Neonatal hepatic drug elimination.

After the transition from in utero to newborn life, the neonate becomes solely reliant upon its own drug clearance processes to metabolise xenobiotics. Whilst most studies of neonatal hepatic drug elimination have focussed upon in vitro expression and activities of drug-metabolising enzymes, the rapid physiological changes in the early neonatal period of life also need to be considered. There are dramatic changes in neonatal liver blood flow and hepatic oxygenation due to the loss of the umbilical blood supply, the increasing portal vein blood flow, and the gradual closure of the ductus venosus shunt during the first week of life. These changes which may well affect the capacity of neonatal hepatic drug metabolism. The hepatic expression of cytochromes P450 1A2, 2C, 2D6, 2E1 and 3A4 develop at different rates in the postnatal period, whilst 3A7 expression diminishes. Hepatic glucuronidation in the human neonate is relatively immature at birth, which contrasts with the considerably more mature neonatal hepatic sulfation activity. Limited in vivo studies show that the human neonate can significantly metabolise xenobiotics but clearance is considerably less compared with the older infant and adult. The neonatal population included in pharmacological studies is highly heterogeneous with respect to age, body weight, ductus venosus closure and disease processes, making it difficult to interpret data arising from human neonatal studies. Studies in the perfused foetal and neonatal sheep liver have demonstrated how the oxidative and conjugative hepatic elimination of drugs by the intact organ is significantly increased during the first week of life, highlighting that future studies will need to consider the profound physiological changes that may influence neonatal hepatic drug elimination shortly after birth.

Differential inhibition of human CYP1A1 and CYP1A2 by quinidine and quinine.

1. The inhibition of recombinant CYP1A1 and CYP1A2 activity by quinidine and quinine was evluated using ethoxyresorutin O-deethylation, phenacetin O-deethylation and propranolol desisopropylation as probe catalytic pathways. 2. With substrate concentrations near the Km of catalysis, both quinidine and quinine potently inhibited CYP1A1 activity with [I](0.5) approximately 1-3 microM, whereas in contrast, there was little inhibition of CYP1A2 activity. The Lineweaver-Burk plots with varying inhibitor concentrations suggested that inhibition by quinidine and quinine was competitive. 3. There was only trace metabolism of quinidine by recombinant CYP1A1, whereas rat liver microsomes as a control showed extensive consumption of quinidine and metabolite production. 4. This work suggests that quinidine is a non-classical inhibitor of CYP1A1 and that it is not as highly specific at inhibiting CYP2D6 as previously thought.

Right heart failure impairs hepatic elimination of p-nitrophenol without inducing changes in content or latency of hepatic UDP-glucuronosyltransferases.

Congestive heart failure has been shown to affect oxidative drug metabolism, however, there has been little study of its effects on drug conjugation. Using the isolated perfused livers from rats with right ventricular failure (RVF) due to pulmonary artery constriction, we studied the effects of RVF on hepatic elimination of p-nitrophenol (PNP) under controlled flow and oxygen delivery conditions. Hepatic clearance of the drug was found to be significantly impaired in RVF as compared with the sham group (0.80 +/- 0.23 versus 1.28 +/- 0.26 ml/min/g of liver). The impairment of PNP clearance in RVF occurred in parallel with significant reduction in metabolic formation clearance of p-nitrophenyl-beta-D-glucuronide; the major metabolite of PNP (0.51 +/- 0.12 versus 1.03 +/- 0.26 ml/min/g of liver). The intrinsic drug-glucuronidation capacity of livers was evaluated by measuring the microsomal content and activity of the UDP-glucuronosyltransferase(s) (UDP-GT) toward p-nitrophenol. There was no significant difference between sham and the RVF groups in either the content or the activity of the UDP-GT. The latency of the UDP-GT enzymes in microsomes was measured and was found to be similar between the two groups. The results of this study show that RVF impairs hepatic elimination of PNP and that this appears to be independent of changes in hepatic perfusion and oxygenation or alterations in hepatic content, activity, and latency of the UDP-GT.

