEMATE: IMPLICATIONS FOR TOXICITY?

A chiral high pressure liquid chromatography method was developed to measure the separate isomers of prilocaine in plasma after administration of the racemate. The concentrations of the isomers in six patients were similar (S(+)/R(-) = 1.06 (SD 0.06)) after brachial plexus block with 1.5% (RS)-prilocaine hydrochloride 35 ml, suggesting that a higher systemic safety margin may not be achieved by substituting racemic prilocaine by one of its isomers. Much higher plasma concentrations of the S(+)- than the R(-)-form after oral administration of 300 mg of the racemate (n = 4) indicated a large difference in intrinsic metabolic clearance of the isomers on first pass through gut, liver or both organs.

The relationship between the pharmacokinetics of ibuprofen enantiomers and the dose of racemic ibuprofen in humans

Ibuprofen is a chiral drug which is used clinically as a racemate. The pharmacological properties of ibuprofen reside almost exclusively with the S(+)-enantiomer. However, a portion of R(-)-ibuprofen is metabolically inverted to its pharmacologically active, mirror-image form. To investigate the influence of increasing dose of racemic ibuprofen on the pharmacokinetics of its individual enantiomers, four healthy male volunteers were given racemic ibuprofen (200, 400, 800, and 1200 mg), orally, on four occasions. The study was conducted using a balanced cross-over design. The extent of absorption of ibuprofen, as assessed by the total urinary recovery of ibuprofen and its metabolites, was extensive and independent of the administered dose. At all four doses, the area under the total and unbound plasma concentration-time curves (AUC and AUCu, respectively), and the unbound fraction in plasma, were significantly greater for the S(+)-enantiomer. With increasing ibuprofen dose, there was a less than proportional increase in the AUC of each enantiomer, while the AUCu for both enantiomers increased in direct proportion to the administered dose. The time-averaged unbound fraction of each enantiomer increased significantly with increasing dose, which caused the non-linearity between AUC and dose. It was predicted that the metabolic intrinsic clearance of each enantiomer, and the fraction of R(-)-ibuprofen which was metabolically inverted to S(+)-ibuprofen, was independent of the administered dose.

Disopyramide pharmacokinetics and metabolism: effect of inducers.

1. The disposition of orally administered disopyramide was studied in a population of smokers (n = 6) and non-smokers (n = 8) before and during phenobarbitone treatment (100 mg daily for 21 days; Cp 21st day = 13.9 +/- 2.0 micrograms ml-1). The comparative inducibility of these populations by phenobarbitone was assessed as was the inductive effect of cigarette smoking, per se. Furthermore, the determinants of the intensity of the inductive effect were examined, as well as the effect of the barbiturate on the binding of disopyramide to alpha 1-acid glycoprotein (AGP). 2. Smokers and non-smokers exhibited similar half-lives (6.48 +/- 1.49 vs 6.66 +/- 1.02 h), apparent total body clearances (0.100 +/- 0.020 vs 0.117 +/- 0.034 l h-1 kg-1), mean renal clearances (0.043 +/- 0.0093 vs 0.057 +/- 0.013 l h-1 kg-1) and apparent intrinsic metabolic clearances (0.057 +/- 0.015 vs 0.060 +/- 0.024 l h-1 kg-1) before phenobarbitone treatment. 3. Both populations responded comparably to barbiturate exposure in that apparent intrinsic metabolic clearance more than doubled. Interestingly, the magnitude of this increase was highly dependent on the observed baseline apparent intrinsic metabolic clearance, (r' = 0.81; P less than 0.001). 4. Phenobarbitone treatment of non-smokers resulted in an increase in the AUC of the active metabolite N-despropyl disopyramide (MND), but not significantly (3.8 +/- 1.6 vs 4.1 +/- 2.3 micrograms ml-1 h). Similar results were observed in smokers (3.5 +/- 1.4 vs 3.9 +/- 2.0 micrograms ml-1 h, respectively). 5. The percent of administered dose recovered in urine as disopyramide in non-smokers was significantly decreased upon phenobarbitone treatment (43 +/- 6% vs 25 +/- 5%), whereas the percent of dose recovered as MND increased significantly in this group (25 +/- 6% vs 31 +/- 5%). The population of smokers responded similarly. 6. At doses typically used to achieve hepatic microsomal enzyme induction in man, phenobarbitone treatment caused no significant change or trend towards a change in serum AGP concentrations as measured using the radial immunodiffusion method in nonsmokers (67.4 +/- 19.9 mg dl-1 vs 68.0 +/- 40.7 mg dl-1) or smokers (64.5 +/- 15.7 vs 67.9 +/- 14.9). Similarly, when AGP concentration was estimated in serum from non-smokers using a nephelometric method no effect attributable to phenobarbitone was observed (47.9 +/- 1.3 vs 47.9 +/- 16.8 mg dl-1). Consistent with this observation, disopyramide free fraction was not affected by barbiturate treatment.

Intrinsic metabolic clearance of parathion and paraoxon by livers from fish and rodents

The specific activities of hepatic enzymes involved in both the activation and detoxification of the anticholinesterase insecticide parathion were significantly greater in both rats and mice compared to either fathead minnows or rainbow trout. Whereas the rates of parathion desulfuration and hydrolysis were comparable, the specific activity for paraoxon hydrolysis was approximately threefold greater than its rate of formation for all species except trout. The kinetic data, however, indicate that the significantly greater K m for paraoxon hydrolysis limits its detoxification. Accordingly, the data are consistent with the progressive accumulation of this toxic metabolite in liver tissue incubated in vitro with parathion. The similar K m for parathion desulfuration and hydrolysis by rodents is consistent with the proposed mechanism of catalysis involving a common S-oxide intermediate. Due to the slow rates of enzyme catalysis, reliable kinetic estimates for the hydrolytic reactions were not possible for both species of fish. Assuming comparable K m values for paraoxon hydrolysis in rodents and fish, the net accumulation of paraoxon in tissue water may be quite similar between species and metabolic transformation may not be an important determinant of the species-selective toxicity of parathion.