Decrease In Intrinsic Clearance Research Articles

Functional characterization of a common CYP4F11 genetic variant and identification of functionally defective CYP4F11 variants in erythromycin metabolism and 20-HETE synthesis

CYP4F11, together with CYP4F2, plays an important role in the synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE) from arachidonic acid. We identified 21 variants by whole exome sequencing, including 4 non-synonymous variants in Korean subjects. The proteins of the wild-type CYP4F11 and the four coding variants (C276R, D315N, D374Y, and D446N) were expressed in Escherichia coli DH5α cells and purified to give cytochrome P450-specific carbon monoxide difference spectra. Wild-type CYP4F2 was also expressed and purified to compare its activity with the CYP4F11 wild-type. Wild-type CYP4F11 exhibited the highest maximal clearance for erythromycin N-demethylase activity followed by the variants D374Y, D446N, C276R, and D315N. In particular, the CYP4F11 D315N protein showed about 50% decrease in intrinsic clearance compared to the wild type. The ability of wild-type CYP4F11 and the variants to synthesize 20-HETE from arachidonic acid was similar; the CYP4F11 D315N variant, however, showed only 68% of wild-type activity. Furthermore, the ability of CYP4F2 to synthesize 20-HETE was 1.7-fold greater than that of CYP4F11. Overall, our results suggest that the metabolism of CYP4F11 substrates may be reduced in individuals carrying the CYP4F11 D315N genetic variant and individuals carrying the common D446N CYP4F11 variant likely exhibit comparable 20-HETE synthesis as individuals expressing wild-type CYP4F11.

The Impact of the Hepatocyte-to-Plasma pH Gradient on the Prediction of Hepatic Clearance and Drug-Drug Interactions for CYP2D6 Substrates.

The proton gradient from the intracellular space to plasma creates an unbound drug gradient for weak acids and bases that could modulate apparent drug clearance and drug-drug interactions. Cytochrome P450 intrinsic clearance and inhibitor potency are routinely determined in vitro at the plasma pH of 7.4 rather than the intrahepatocyte pH of 7.0. We determined the impact of pH on in vitro enzyme kinetic parameters and inhibition potency for substrates (bufuralol, dextromethorphan), reversible inhibitors (quinidine, amiodarone, desethylamiodarone, clozapine), and mechanism-based inhibitors (paroxetine, desethylamiodarone) of the major drug metabolizing-enzyme CYP2D6. The lower intracellular pH 7.0 compared with pH 7.4 resulted in a 60 and 50% decrease in intrinsic clearance for the substrates bufuralol and dextromethorphan, respectively. Reversible inhibition constants for three of the four inhibitors tested were unaffected by pH, whereas for the inhibitor quinidine, a 2-fold increase in the inhibition constant was observed at pH 7.0. For time-dependent inhibitors desethylamiodarone and paroxetine, changes in time-dependent inhibition parameters were different for each inhibitor. These results were incorporated into physiologically based pharmacokinetic models indicating that the changes in in vitro parameters determined at pH 7.0 offset the effect of increased unbound intracellular concentrations on apparent clearance and extent of drug-drug interactions. However, this offset between concentration and enzyme activity cannot be generalized for all substrates, inhibitors, and enzymes, as the effect of a lower pH in vitro varied significantly; therefore, it would be prudent to determine in vitro enzyme parameters at the hepatocyte-appropriate pH 7.0.

Analysis of genetic polymorphism and biochemical characterization of a functionally decreased variant in prostacyclin synthase gene (CYP8A1) in humans

Prostacyclin synthase (CYP8A1) is an enzyme responsible for the biosynthesis of prostacyclin (PGI2) which inhibits platelet activation and exhibits anti-inflammatory effect. The objectives of this study were to identify CYP8A1 genetic variants and characterize functional consequences of CYP8A1 variants. In total, 27 variants including four previously unidentified single-nucleotide polymorphisms (SNPs) were identified by direct DNA sequencing in Koreans (n=48). Among them, CYP8A1 A447T and E314Stop were newly assigned as CYP8A1∗5 and CYP8A1∗6 by the Human Cytochrome P450 Allele Nomenclature Committee, respectively. CYP8A1∗5 was found in the heme binding area in three individuals as a heterozygous mutation. To investigate the functional change of CYP8A1∗5, CYP8A1∗5 and wild-type CYP8A1 protein were overexpressed in an Escherichia coli expression system and purified. Metabolism of PGH2 by the CYP8A1∗5 protein exhibited significantly decreased activity, resulting in a 45% decrease in Vmax and a 1.8-fold decrease in intrinsic clearance compared to the wild-type. Based on the predicted crystal structure of CYP8A1∗5 using the Molecular Operating Environment platform, the distance from CYP8A1 Cys441 to the heme was altered with a significantly changed binding free energy for the mutant protein. Further studies would be needed to determine the effect of CYP8A1∗5 on PGI2 levels in humans.