Role of cytochrome P450 2D6 (CYP2D6) in the stereospecific metabolism of E- and Z-doxepin.

The tricyclic antidepressant, doxepin, is formulated as an irrational mixture of E (trans) and Z (cis) stereoisomers (85%: 15%). We examined the stereoselective metabolism of doxepin in vitro, with the use of human liver microsomes, recombinant CYP2D6 and gas chromatography-mass spectrometry. In human liver microsomes over the concentration range 5-1500 microM, the rate of Z-doxepin N-demethylation exceeded that of E-doxepin above 100 microM in two of three livers. Eadie-Hofstee plots were curvilinear indicating the involvement of several enzymes in N-demethylation. Coincubation of doxepin with 7,8-naphthoflavone and ketoconazole reduced the rates of N-demethylation of E- and Z-doxepin by 30-50% and 40-60%, respectively, suggesting the involvement of CYP1A and CYP3A4, whilst quinidine had little effect on N-demethylation. In contrast, doxepin hydroxylation was exclusively stereo-specific; E-doxepin and E-N-desmethyldoxepin were hydroxylated with high affinity in liver microsomes and by recombinant CYP2D6 (Km in the range of 5-8 microM), but there was no evidence of Z-doxepin hydroxylation. In 'metabolic consumption' experiments with liver microsomes (having measurable CYP2D6 activity) and initial substrate concentration of 1 microM, the consumption of E-doxepin was greater (P < 0.05, n = 5) than that of Z-doxepin. Quinidine inhibited the consumption of E-doxepin but did not affect the consumption of Z-doxepin. With N-desmethyldoxepin, quinidine inhibited the consumption of E-N-desmethyl-doxepin whereas Z-N-desmethyldoxepin appeared to be a terminal oxidative metabolite. In summary, CYP2D6 is a major oxidative enzyme in doxepin metabolism; predominantly catalysing hydroxylation with an exclusive preference for the E-isomers. The relatively more rapid metabolism of E-isomeric forms, and the limited metabolic pathways for the Z-isomers may explain the apparent enrichment of Z-N-desmethyldoxepin that is observed in vivo.

Impaired elimination of propranolol due to right heart failure: drug clearance in the isolated liver and its relationship to intrinsic metabolic capacity.

It is unclear if reduced hepatic drug elimination in congestive heart failure is primarily due to impairment of enzyme function as a result of tissue hypoxia, to the direct effects of hepatic congestion, or to changes intrinsic to the liver, such as reductions in enzyme content and activity. We therefore compared propranolol clearance in perfused rat livers from animals with right ventricular failure (RVF) with that from control animals. Despite the fact that both groups were perfused at comparable flow rates, perfusion pressures, and levels of oxygen delivery, hepatic extraction of propranolol was significantly reduced in RVF livers (0.688 +/- 0.122 versus 0.991 +/- 0.006 ml/min/g of liver in controls, P <.001). This effect was reflected in a 97% reduction in propranolol intrinsic clearance in RVF livers (5 +/- 4 versus 172 +/- 82 ml/min/g of liver in controls, P <.01). In RVF livers, total hepatic CYP expression was reduced by 19% compared with controls, whereas cytochrome P450 isoenzymes 1A1/2 and 2D1 were reduced by 41 and 26%, respectively. Despite the 97% reduction in propranolol intrinsic clearance in perfused RVF liver, intrinsic clearance in microsomal preparations from the same livers was reduced by only 48% compared with controls (P <.05). These findings suggest that impaired propranolol clearance in RVF is not primarily accounted for by reduced hepatic oxygen delivery or by changes in hepatic content and activity of drug-metabolizing enzymes.

Neonatal hepatic propranolol elimination: studies in the isolated perfused neonatal sheep liver.

