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.

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.

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.

Differential effects of quinidine on the disposition of nifedipine, sparteine, and mephenytoin in humans.

The effects of quinidine on oxidative routes of drug metabolism mediated by different forms of cytochrome P450 were investigated in 10 healthy subjects. Each subject was studied on three different occasions and separately received oral administration of (1) a "cocktail" of nifedipine (5 mg), sparteine sulfate (90 mg), and mephenytoin (100 mg), (2) quinidine sulfate (200 mg), and (3) quinidine sulfate followed by the "cocktail" 1 hour later. Quinidine pretreatment significantly inhibited the aromatization of nifedipine to its major first-pass pyridine metabolite (M-0) and prolonged the elimination half-life of the calcium channel antagonist, both by about 40%. More marked inhibition of metabolism was observed with sparteine, and the formation of dehydrosparteine was abolished. A significant correlation was found between the 0-8-hour urinary ratio and the plasma concentration ratio of sparteine to dehydrosparteine obtained 4 hours after drug administration. No quinidine-induced changes were observed in the 4-hydroxylation of mephenytoin. The interaction between quinidine and nifedipine supports the involvement of a common P450 (P450IIIA4) in the metabolism of the two drugs.

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 (Cin) phenol concentration of 1 microgram/mL in controls, 52% was metabolized to sulphate conjugate and 37% to glucuronide conjugate at steady state. At this Cin, MI had no effect on phenol clearance (CL) (control: 9.63 +/- 0.38 vs MI: 9.65 +/- 0.36 mL/min; P greater than 0.05) or on the formation clearance (CLm) of the glucuronide or sulphate conjugates of phenol. When phenol Cin was increased 10-fold to 10 micrograms/mL, 6% was metabolized to sulphate conjugate and 94% to glucuronide conjugate. At this Cin phenol CL was decreased significantly (control: 9.44 +/- 0.46 vs MI: 7.09 +/- 1.51 mL/min; P less than 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 CLm of the glucuronide conjugate (control: 8.88 +/- 0.96 vs 5.98 +/- 1.87 mL/min; P less than 0.05), whereas the CLm of the sulphate conjugate was unchanged. There was a negative correlation between phenol glucuronide CLm and the severity of the erythrocytic parasitaemia (r2 = 0.75, P less than 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.

The effect of hypoxia and acidosis on propranolol clearance in the isolated perfused rat liver preparation.

The effect of hypoxia and acidosis on the elimination of an oxidatively metabolized drug, S-propranolol, was examined in the single-pass isolated perfused rat liver (IPRL). The experiments (N = 6) consisted of four consecutive 30 min phases: normal pH (pH 7.4)/normal oxygen delivery, normal pH/hypoxia, hypercapnic acidosis (pH 7.1)/normal oxygenation and hypercapnic acidosis/hypoxia. Hypoxia and acidosis were produced by equilibrating the perfusate with appropriate mixtures of O2, N2 and CO2. With normal oxygen delivery there was no difference in hepatic clearance of propranolol between normal pH and acidosis (9.65 +/- 0.34 and 9.78 +/- 0.11 mL/min, respectively. P < 0.05). During hypoxia, propranolol clearance was impaired to a similar extent under both pH conditions (7.41 +/- 0.97 and 8.06 +/- 0.81 mL/min, respectively, P > 0.05). Therefore, respiratory acidosis does not affect the clearance of propranolol by the IPRL, nor does it influence the sensitivity of propranolol clearance to hypoxia. Neither acidosis nor hypoxia resulted in a significant reduction in bile flow compared with the normal pH/normal oxygen phase and there was no correlation between bile flow and perfusate bicarbonate concentration (P > 0.05).

Potent inhibition of yeast-expressed CYP2D6 by dihydroquinidine, quinidine, and its metabolites.