CYP3A4*16andCYP3A4*18Alleles Found in East Asians Exhibit Differential Catalytic Activities for Seven CYP3A4 Substrate Drugs

CYP3A4, the major form of cytochrome P450 (P450) expressed in the adult human liver, is involved in the metabolism of approximately 50% of commonly prescribed drugs. Several genetic polymorphisms in CYP3A4 are known to affect its catalytic activity and to contribute in part to interindividual differences in the pharmacokinetics and pharmacodynamics of CYP3A4 substrate drugs. In this study, catalytic activities of the two alleles found in East Asians, CYP3A4*16 (T185S) and CYP3A4*18 (L293P), were assessed using the following seven substrates: midazolam, carbamazepine, atorvastatin, paclitaxel, docetaxel, irinotecan, and terfenadine. The holoprotein levels of CYP3A4.16 and CYP3A4.18 were significantly higher and lower, respectively, than that of CYP3A4.1 when expressed in Sf21 insect cell microsomes together with human NADPH-P450 reductase. CYP3A4.16 exhibited intrinsic clearances (V(max)/K(m)) that were lowered considerably (by 84-60%) for metabolism of midazolam, carbamazepine, atorvastatin, paclitaxel, and irinotecan compared with CYP3A4.1 due to increased K(m) with or without decreased V(max) values, whereas no apparent decrease in intrinsic clearance was observed for docetaxel. On the other hand, K(m) values for CYP3A4.18 were comparable to those for CYP3A4.1 for all substrates except terfenadine; but V(max) values were lower for midazolam, paclitaxel, docetaxel, and irinotecan, resulting in partially reduced intrinsic clearance values (by 34-52%). These results demonstrated that the impacts of both alleles on CYP3A4 catalytic activities depend on the substrates used. Thus, to evaluate the influences of both alleles on the pharmacokinetics of CYP3A4-metabolized drugs and their drug-drug interactions, substrate drug-dependent characteristics should be considered for each drug.

Liver Fibrosis Impairs Hepatic Pharmacokinetics of Liver Transplant Drugs in the Rat Model

This study aims to investigate hepatic pharmacokinetics of the four most common drugs (metoprolol, omeprazole, spironolactone, and furosemide) given to patients undergoing liver transplantation before surgery. The investigation was carried out in CCl(4)-induced fibrotic perfused rat livers and the results were compared to those in normal rat liver. Drug outflow fraction-time profiles were obtained after bolus injection into a single-pass-perfused normal or fibrotic rat liver. The pharmacokinetic parameters were estimated using previously developed barrier-limited and space-distributed models. The results showed a marked increase in the liver fibrosis index for CCl(4)-treated rats compared to controls (p<0.05). The extraction ratios (E) for all drugs were significantly lower (p<0.05) in fibrotic than in normal livers and the decrease in E was consistent with the decrease in intrinsic clearance and permeability-surface area product. In addition, other than for furosemide, the mean transit times for all drugs were significantly longer (p<0.01) in the fibrotic livers than in normal livers. Pharmacokinetic model and stepwise regression analyses suggest that these differences arise from a reduction in both the transport of drugs across the basolateral membrane and their metabolic clearance and were in a manner similar to those previously found for another group of drugs.

Catalytic activities of human cytochrome P450 2C9*1, 2C9*3 and 2C9*13

Cytochrome P450 2C9 (CYP2C9) is a geneticly polymorphic enzyme responsible for the metabolism of some clinically important drugs. CYP2C9*13 is an allele identified in a Chinese poor metabolizer of lornoxicam which has a Leu90Pro amino acid substitution. This paper reports on a study aimed at comparing the catalytic properties of CYP2C9*13 with those of the wild-type CYP2C9*1 and mutant CYP2C9*3 (Ile359Leu) in the COS-7 expression system using various substrates. CYP2C9*3 and *13 produced far lower luminescence than CYP2C9*1 in luciferin H metabolism. CYP2C9*13 exhibited an 11-fold increase in Km but no change in Vmax with tolbutamide as the substrate, a five-fold increase in Km and an 88.8% reduction in Vmax with diclofenac. These data indicate that CYP2C9*13 exhibits reduced metabolic activity toward all studied CYP2C9 substrates. The magnitude of the CYP2C9*13-associated decrease in intrinsic clearance (Vmax/Km) is greater than that associated with CYP2C9*3.