Using the isolated perfused neonatal sheep liver model, we examined the disposition of propranolol (n = 8, age 0.25-10 days) and compared our findings with our previous study from the perfused near-term fetal sheep liver (Ring JA, et al. 1995. Drug Metab Dispos 23:190-196). Within 45 min of dosage, perfusate propranolol levels had fallen by three orders of magnitude to be less than the limit of detection. Perfusate disappearance curves were monoexponential in six experiments and biexponential in two experiments. The mean shunt-corrected hepatic extraction ratio was 0.92 +/- 0.09, much greater than that seen in the fetal sheep liver (0.26 +/- 0.13, P < 0.0001) but still less than values in the adult sheep (0.97). At the conclusion of the perfusion, 4-hydroxypropranolol was the major metabolite present and 5-hydroxypropranolol and N-desisopropylpropranolol were minor metabolites. We conclude that the isolated perfused neonatal sheep liver is a useful model with which to study the maturation of neonatal hepatic drug oxidation. Our study shows that propranolol is rapidly eliminated by the neonatal liver to form several metabolites at rates far greater than in the fetal liver, but rates of elimination have not yet reached that reported in the adult sheep liver.

Conjugation of para-nitrophenol by the isolated perfused neonatal sheep liver.

We examined the metabolism of para-nitrophenol (PNP) in the isolated perfused neonatal sheep liver (n = 8, 0.25-11 days) and compared the findings with our previous data from the perfused near-term fetal sheep liver (Ring, J. A., et al. Drug Metab Dispos 1996, 24, 1378). A three-step dosage regimen was used (72, 144, and 288 micromol of PNP). At the end of each dosage phase, PNP had fallen below detectable levels, and 101 +/- 16% of the dose was accounted for as PNP conjugates. Elimination of PNP from perfusate varied with dose. Elimination was first order with the 72-micromol dose; with the 144-micromol dose, elimination was first order in four livers but Michaelis-Menten kinetics in the remaining four. With all the 288-micromol doses, elimination was Michaelis-Menten and gave the following biochemical parameters: K(m) = 255 +/- 138 microM (fetal = 14.7 microM, P < 0.01), V(max) = 515 +/- 285 nmol/min/g liver (fetal = 34.3 nmol/min/g liver, P < 0.01), and intrinsic hepatic clearance = 2.36 +/- 1.21 mL/min/g liver (fetal = 4.74 mL/min/g liver, P > 0. 05). The mean shunt-corrected hepatic extraction ratio of PNP was 0. 82 (range, 0.40-1.0) and strongly correlated with neonatal age (r = 0.90, P < 0.05). We conclude that PNP is highly extracted by the isolated perfused neonatal sheep liver at much higher efficiency than in the near-term fetal sheep, reflecting a maturation of conjugation that progresses further in the early neonatal period.

Uptake and excretion of sodium taurocholate by the isolated perfused neonatal sheep liver.

We present a model for perfusion of the isolated perfused neonatal sheep liver which allows examination of drug disposition by the intact organ. We studied the disposition of sodium taurocholate (TC) in seven neonatal lambs (ages 2-11 days) and compared the results with earlier data from the perfused fetal sheep liver (Ring, J. A. et al. Biochem. Pharmacol. 1994, 48, 667-674). Measurements of perfusion pressure, oxygen consumption, lactate:pyruvate ratio, bile flow, and liver histology indicated that the preparation was both viable and stable over a 2 h period. [14C]-labeled TC was added to the reservoir by constant infusion (30 micromol/h) and the ductus venosus shunt quantitated by injection of [153Gd]-labeled microspheres. Shunt-corrected hepatic extraction ratio of TC was 0. 56 +/- 0.14 (fetal 0.23 +/- 0.16, p < 0.005) and clearance of TC was 0.92 +/- 0.35 mL/min/g liver (fetal 0.44 +/- 0.23 mL/min/g, p < 0. 01). We conclude that the isolated perfused neonatal sheep liver is a useful experimental model which will facilitate the study of the developmental physiology and pharmacology of the liver. There is considerable maturation of the biliary excretion of TC between the late fetal and early neonatal periods in the lamb.

Fetal hepatic drug elimination.

