Prediction of hepatic clearance from microsomes, hepatocytes, and liver slices.
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Biomedical subjects
J B Houston
Publications and source records attributed to J B Houston.
Kinetics of drug metabolism in rat liver slices. III. Relationship between metabolic clearance and slice uptake rate.
The time course for distribution of five compounds (caffeine, tolbutamide, phenytoin, ondansetron, and diazepam) was studied in precision-cut rat liver slices. Transport of these compounds differed greatly, with caffeine being distributed rapidly, but not accumulating above the media concentration. Although tolbutamide similarly was not accumulated within the tissue, its uptake rate was slower. The rate of phenytoin, ondansetron, and diazepam distribution (with appropriate corrections for metabolism) was slower still; yet, these drugs were accumulated within the cells of the slice to a concentration approximately 15-fold that of the media. Examination of the physicochemical properties of these compounds demonstrated that the extent of accumulation positively correlated with lipophilicity, whereas the rate of uptake was not statistically correlated with log D. The extent of accumulation within the slice was assessed by an apparent volume of distribution parameter (ranging from 26 to 195 microliters/slice). Using cell:media partition coefficients determined in hepatocytes and the intra- and extracellular spaces within the slice (as measured with the markers tritiated water and sucrose), it was possible to predict apparent volumes of distribution for each drug in the liver slice. Comparison of observed and predicted apparent volumes of distribution gave ratios of 0.34-1. Intrinsic clearance values for these five drugs are available for slices and cells (slice: cell intrinsic clearance ratios 0.05-0.43; Worboys et al., Drug Metab. Dispos. 24, 676-681, 1996). Drugs that demonstrate low intrinsic clearance ratios also have low apparent volume ratios, thus indicating that reduced drug uptake and clearance in slices, relative to hepatocytes, are interdependent. Both phenomena may be rationalized by the existence of a drug concentration gradient within the slice. At very high drug concentrations, Vmax operates, and the consequence of the gradient is minimal. Therefore, it is possible to speculate upon the fraction of hepatocytes within the slice contributing to clearance by considering Vmax values. For six pathways of metabolism, Vmax in slices averages 35% of the corresponding parameter in isolated hepatocytes. This is most likely due to limited oxygen and compromised metabolic function of the core cells. These distribution phenomena severely complicate the possibility of using a scaling factor based on the theoretical number of slices obtainable from a liver to predict in vivo intrinsic clearance.
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Scaling factors to relate drug metabolic clearance in hepatic microsomes, isolated hepatocytes, and the intact liver: studies with induced livers involving diazepam.
Microsomal protein recovery and hepatocellularity have been determined and investigated as scaling factors for interrelating clearance by hepatic microsomes, freshly isolated hepatocytes and whole liver from untreated (UT) rats and rats treated with either the cytochrome P450 inducer phenobarbital (PB) or dexamethasone (DEX). Hepatocellularity in UT rats (1.1 x 10(8) hepatocytes/g liver) was not significantly different after either PB or DEX induction (1.1 and 1.3 x 10(8) hepatocytes/g liver, respectively). However the microsomal protein recovery index, which provides a scaling factor that is inversely related to the efficiency of the microsomal preparation procedure, was 47 mg/g liver in both PB and DEX microsomes and differs from UT rats (60 mg/g liver). These contrasting findings are consistent with the interlaboratory trends in the literature, indicating that, although hepatocellularity estimates are in good accord, microsomal recovery can vary 2-fold; this has implications for scaling. The oxidation of diazepam to its three primary metabolites was measured in PB and DEX microsomes and hepatocytes and the scaling factors were applied to these data and previously reported UT data. Marked changes in kinetics occur on induction resulting in a shift in the major pathway. In particular, 3-hydroxylation is induced over 20-fold by DEX. Diazepam CL(int) was determined in vivo after administration of a bolus dose into the hepatic portal vein of UT, PB, and DEX rats; values of 127, 191, and 323 ml/min/SRW (where SRW is a standard rat weight of 250 g), respectively, were obtained. Using these scaling factors, the hepatocyte predictions of CL(int) were excellent (99, 144, and 297 ml/min/SRW for UT, PB, and DEX, respectively), whereas only the DEX prediction (248 ml/min/SRW) was accurate for the microsomal system, with a substantial underprediction for UT and PB (46 and 68 ml/min/SRW, respectively). Evidence is presented for product inhibition, resulting from accumulation of primary metabolites within the microsomal preparation, as the mechanism responsible for this underprediction. These results illustrate that the scaling factor approach is applicable to induced livers in which both cytochrome P450 complement and zonal distribution are altered. These data, together with our previous studies, demonstrate that CL(int) in cells (2.4-297 ml/min/SRW), microsomes (2.7-248 ml/min/SRW), and in vivo (1.5-323 ml/min/SRW) are related in a linear fashion and hence inherently both in vitro systems are of equal value in predicting in vivo CL(int).