The effect of genetic polymorphisms in CYP2C9 on sulphamethoxazole N-hydroxylation.

Sulphamethoxazole undergoes CYP2C9-mediated bioactivation to a hydroxylamine. In this study, we investigated the effect of the CYP2C9Arg144 to Cys (CYP2C9*2) and CYP2C9Ile359 to Leu (CYP2C9*3) polymorphisms on sulphamethoxazole N-hydroxylation. Human livers were genotyped using polymerase chain reaction amplification and restriction fragment length polymorphism analysis. Formation of sulphamethoxazole hydroxylamine and methylhydroxy tolbutamide in microsomes prepared from cell lines and the genotyped human livers was determined by high-pressure liquid chromatography. Microsomes prepared from the cell line expressing the allelic variants CYP2C9-Cys144 and CYP2C9-Leu359 displayed a threefold and 20-fold decrease in intrinsic clearance (Cl(int)) for sulphamethoxazole, respectively, when compared with the wild-type, CYP2C9-Arg144. A significant decrease (P < 0.05) in Cl(int) was also observed with tolbutamide for both mutations. Of the 26 human livers genotyped, 61.5% were homozygous wild-type, 26.9% were heterozygotes for CYP2C9*2 and 15.4% were heterozygotes for CYP2C9*3. No homozygous mutant livers were detected. There was a good correlation between sulphamethoxazole N-hydroxylation and tolbutamide methyl hydroxylation (r = 0.825). However, there was no difference in the kinetic parameters for either sulphamethoxazole N-hydroxylation or tolbutamide methyl hydroxylation between the wild type livers (n = 6) and either the livers heterozygous for the CYP2C9*2 (n = 5) or the livers heterozygous for the CYP2C9*3 mutation (n = 3). The CYP2C9*2 and CYP2C9*3 polymorphisms may have some influence on the bioactivation of sulphamethoxazole, particularly in individuals who are homozygous mutants, and this could act as a protective factor against sulphamethoxazole hypersensitivity. However, given the rarity of homozygous mutants, it is likely that other metabolic and immunological risk factors will dominate individual susceptibility.

Effects of hyperlipidemia on the pharmacokinetics of nifedipine in the rat.

The effect of hyperlipidemia on nifedipine pharmacokinetics was studied. The mechanisms by which hyperlipidemia affects pharmacokinetics of drugs are mainly undetermined. Hyperlipidemia may decrease the fraction of unbound drug in plasma and/or decrease intrinsic ability of the cytochrome P-450 systems due to excess membrane cholesterol. Hyperlipidemia is a primary risk factor for coronary artery disease leading to hypertension and ischemic heart disease, for which nifedipine, a calcium channel blocker, is used. Poloxamer 407 (P407)-induced hyperlipidemic rat model was used to study the effects of hyperlipidemia on the pharmacokinetics of nifedipine (6 mg kg-1 given i.v., i.p. and p.o.). Total plasma cholesterol levels increased from 0.82-2.02 to 5.27-11.05 mmol L-1 48 h post P407 administration (1 g kg-1, i.p.). Protein binding studies were conducted by an ultrafiltration method. Hyperlipidemia significantly decreased CLTB by 38% and CLTB/F by 45 and 42% following po and i.p. doses, respectively, thereby increasing AUC0-infinity, Cmax and half-life. Absolute bioavailability and Vdss remained unchanged. AUC0-infinity was affected to the same extent in each route of administration, therefore, the effect was mainly systemic rather than presystemic. Hyperlipidemia significantly lowered the fraction unbound in plasma by approximately 31%. The altered pharmacokinetics of nifedipine by P407-induced HYPERLIPIDEMIA may be, at least in part, due to the decrease in fraction unbound in plasma. A decrease in intrinsic clearance, however, cannot be ruled out.