The majority of studies of fetal hepatic elimination have concentrated on the expression and activity of the metabolizing enzymes, but the unique physiologic milieu of the fetal liver should also be considered. The basic structure of the liver is formed by the end of the first trimester. The fetal hepatic circulation differs substantially from that of the adult in that there is an extra input vessel, the umbilical vein, and there is shunting of 30-70% of hepatic blood flow via the ductus venosus. The left and right lobes of the fetal liver seem to function independently with respect to a variety of biochemical parameters, due at least in part to the lower oxygen supply to the right lobe. The zonation of drug-metabolizing enzymes along the hepatic acinus, which is prominent in the adult liver, is absent in the fetal liver. Unlike rodent species, the human fetal liver has a significant capacity for drug metabolism. Of the oxidative enzymes, CYP3A7 accounts for up to 50% of total fetal hepatic cytochrome P450 content. Expression of this enzyme decreases dramatically after birth. CYP1A1 and CYP2D6 have also been detected in human fetal liver, but whether CYP2E1 is expressed remains controversial. Several other cytochrome P450s have been identified and await characterization. Fetal hepatic drug conjugation may prolong fetal exposure to the metabolites produced, which, being more water soluble, do not readily cross the placenta back to the mother and, if excreted in fetal urine, can be recycled in the fetus via amniotic fluid and fetal swallowing. Limited activity of glucuronidation enzymes has been demonstrated in human fetal liver in contrast to the activity of sulfation enzymes, which is significant. Limited in vivo studies in fetal sheep have demonstrated significant fetal hepatic drug elimination, and this has been confirmed in studies of the isolated perfused fetal sheep liver. Our understanding of fetal hepatic elimination processes has advanced steadily over the years. Future developments, however, should consider more fully the influence of the unique physiological milieu of the fetal liver, in addition to the expression and activity of drug metabolizing enzymes.

Stereoselective measurement of E- and Z-doxepin and its N-desmethyl and hydroxylated metabolites by gas chromatography-mass spectrometry.

A stereoselective method of analysis of the antidepressant drug doxepin (DOX, an 85:15% mixture of E-Z stereoisomers), its principal metabolites E- and Z-N-desmethyldoxepin (desDOX) and ring-hydroxylated metabolites in microsomal incubation mixtures is described. DOX and its metabolites were extracted from alkalinised incubation mixtures by either: 9:1 hexane-propan-2-ol (method 1) or 1:1 hexane-dichloromethane (method 2), derivatised with trifluoroacetic anhydride and analysed by GC-MS with selected ion monitoring. Both methods were suitable for the analysis of individual desDOX isomers as indicated by correlation coefficients of > or = 0.999 for calibration curves constructed between 50 and 2500 nM, and good within-day precision at 125 nM (C.V. < or = 14%) and 1000 nM (C.V. < or = 8%). Method 1, however, was unsuitable for the analysis of ring-hydroxylated metabolites of DOX, whereas the hydroxylated metabolites of E-DOX and E-desDOX (generated in situ) were extracted by method 2 with a C.V. of ca. 13%. This is the first assay method that permits the simultaneous measurement of desDOX and hydroxylated metabolites of DOX in microsomal mixtures.

Metabolism of dexfenfluramine in human liver microsomes and by recombinant enzymes: role of CYP2D6 and 1A2.