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Diazepam-omeprazole inhibition interaction: an in vitro investigation using human liver microsomes.
1. The metabolism of diazepam to its primary metabolites 3-hydroxydiazepam (3HDZ) and nordiazepam (NDZ) was evaluated in human liver microsomes. The 3HDZ pathway was the major route of metabolism representing 90% of total metabolism with a Vmax/Km ratio of 0.50-7.26 microliters min-1 mg-1 protein. 2. Inhibition of the two metabolic pathways of diazepam by omeprazole was investigated. The NDZ pathway was not affected by omeprazole whilst a Ki of 201 +/- 89 microM was obtained for the 3HDZ pathway (Km/Ki ratio of 3.0 +/- 0.9). 3. Inhibitory effects of omeprazole sulphone on the 3HDZ and NDZ pathways were also investigated. Omeprazole sulphone inhibited both pathways with similar Kis of 121 +/- 45 and 188 +/- 73 microM respectively (Km/Ki ratios of 5.2 +/- 2.3 and 3.3 +/- 1.5 respectively). 4. These in vitro data provide direct evidence for cytochrome P450 inhibition as the mechanism for the well documented diazepam-omeprazole clinical interaction and indicate that omeprazole sulphone, as well as the parent drug, contribute to the inhibition effect.
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Metabolite kinetics of ondansetron in rat. Comparison of hepatic microsomes, isolated hepatocytes and liver slices, with in vivo disposition.
1. The kinetics of hydroxylation and N-demethylation of ondansetron have been determined in freshly isolated hepatocytes, hepatic microsomes and precision-cut liver slices from the male Sprague-Dawley rat. In vivo studies have also been carried out to characterize the pharmacokinetics of ondansetron and in vitro data have been assessed for their value as predictors of hepatic clearance. 2. In the three in vitro systems, the formation of hydroxylated and demethylated metabolites were characterized as a function of substrate concentration by a high-affinity, low-capacity site and a low-affinity, high-capacity site which was not saturated over the concentration range studied (2.5-500 microM). Slices gave consistently higher Km's (20 and 30 microM for hydroxylation and demethylation respectively) than hepatocytes (3 and 13 microM respectively) and microsomes (2 and 5 microM respectively.) The rank order of Vmax and CL(int) was the same for each system; hydroxylation rates exceeding demethylation rates. Although two hydroxylations (7- and 8-hydroxy metabolites) occurred exclusively in microsomes, these are believed to originate from a common precursor. 3. The high CL(int) of ondansetron (150 ml/min/SRW, where SRW is a standard rat weight of 250g) is well predicted by scaling either microsomal clearance for microsomal protein recovery or hepatocyte clearance for hepatocellularity (212 and 135 ml/min/SRW respectively). In contrast, the use of liver slice data scaled to a whole liver substantially underestimates CL(int) (9 ml/min/SRW).