Effects of Temperature and Propofol on Hepatic Blood Flow

In Response: We used the method of Soons et al. [1] to evaluate indocyanine green (ICG) clearance and estimated hepatic blood flow and thus assumed an ICG extraction ratio of 0.7 in each participant. This method assumes that the intrinsic clearance of ICG remains constant, a reasonable assumption since the high extraction ratio of ICG makes it relatively insensitive to changes in intrinsic clearance (ICG clearance depends far more on hepatic blood flow than on intrinsic clearance) [2]. Dr. Carmichael is concerned that the observed change in ICG clearance, which we concluded to be primarily due to a reduction in hepatic blood flow, might instead result from reduced intrinsic clearance of ICG, based on the report of Lange et al. [3]. Unfortunately, the study of Lange et al. [3] suffers major methodologic weaknesses, reducing confidence in their estimations of the ICG extraction ratio, intrinsic clearance, and the effect of propofol on these values. The combination of a long ICG infusion (approximately 2 h) and ICG analysis by spectrophotometry rather than high-performance liquid chromatography probably overestimated the concentration of ICG since spectrophotometry does not distinguish between the parent compound and its metabolites, which are eliminated much more slowly [1,4]. High-performance liquid chromatography methods, in contrast, do not share this problem because the parent compound is separated by chromatography from the metabolites. The extent to which the spectrophotometric method may overestimate ICG concentration increases with the duration of infusion and can be as high as 40% under the conditions of Lange et al.'s study [4]. An overestimation of ICG concentration by even 20% is sufficient to produce an erroneous apparent 16% decrease in ICG extraction ratio (assuming a starting value of 0.7) and an erroneous apparent 27% decrease in intrinsic clearance of ICG with the equations used by Lange et al. [3]. All of the apparent changes in these variables reported by Lange et al. at sternotomy may result from suboptimal methodology. Lange et al.'s assessment made immediately after induction does suggest reduced ICG extraction and intrinsic clearance. However, the study design makes it difficult to assume that the effects are due to propofol rather than the other concurrently administered drugs (fentanyl and pancuronium) or are from the anesthetic state per se. In contrast, our volunteers were anesthetized with isoflurane and nitrous oxide for at least 3 h before administration of ICG, thus allowing ICG clearance changes due to the propofol infusion to be separated from those resulting from anesthesia itself or other concurrently administered drugs. In short, the evidence that propofol changes the hepatic extraction ratio and intrinsic clearance of ICG is far from compelling. We thus stand by our conclusions, albeit with the assumptions and caveats described at length in our article. In any case, whether the actual mechanism is reduction in hepatic blood flow or intrinsic clearance (and extraction ratio), it is obvious that propofol affects hepatic elimination of drugs, including its own elimination. Kate Leslie, MB,BS, FANZCA Daniel I. Sessler, MD Andrew R. Bjorksten, PhD Azita Moayeri, BA Department of Anaesthesia The Royal Melbourne Hospital Victoria 3050, Australia

Effect of malaria on phenol conjugation pathways in perfused rat liver

The effect of malaria infection (MI) on sulphation and glucuronidation of phenol was investigated in single-pass perfused livers from rats infected with the rodent malaria parasite Plasmodium berghei. At a hepatic inflow ( C in) phenol concentration of 1 μg/mL in controls, 52% was metabolized to sulphate conjugate and 37% to glucuronide conjugate at steady state. At this C in, MI had no effect on phenol clearance ( CL) (control: 9.63 ± 0.38 vs MI: 9.65 ± 0.36 mL/min; P > 0.05) or on the formation clearance ( CL m) of the glucuronide or sulphate conjugates of phenol. When phenol C in was increased 10-fold to 10 μg/mL, 6% was metabolized to sulphate conjugate and 94% to glucuronide conjugate. At this C in phenol CL was decreased significantly (control: 9.44 ± 0.46 vs MI: 7.09 ± 1.51 mL/min; P < 0.05) and represented a decrease in intrinsic clearance (sinusoidal perfusion model) of at least 55%. This decrease was accounted for entirely by the decrease in the CL m of the glucuronide conjugate (control: 8.88 ± 0.96 vs 5.98 ± 1.87 mL/min; P < 0.05), whereas the CL m of the sulphate conjugate was unchanged. There was a negative correlation between phenol glucuronide CL m, and the severity of the erythrocytic parasitaemia ( r 2 = 0.75, P < 0.05). The dose-dependent reduction in phenol glucuronidation in MI may be due to reduced availability of the cosubstrate uridine diphosphoglucuronic acid (UDPGA), because previous studies have shown that UDPGA availability depends on glycogen stores, which are known to be reduced in MI. These data suggest that sulphate conjugation is preserved in MI and that glucuronidation is preserved at low doses of substrate. At high substrate doses, glucuronidation is impaired in MI and the impairment correlates with the severity of the infection.