Dexfenfluramine has been widely used as an appetite suppressant in the treatment of obesity. It was recently shown that the apparent non-renal clearance of dexfenfluramine was significantly lower in poor metabolizers than in extensive metabolisers of debrisoquine which suggested the involvement of the polymorphically expressed enzyme, CYP2D6, in dexfenfluramine metabolism. In this study, human liver microsomes and yeast-expressed recombinant enzymes were used to examine dexfenfluramine metabolism in vitro. In human liver microsomes, the major product of dexfenfluramine was nordexfenfluramine with lesser amounts of a novel metabolite, N-hydroxynordexfenfluramine, and ketone and alcohol derivatives being formed. Eadie-Hofstee plots (v against v/[s]) of nordexfenfluramine formation between 1 and 1000 microM substrate concentration were biphasic in three of four liver microsome samples examined, with mean Km values of 3 and 569 microM for the high and low affinity enzymes, respectively. At a substrate concentration (0.5 microM) around the known therapeutic plasma concentration, there was negligible inhibition of microsomal dexfenfluramine N-dealkylation by sulphaphenazole and ketoconazole, but between 33 and 100% inhibition by quinidine, and 0-58% inhibition by 7,8-naphthoflavone in seven liver samples. In human liver microsomes, there was also a significant correlation (rs= 0.79, n = 10, P < 0.01) between dextromethorphan O-demethylation and dexfenfluramine (at 1 microM) N-dealkylation activities. Dexfenfluramine was a specific inhibitor (IC50 46 microM) of CYP2D6-mediated dextromethorphan O-demethylation in human liver microsomes but did not appreciably inhibit six other cytochrome P450 isoform-selective activities for CYP1A2, 2A6, 2C9, 2C19, 2E1 and 3A activities in human liver microsomes. Yeast-expressed recombinant human CYP2D6 metabolized dexfenfluramine with high affinity (Km 1.6 microM, Vmax 0.18 nmol min(-1) nmol P450(-1)) to nordexfenfluramine which was the sole product observed. Recombinant CYP1A2 was a lower affinity enzyme (Km 301 microM, Vmax 1.12 nmol min(-1) nmol P450(-1)) and produced nordexfenfluramine with small amounts of N-hydroxynordexfenfluramine. This is the first detailed study to examine the in-vitro metabolism of dexfenfluramine in human liver microsomes and by recombinant human P450s. We were able to identify CYP2D6 (high affinity) and CYP1A2 (low affinity) as the major enzymes catalysing the N-dealkylation of dexfenfluramine in human liver microsomes.

A priming dose of propranolol reduces the availability of a subsequent dose in the isolated perfused rat liver preparation.

1. A 50 microL bolus dose containing (+/-)-propranolol hydrochloride (200 microg) and [14C]-sucrose, or antipyrine (2 mg) and [14C]-sucrose, or [14C]-taurocholate sodium was injected into the portal vein of the isolated perfused rat liver preparation and perfusate outflow samples were collected frequently for the next 30 min. After a 20 min washout period this procedure was repeated. 2. [14C]-Sucrose, antipyrine and [14C]-taurocholate each eluted as a single peak at 18, 31 and 28 s, respectively, after each dose. In contrast, propranolol eluted with two peaks at approximately 18 and 128 s after dosing. 3. There was no significant difference in dose-corrected area under the outflow curve (AUC) for [14C]-sucrose, antipyrine or [14C]-taurocholate between the first and second doses whereas the mean propranolol AUC for the second dose was only 0.577+/-0.439 that for the first dose (P<0.05). 4. Unmetabolized propranolol accounted for more than 80% of the drug in hepatic tissue for the first and second doses at 18 s and greater than 50% at 128 s, and there was no significant difference in these values at each time between the first and second doses. 5. These findings suggest that for an avidly extracted drug, such as propranolol, systemic availability of orally administered drug will be highly dependent on factors that influence the hepatic tissue binding of the drug.

Propranolol elimination by right and left fetal liver: studies in the intact isolated perfused fetal sheep liver.