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Comparison of ketoconazole and fluconazole as cytochrome P450 inhibitors. Use of steady-state infusion approach to achieve plasma concentration-response relationships.
The ability of two azole antifungal agents, ketoconazole and fluconazole, to inhibit hepatic cytochrome P450 activity in vivo in the rat has been determined. To make a valid comparison, differences in pharmacokinetic properties between the azoles were accounted for by using an infusion approach to maintain steady-state plasma concentrations over a range of 1-48 mg/liter. Both compounds showed a maximum inhibitory effect, assessed by a reduction in antipyrine clearance, of approximately 75%. The relationship between steady-state plasma concentration and the degree of inhibition of antipyrine clearance was nonlinear for both azoles. However, the inhibitory effect resulted at lower concentrations for ketoconazole than for fluconazole. Analysis of these data provided Ki values of 3 and 10 microM, for ketoconazole and fluconazole, respectively, based on plasma concentration of azole. This difference in activity is 2 orders of magnitude greater when Ki values are expressed in terms of unbound concentration in the blood, which may be more representative of hepatic tissue concentrations. Ki values based on unbound drug concentration are 0.07 and 8.7 microM for ketoconazole and fluconazole, respectively. These data confirm the conclusions based on in vitro findings that ketoconazole is a more inhibitory of mammalian cytochrome P450 isoenzymes than fluconazole.
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Kinetics of drug metabolism in rat liver slices. II. Comparison of clearance by liver slices and freshly isolated hepatocytes.
The kinetics of metabolism of diazepam, phenytoin, and caffeine have been determined in rat liver slices of 260 microns thickness. The formation of 4'-hydroxy metabolites of diazepam and phenytoin are described by one- and two-site Michaelis-Menten equations, respectively. The other oxidative pathways for diazepam are less readily saturable than the 4'-hydroxylation. This kinetic behavior is consistent with that previously reported for other in vitro systems. In contrast, the metabolism of caffeine, assessed by total metabolism, showed differences from that observed with other systems, and this is believed to result from the incubation conditions used. By determining the hepatocellularity of the standard slice used, CLint (Vmax/KM) data were expressed per million cells and compared with the same parameters derived from incubations with freshly isolated hepatocyte suspensions. Data on diazepam, phenytoin, and caffeine were combined with previously published data on tolbutamide, ethoxycoumarin, and ondansetron to give a total of nine CLint values representing different pathways. CLint values in slices are consistently less than those in hepatocytes (ratio differing from 0.4 to 0.05). The CLint ratio decreased in a regular fashion as the hepatocyte CLint increased from 1.4 microliters/min (caffeine) to 105 microliters/min (ondansetron). It was also observed that the KM value for a particular pathway in slices always exceeded the corresponding value in isolated hepatocytes (ratios differing from 1.09 to 7.59). The KM ratio was positively related to hepatocyte CLint. These observations are consistent with the delayed accessibility of substrate to all the cells within a slice. It is proposed that the parallel processes of drug transport and metabolism within the slice do not allow a distribution equilibrium to be achieved between all the cells within a slice and the incubation media.
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Assessment of intestinal permeability changes induced by nonsteroidal anti-inflammatory drugs in the rat.
Intestinal permeability was investigated as an alternative to intestinal ulceration for measuring nonsteroidal anti-inflammatory drug (NSAID) gut damage in the rat and developed as a method for routine measurement. NSAID dose-response curves produced using the two indices of damage showed that intestinal permeability is as sensitive and reproducible as ulceration, although changes could not be detected before visible ulceration occurred. Lactulose, [51Cr]-EDTA and [14C]-carboxyinulin were compared as possible in vivo markers of rat intestinal permeability. Measurement of [51Cr]-EDTA permeation was found to be the most sensitive and reproducible method. Dose-response curves produced by measuring [51Cr]-EDTA permeation were used to compare the potency of the two NSAIDs piroxicam and (S+) ibuprofen; piroxicam was found to be 10 times more potent in increasing intestinal permeability than (S+)-ibuprofen. These studies show that intestinal permeability measurement is a useful alternative to other methods of assessing NSAID adverse effect and is easily and rapidly performed.