Pharmacokinetics of remoxipride in elderly psychotic patients

The pharmacokinetics of remoxipride when given as single doses of 50 mg and repeated doses of 50 mg, 100 mg, and 200 mg twice daily to 10 elderly psychotic patients (71-89 years) were compared with the findings of two other studies to reveal any age-related differences. The three studies comprised a total of 38 patients in three distinct age groups: elderly (71-89 years), middle-aged (46-69 years) and young (19-36 years). AUC, Cmax and Cmin of both total and unbound remoxipride increased with increasing age. The unbound fraction was similar in the three age groups. The half-life was prolonged in the elderly, most probably caused by a decrease in intrinsic clearance. A two-fold increase in AUC of both total and unbound concentrations was observed in the elderly group compared to the young, suggesting that patients over 70 years in general require half the dose needed by young adult patients. In general, the pharmacokinetics of remoxipride in the elderly are linear.

Halothane inhibition of propranolol metabolism is stereoselective.

Propranolol, like many drugs, is used clinically as a racemic mixture. The major pharmacodynamic effects of propranolol, however, are mediated by the (-)-isomer, which is 100 times as potent as the (+)-isomer. The two isomers also differ in their pharmacokinetic characteristics. To determine whether halothane anesthesia stereo-selectively inhibits the metabolism of racemic propranolol, eight male mongrel dogs were studied. On the first day of the study, 40 mg racemic propranolol was infused into the portal vein and arterial blood samples were obtained over the following 4 h for the measurements of (+)- and (-)-propranolol concentrations by HPLC. The study was repeated 24 h later during 2 MAC halothane anesthesia, when the intrinsic clearance of total propranolol was decreased by 67.5 +/- 5%, from 6.14 +/- 1.1 1/min to 1.84 +/- 0.4 1/min (P less than 0.05). The decrease in intrinsic clearance was stereoselective, (-)-propranolol being affected to a greater extent than (+)-propranolol; thus, the decrease in the clearance of (-)-propranolol, from 10.96 +/- 2.71/min to 2.6 +/- 0.71/min (73 +/- 5%) was significantly (P less than 0.05) greater than the decrease in the clearance of (+)-propranolol, (62 +/- 3%) from 4.3 +/- 0.8 1/min to 1.5 +/- 0.3 1/min. Furthermore, the ratio of the intrinsic clearance of (-)-propranolol to the intrinsic clearance of (+)-propranolol was significantly (P less than 0.05) reduced by halothane anesthesia, from 2.42 +/- 0.29 to 1.69 +/- 0.11. Stereoselective inhibition of propranolol metabolism results in proportionally higher concentrations of (-)-propranolol during halothane anesthesia than are present in awake dogs.(ABSTRACT TRUNCATED AT 250 WORDS)

COMPARISON OF THE EFFECTS OF ISOFLURANE OR FENTANYL-NITROUS OXIDE ANAESTHESIA ON PROPRANOLOL DISPOSITION IN DOGS

The disposition of propranolol was studied, using dual-route administration, in two groups of six dogs. Each dog was studied on three consecutive days: day 1 awake, day 2 during anaesthesia, and day 3, 24 h after anaesthesia. Anaesthesia was with isoflurane 2.0 MAC (in oxygen) in one group and with a fentanyl-nitrous oxide-atracurium regimen in the other group. In the group receiving fentanyl, anaesthesia caused a significant decrease (63%) in intrinsic clearance from the day 1 value (P less than 0.05) and a 45% decrease in systemic clearance (P less than 0.05). Hepatic plasma flow decreased by 27% (ns). A similar pattern was found with isoflurane: intrinsic clearance decreased by 53% (P less than 0.05) and systemic clearance by 40% (P less than 0.05). Hepatic plasma flow decreased by 40% (ns). In both groups, the values 24 h after anaesthesia were not significantly different from those obtained on day 1. Anaesthesia with either fentanyl-nitrous oxide-atracurium or isoflurane has a marked, but short-lasting effect on the disposition of propranolol, in part as a result of a decrease in intrinsic clearance.