The inhibitory effects of dihydroquinidine, quinidine and several quinidine metabolites on cytochrome P450 2D6 (CYP2D6) activity were examined. CYP2D6 heterologously expressed in yeast cells O-demethylated dextromethorphan with a mean Km of 5.4 microM and a Vmax of 0.47 nmol/min/nmol. Quinidine and dihydroquinidine both potently inhibited CYP2D6 metabolic activity (mean Ki = 0.027 and 0.013 microM, respectively) in yeast microsomes and in human liver microsomes. The metabolites, 3-hydroxyquinidine, O-desmethylquinidine and quinidine N-oxide also inhibited CYP2D6, but their Ki values (0.43 to 2.3 microM) were one to two orders of magnitude weaker than the values for quinidine and dihydroquinidine. There was a trend towards an inverse relationship between Ki and lipophilicity (r = -0.90, N = 5, P = 0.07), as determined by the retention-time parameter k' using reverse-phase HPLC. Thus, although the metabolites of quinidine have the capacity to inhibit CYP2D6 activity, quinidine and the impurity dihydroquinidine are the important inhibitors of CYP2D6.

Hepatic uptake and excretion of [14C]sodium taurocholate by the isolated perfused fetal sheep liver.

We have developed an in situ isolated perfused fetal sheep liver preparation to study fetal hepatic function free from the confounding influences of the mother and other fetal organs, and we have used the preparation to study the fetal hepatic clearance and biliary excretion of sodium taurocholate (TC). The viability and stability of this model were established by monitoring perfusion pressure, oxygen consumption, perfusate enzymes and electrolytes, the perfusate concentration ratio of lactate to pyruvate, bile flow, and liver histology. Perfusate delivery was 300 mL/min with a mean value of 3.94 mL/min/g liver (range: 2.46-6.72 mL/min/g liver). Gadolinium radiolabeled 15 microns microspheres were used to quantify the ductus venosus shunt through the liver and to determine relative flow rates between right and left hepatic lobes. TC was added to the reservoir either as a [14C]TC tracer bolus dose (2 microCi, N = 5) followed by a constant infusion of unlabeled TC, or as an initial bolus of [14]TC (54 mumol) followed by a [14C]TC constant infusion (30 mumol/hr, specific activity 30 microCi/mmol; N = 3). Perfusate samples were taken from the reservoir every 15 min and bile was collected in 30 min aliquots. Perfusion pressure (7.9 +/ 0.30 mmHg), perfusate potassium and oxygen consumption (0.9 +/- 0.07 mumol/min/g liver) were constant throughout, and the perfusate lactate/pyruvate concentration ratio was low (< 20). Liver histology showed no hypoxic changes. Bile flow fell slightly over the 150 min experiment time from 0.6 to 0.5 muL/min/g liver. These data indicate preparation viability and stability. The extent of the ductus venosus shunt was 16-66% (mean 35 +/- 6%) of umbilical vein flow, which correlated inversely with fetal gestational age (r = 0.94, P < 0.001). Relative flow to right and left lobes of liver was 1:1.4. In bolus dose experiments, TC t1/2 was 81.6 +/- 26 min, clearance (Cl) was 35.0 +/- 22.6 mL/min, shunt corrected extraction (E*) was 0.29 +/- 0.17 and biliary clearance (ClB) was 35.5 +/- 19.5 mL/min. In constant infusion experiments the corresponding results were Cl: 34.7 +/- 18.2, E*: 0.23 +/- 0.16, and ClB 32.7 +/- 17.7. The cumulative biliary excretion of [14C]TC in bolus dose experiments was 86.5 +/- 8.7% of the dose, and in constant infusion experiments, concentration of TC in bile was on average over 800 times that in plasma.(ABSTRACT TRUNCATED AT 400 WORDS)

The effect of hypoxia on propranolol clearance during antegrade and retrograde flow in the isolated perfused rat liver preparation.