Propranolol extraction in vivo by the left lobe of the fetal sheep liver is greater than that by the right lobe, and this may be due to the fact that oxygenation of the left lobe is greater than that of the right lobe. To explore this hypothesis, we studied the elimination of (R)-(+)-propranolol (PROP) by right and left lobes of the intact isolated perfused fetal sheep liver model, in which there is equal oxygenation of both liver lobes. After isolation of the liver, near-term fetal sheep livers (n = 11) were perfused (2.68 +/- 1.05 ml/g liver/min) in situ via the umbilical vein in a 1-liter recirculating system. PROP was infused (1.2 mg/hr) into the reservoir after an initial bolus dose (2.3 mg). Perfusate samples were taken from the common and right and left hepatic veins every 10 min for determination of PROP concentrations and oxygen consumption over the 180-min experimental period. Mean ductus venosus shunt through the liver was 42 +/- 21% of perfusate flow. Oxygen consumption was not significantly different between the left and right lobes of the liver (0.79 +/- 0.46 and 0.67 +/- 0.44 micromol/g liver/min, respectively, P > .05), nor was there any significant difference between lobes in PROP hepatic extraction at steady state (0.25 +/- 0.20 and 0.25 +/- 0.23, respectively, P > .05). This supports the hypothesis that the difference between lobes in PROP extraction in vivo may be due to the difference in degree of oxygenation of the left and right lobes that is known to be present in vivo.

Measurement of dexfenfluramine metabolism in rat liver microsomes by gas chromatography-mass spectrometry.

A specific and useful method was developed for the determination of dexfenfluramine metabolism by microsomal systems utilising GC-MS. The synthesis of two metabolites 1-(3-trifluoromethylphenyl)propan-2-ol ('alcohol') and 1-(3-trifluoromethylphenyl)-1,2-propanediol ('diol') via straightforward routes, were confirmed by MS and NMR spectra. The conditions for extraction from alkalinised microsomal mixtures of the metabolites nordexfenfluramine, 1-(3-trifluoromethylphenyl)propan-2-one ('ketone'), alcohol and diol, their conversion to trifluoroacetate derivatives and analysis by GC-MS-SIM are described. Calibration curves were constructed between 48 and 9662 nM and fitted to quadratic equations (r2>0.999). The method precision was good over low (121 nM) medium (2415 nM) and above medium (9662 nM) concentrations for all metabolites; the within- and day-to-day coefficients of variation ranged between 2.5-12.4% and 6.7-17.5%, respectively. The accuracy, measured as bias, was very good both within- and day-to-day (range: -0.4-12.6%, 0.8-18.9%). For most metabolites, the C.V. for the assay and bias increased at 121 nM. Dexfenfluramine metabolism by rat liver microsomes was investigated using the assay method and showed a concentration dependent increase in nordexfenfluramine and ketone metabolites over the substrate range of 5-200 microM.

Effects of acute myocardial infarction on theophylline elimination in rats.

We investigated the effect of acute myocardial infarction (MI) on the hepatic clearance of theophylline in rats using the coronary artery ligation model. After 48 hr of ligation, there were significant changes in left ventricular performance in the MI rats, compared with controls, as indicated by elevated left ventricular end-diastolic pressure, reduced mean arterial pressure, and reduced left ventricular systolic pressure (20 +/- 2 vs. 12 +/- 3 mm Hg, p < 0.01; 90 +/- 6 vs. 101 +/- 6 mm Hg, p < 0.01; and 100 +/- 8 vs. 114 +/- 8 mm Hg, p < 0.01, respectively). Despite these changes, MI rats were able to maintain their cardiac output at rest (77.9 +/- 6.8 vs. 77.2 +/- 9.2 ml/min), and there was no change in mean central venous pressure (3 +/- 1 vs. 2 +/- 1 mm Hg). Although hepatic perfusion and oxygenation were preserved (17.3 +/- 2.2 vs. 18.7 +/- 3.3 ml/min and 127 +/- 27 vs. 125 +/- 19 mumol/min respectively), clearance of theophylline was reduced by 23% in the MI rats, compared with controls (0.86 +/- 0.20 vs. 1.12 +/- 0.17 ml/min, p = 0.01). There was no significant correlation between theophylline clearance and the infarct size (r = -0.038, p > 0.05). These findings demonstrate that the elimination of theophylline is impaired after acute MI, independent of any changes in hepatic perfusion or oxygenation.

Conjugation of para-nitrophenol by isolated perfused fetal sheep liver.