We investigated, using the single-pass isolated perfused rat liver preparation, whether the centrilobular location of hepatic oxidative drug metabolism could be a contributing factor to the marked sensitivity of drug oxidation to hypoxia. Livers (N = 7) were each perfused for 130 min with 2 micrograms/mL (+)-propranolol, a drug metabolized almost entirely by oxidation in the rat. The direction of flow was reversed after 60 min, the order of flow direction being randomized. Normal oxygenation was used during the first 30 min of antegrade and of retrograde perfusion, but in the second 30 min perfusate was equilibrated with a N2/O2 mixture designed to reduce hepatic oxygen delivery by half. During normal oxygenation there was no significant difference between antegrade and retrograde perfusion in hepatic oxygen delivery and physiological parameters such as oxygen consumption and extraction, perfusion pressure and bile flow. During hypoxia, mean oxygen delivery was slightly lower with retrograde perfusion (retrograde: mean = 2.37 mumol/min/g liver, range = 1.56-3.17; antegrade: mean = 2.90 mumol/min/g liver, range = 1.96-4.08; P = 0.04), but there was no significant difference in physiological parameters within each liver (P > 0.05). Propranolol clearance during normal oxygenation was similar to the perfusion rate (10 mL/min) and was the same for both directions of perfusion (antegrade 9.88 +/- 0.07 mL/min, retrograde 9.88 +/- 0.13 mL/min, P > 0.05). Hypoxia reduced propranolol clearance substantially, but the decrease was significantly greater with antegrade perfusion (5.65 +/- 1.89 mL/min) than with retrograde perfusion (6.76 +/- 1.95 mL/min, P = 0.014). Oxidative drug metabolism is located primarily in the centrilobular zone and sinusoidal oxygen concentration is lowest in the "downstream" zone with both antegrade and retrograde perfusion. These findings suggest that the centrilobular location of propranolol metabolism may influence the effect of hypoxia on propranolol elimination, but is not a major contributor to the marked sensitivity of propranolol elimination to hypoxia antegrade perfusion.

Transfer of acipimox across the isolated perfused human placenta.

The placental transfer of the new lipid-lowering agent, acipimox was investigated in the isolated perfused human placenta. Placentas obtained at caesarean section were perfused for 120 min, with both maternal and fetal circuits in closed recycling mode. Acipimox was added to either the maternal circuit alone (five experiments) or to both maternal and fetal circuits simultaneously (five experiments) to achieve initial concentrations of 5 micrograms/ml. Antipyrine (20 micrograms/ml) and l-(14C)-leucine (250 microM) were added in like fashion as reference compounds. Two hours after addition to the maternal circuit alone antipyrine was close to equilibrium across the placenta, but equilibration of acipimox was incomplete (fetal/maternal ratio = 0.58 +/- 0.11). Maternal to fetal placental clearance of acipimox (0.80 +/- 0.18 ml/min) was 25 per cent of antipyrine clearance. After simultaneous administration to both maternal and fetal circuits the l-(14C)-leucine fetal/maternal ratio was 1.44 +/- 0.13 at 120 min, whereas maternal and fetal concentrations of acipimox and antipyrine were at equilibrium for the duration of the experiment (fetal/maternal ratio of acipimox at 120 min = 1.10 +/- 0.06). This study shows that acipimox is transferred across the human placenta by diffusion at a slow rate. The low permeability of the placenta may afford some protection to the fetus from acipimox administered to the mother in vivo.

Determinants of placental drug transfer: studies in the isolated perfused human placenta.

There is little information on the effects of maternal and fetal placental blood flow rates, which can change independently, on the placental transfer rate of drugs of different placental permeabilities. We examined the effects of varying maternal and fetal perfusion flow rates on the placental transfer of three model compounds; antipyrine (high permeability), diclofenac (intermediate permeability), and cimetidine (low permeability) in the single-pass, dual-perfused lobule of the isolated human placenta. In variable flow ratio experiments (n = 9) fetal perfusate flow rate was held constant while a different maternal flow rate was used in each of five 25-min phases such that the maternal/fetal flow ratio ranged from 0.16 to 3.3. In constant flow ratio experiments (n = 4), the flow ratio was kept at 2.0, while maternal and fetal flow rates were varied from 4-18 and 2-9 mL/min, respectively. In the variable flow ratio experiments, the fetal transfer fraction (fetal venous/maternal arterial drug concentrations) varied approximately fivefold among the five phases for each of the three drugs. Therefore, placental transfer was flow dependent regardless of placental drug permeability. By contrast, in the constant flow ratio experiments, fetal transfer fraction was unchanged throughout the five phases for each of the three drugs. Of the various kinetic models that have been formulated to account for the different possible vessel geometries, the double pool flow model, which is a venous equilibrium model and predicts the least efficient drug transfer rate of those proposed, together with a small maternal arteriovenous shunt, produced the best fit overall.(ABSTRACT TRUNCATED AT 250 WORDS)

Product inhibition and dose-dependent bioavailability of propranolol in the isolated perfused rat liver preparation.