Using our recently described, isolated perfused fetal sheep liver model, we have studied the metabolism and disposition of para-nitrophenol (PNP) in intact fetal liver. Fetal sheep (mean gestational age, 137 +/- 7 days; range, 127-145 days; n = 8) were delivered under anesthesia near term, and the livers were isolated and perfused in situ, via the umbilical vein, in an oxygenated 1-liter recirculating system, at pH 7.40 at 37 degrees C. The perfusate delivery rate was 4.39 +/- 1.46 ml/g liver/min. Either a 14-micromol (n = 4), 72-micromol (n = 3), or 144-micromol (n = 5) bolus dose of PNP was added to the reservoir. Samples were taken from the reservoir every 5-10 min, and all bile was collected at 15-30-min intervals. Elimination of PNP from perfusate demonstrated Michaelis-Menten kinetics, and the calculated pharmacokinetic parameters for PNP elimination were KM = 13.0 +/- 9.66 microM, Vmax = 32.1 +/- 22.4 nmol/min/g liver, and intrinsic clearance = 3.39 +/- 2.54 ml/min/g liver. At the end of the 120-min perfusion period, PNP could be accounted for entirely as PNP-sulfate (PNP-S) and PNP-glucuronide (PNP-G). The perfusate ratio of PNP-S to PNP-G at 120 min was 2.21 +/- 0.88 at the 14-micromol dose, 0.86 +/- 0.56 at the 72-micromol dose, and 0.31 +/- 0.17 at the 144-micromol dose, because of saturation of sulfate production with increasing dose. PNP-S and PNP-G were eliminated into bile in small amounts (<3% of dose), and the PNP-S/PNP-G ratio in bile was 1. We conclude that near-term fetal sheep liver can metabolize PNP to PNP-G and PNP-S with efficiencies that may be comparable to those of adults, that, as in adults, sulfation is of low capacity, relative to glucuronidation, and that, unlike adults, fetuses have little capacity to transport the PNP-G formed in the hepatocytes into bile.

(S)-4'-hydroxypropranolol causes product inhibition and dose-dependent bioavailability of propranolol enantiomers in the isolated perfused rat liver and in rat liver microsomes.

1. Previous evidence suggests that the dose-dependent bioavailability of racemic propranolol may be partly due to product inhibition. We have examined this further by studying the individual enantiomers of propranolol in the perfused rat liver (IPRL) and in rat liver microsomes. 2. In recirculating IPRL experiments, (R)-propranolol (n = 7) or (S)-propranolol (n = 4) were infused at rates of 75, 150 and 231 nmol/min for three sequential 36-min phases. In single-pass experiments, (R)-propranolol (n = 4) or (S)-propranolol (n = 4) were administered at rates of 80, 136 and 239 nmol/min for three sequential 30-min phases. Steady-state bioavailability increased 10-20-fold over this dose range with both enantiomers in both recirculating and single-pass experiments. At the higher administration rates of (S)-propranolol, bioavailability in recirculating experiments was significantly greater than that in single-pass experiments, whereas there was no significant difference for (R)-propranolol. This suggests product inhibition of (S)- but not (R)-propranolol metabolism. 3. Of the metabolites examined, racemic 4'-hydroxypropranolol (4-OHP) inhibited the formation of 4-OHP, 5'-hydroxypropranolol (5-OHP) and desisopropylpropranolol (DIP) from (S)-propranolol and (R)-propranolol in microsomal studies (IC50 20 microM). Tissue levels of (S)-4-OHP in recirculating experiments (28.0 microM) at the highest dose (239 nmol/ min) of (S)-propranolol were greater than its IC50 of 20 microM, suggesting that 4-OHP is the inhibiting metabolite in the intact liver. The absence of evidence for product inhibition with (R)-propranolol in perfused livers suggests that (S)-4-OHP inhibits 4-hydroxylation of each isomer but (R)-4-OHP does not. 4. We conclude that in the recirculating IPRL, product inhibition of propranolol metabolism is evident with the (S)-isomer, but not he (R)-isomer, and that the inhibiting metabolite is (S)-4-OHP.