We investigated in the isolated perfused rat liver (IPRL) whether product inhibition of metabolism contributes to the dose-dependent bioavailability of propranolol, a drug with a high, but saturable, hepatic first-pass effect. (+/-)-Propranolol was infused in the IPRL, using a recirculating design, for three 36-min periods (n = 9). Mean steady-state reservoir, i.e. hepatic inflow concentrations (Cin), were 4.97, 10.4, and 20.4 microM, respectively. Mean reservoir concentrations of the metabolites 4'-hydroxypropranolol, 5'-hydroxypropranolol, N-desisopropylpropranolol, and naphthoxylactic acid (NLA), a major side-chain-oxidation metabolite, increased disproportionately with propranolol dose, but their production rate did not reach steady state. In separate experiments (n = 4), perfusate containing 7.1, 12.8, and 21.6 microM (+/-)-propranolol, corresponding to administration rates of 114, 205, and 346 nmol/min, respectively, was passed through the liver for 30 min each using a single-pass design. The bioavailability (hepatic outflow concentration/Cin) of propranolol increased with Cin from 0.012 to 0.150 to 0.288 in the recirculating IPRL. In the single-pass IPRL the increase (0.0077 in 0.0669 to 0.136) was significantly less (P < 0.001). The greater bioavailability of propranolol in recirculating experiments was attributed to product inhibition since metabolites do not accumulate with the single-pass design. NLA did not appear to be the inhibiting metabolite because in further single-pass experiments with propranolol Cin of 21.6 microM the presence of NLA (21.6 microM) in perfusate had no effect on propranolol bioavailability (n = 7) compared with control experiments (n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Disposition of the diastereoisomers quinine and quinidine in the ovine fetus.

The disposition of the diastereoisomers quinine and quinidine was investigated in the near-term pregnant ewe. Five sheep were administered quinine and quinidine separately in random order by a combination of bolus and 30-h iv infusion. On a subsequent occasion, four of the five sheep were also administered the two drugs simultaneously. After separate dosage, systemic clearance of quinine tended to be greater than that of quinidine (714 +/- 299 versus 422 +/- 146 mL/min, p = 0.08). Maternal renal clearance exhibited no stereoselectivity and represented less than 2% of total clearance. Simultaneous administration did not alter the disposition of either drug in the mother. After separate dosage, fetal total concentrations (Cf) of quinine and quinidine were substantially lower than maternal total concentrations, as reflected in Cf:Cm ratios of 0.15 +/- 0.06 versus 0.10 +/- 0.08, respectively. Similarly, fetal unbound concentrations (Cfu) were substantially lower than maternal unbound concentrations (Cmu; Cfu/Cmu = 0.46 +/- 0.09 for quinine and 0.23 +/- 0.09 for quinidine). This indicates the presence of fetal elimination of both isomers. Fetal renal clearances of quinine and quinidine were similar (0.34 +/- 0.24 mL/min versus 0.38 +/- 0.24 mL/min) and less than that of endogenous creatinine, indicating the absence of net renal tubular secretion. After simultaneous dosage of quinine and quinidine, Cf:Cm (0.48 +/- 0.24 and 0.31 +/- 0.19, respectively) and Cfu:Cmu (0.73 +/- 0.14 and 0.52 +/- 0.20, respectively) were greater than for separate dosages. Fetal renal clearance of both drugs was unchanged, suggesting that the higher Cfu:Cmu ratios after simultaneous dosage were due to mutual inhibition of the fetal metabolism of these drugs.

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.

Studies on the mechanisms of action of activated charcoal on theophylline pharmacokinetics.

Oral administration of repeated doses of activated charcoal to volunteers and dogs significantly increased the systemic clearance of intravenously administered theophylline and decreased its elimination half-life. This effect is most likely to be due to theophylline entering the gut and being adsorbed onto the charcoal. The mechanism by which intravenously administered theophylline enters the gut has been examined. Its biliary excretion after intravenous administration to patients with T-tube biliary drainage accounted for 0.28% of the dose and a similarly small biliary excretion was found in dogs. In the latter total biliary diversion had no effect on the clearance or half-life of theophylline after intravenous administration. In two dogs the theophylline content of jejunal aspirate was comparable with that of simultaneously withdrawn venous plasma samples. These results suggest that the presence of charcoal in the gut represents a sink adsorbing theophylline entering the lumen by diffusion across the intestinal wall, and by this mechanism it increases clearance of the drug even after intravenous administration.

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.

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.