Propranolol hydroxylation and N-desisopropylation by cytochrome P4502D6: studies using the yeast-expressed enzyme and NADPH/O2 and cumene hydroperoxide-supported reactions.

We have studied the enantioselectivity and regioselectivity of ring-hydroxylation and N-desisopropylation of R(+)- and S(-)-propranolol in microsomes from yeast expressing cytochrome P4502D6 (CYP2D6), using both NADPH and molecular oxygen (NADPH/O2) and cumene hydroperoxide-supported reactions. With NADPH/O2-supported reactions, CYP2D6 catalyzed 4- and 5-ring-hydroxylation, as well as N-desisopropylation of propranolol, although Vmax was considerably greater for ring-hydroxylation, compared with N-desisopropylation. The R/S ratios for KM and Vmax were less than unity for all three pathways. In contrast, using cumene hydroperoxide-supported reactions, CYP2D6 catalyzed 4- and 5-ring-hydroxylation, and there was negligible N-desisopropylation of propranolol. The R/S ratio for KM was less than unity, but the R/S ratio for Vmax was close to unity. The cumyl group of cumene hydroperoxide did not seem to be a selective inhibitor of N-desisopropylation, because i) cumyl alcohol (a nonalkylhydroperoxide analog of cumene hydroperoxide) did not inhibit N-desisopropylation in NADPH/O2-supported reactions, and ii) the use of t-butyl hydroperoxide (a noncumyl alkylhydroperoxide) to support CYP2D6 catalysis resulted in ring-hydroxylation, but not N-desisopropylation. At a propranolol concentration near KM, quinidine inhibited both ring-hydroxylation and N-desisopropylation in an equipotent manner in NADPH/O2-supported reactions. However, in cumene hydroperoxide-supported reactions, the IC50 of inhibition of ring-hydroxylation by quinidine was an order of magnitude less potent than in NADPH/O2-supported reactions. Our study shows that recombinant CYP2D6 cannot only catalyze 4- and 5-ring-hydroxylation of propranolol, but also N-desisopropylation. The lack of propranolol N-desisopropylation observed in cumene hydroperoxide-supported reactions highlights the need for caution when using alkyhydroperoxides to study CYP2D6 catalysis.

Effect of erythrocyte binding on elimination of harmol by the isolated perfused rat liver.

The effect on the hepatic elimination rate of drug bound to erythrocytes and to albumin was compared with harmol, a relatively hydrophilic drug of high hepatic intrinsic clearance, in the single-pass isolated perfused rat liver preparation (n = 12). The steady-state hepatic extraction ratio (E) of harmol (50 microM) was measured during three consecutive 35-min periods with three different perfusates: Krebs-Henseleit buffer, buffer containing bovine serum albumin (2%), and buffer containing washed human erythrocytes (10%) perfused at 5 mL/min/g liver in randomized order. The mean unbound fraction (fu) of harmol in the latter two perfusates was 0.55 +/- 0.07 and 0.62 +/- 0.08, respectively, and the mean E for the three perfusates were 0.85 +/- 0.06, 0.62 +/- 0.07, and 0.71 +/- 0.08, respectively. The sinusoidal model fitted the relationship between E and fu better than the venous equilibrium model. Four further experiments, with perfusates of buffer, buffer + 2% albumin, and buffer + 4% albumin, confirmed that harmol elimination conformed to the sinusoidal model. For each of the 12 experiments that used erythrocyte perfusate, E and fu data from each of the two non-erythrocyte perfusates were used to predict E for the erythrocyte perfusate at the observed fu of 0.62, with the sinusoidal model. There was no significant difference between the observed (0.71 +/- 0.08) and predicted (0.68 +/- 0.10) E values (p > 0.05). This result suggests that release of harmol from erythrocytes is not a rate-limiting factor in the hepatic elimination of harmol, and that plasma membrane permeability does not contribute readily to a red cell carriage effect, at least with moderately polar and small molecules.