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Regional drug delivery II: relationship between drug targeting index and pharmacokinetic parameters for three non-steroidal anti-inflammatory drugs using the rat air pouch model of inflammation.
PURPOSE: To quantify the advantage gained by direct administration to a target site for two non-steroidal anti-inflammatory drugs (NSAIDs) piroxicam and diclofenac in the rat air pouch model of inflammation. To derive a model relating drug targeting index (DTI) to the pharmacokinetic parameters of the target and systemic sites, and to compare predictions with observations. METHODS: DTI was calculated based on area under the concentration time curve at target (pouch) and systemic site (venous blood) following administration into and sampling from both sites. A model was derived relating DTI to systemic clearance, target permeability, plasma protein binding and fraction of the targeted dose that is systemically available. RESULTS: Both NSAIDs exhibited linear pharmacokinetics over the dose ranges studies. They differed primarily in total body clearance which was approximately 16 fold greater for diclofenac (213 ml hr-1 per 250 g) than piroxicam (13 ml hr-1 per 250 g). Observed DTIs (11, 114 and 276 for piroxicam, S[+]ibuprofen [studied previously] and diclofenac) were ranked in order of total body clearance but were approximately 7.5 fold lower than predicted (101, 700 and 2214 respectively). CONCLUSIONS: The discrepancy was explained by the influx of the plasma binding protein, albumin, into the target site due to increased vascular permeability associated with the inflammatory response. The originally derived equation for DTI, which assumed only unbound drug diffuses across the target site, was modified to take into account the simultaneous flux of bound drug.
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Effect of omeprazole on diazepam disposition in the rat: in vitro and in vivo studies.
PURPOSE: The inhibitory effects of omeprazole on diazepam metabolism in vitro and in vivo are compared in the rat. METHODS: 3-hydroxylation and N-demethylation of diazepam was investigated in the presence of a range of omeprazole concentrations (2-500 microM) in hepatic microsomes and hepatocytes. Zero order infusions together with matched bolus doses of omeprazole were used to achieve a range of steady state plasma concentrations (10-50mg/L) and to study the diazepam-omeprazole interaction in vivo. RESULTS: The 3-hydroxlation pathway was more prone to inhibition (KIs 108 +/- 30 and 28 +/- 11 microM in microsomes and hepatocytes, respectively) than the demethylation pathway (KIs of 226 +/- 76 and 59 +/- 27 microM in microsomes and hepatocytes, respectively). In both in vitro systems, the mechanism of inhibition was competitive with Km/KI ratios larger than 1 for the 3HDZ pathway and smaller than 1 for the NDZ pathway. There was an omeprazole concentration dependent decrease in diazepam clearance in vivo which could be modelled using a simple inhibition equation with a KI of 57 microM (19.8mg/L). In contrast there was no statistically significant change in the steady state volume of distribution for diazepam in the presence of omeprazole. CONCLUSIONS: The in vivo KI for the omeprazole: diazepam inhibition interaction shows closer agreement with the KI values obtained in hepatocytes than with those observed in microsomes.
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Regional drug delivery I: permeability characteristics of the rat 6-day-old air pouch model of inflammation.
PURPOSE: To determine the permeability characteristics of the rat air pouch model of inflammation using permeability extremes within which the NSAIDs S[+] ibuprofen, piroxicam and diclofenac could be evaluated. METHODS: Permeability was calculated using concentration data obtained following intrapouch and intravenous administration of [3H]-water, [14C]-urea, [14C]-inulin and [125I]-albumin and compared to similar data obtained for the three NSAIDs. RESULTS: Similar permeability values (5-6.5 ml hr-1) were obtained for the three NSAIDS which fell between the permeability extremes of the molecular weight markers [3H]-water (9.7 ml hr-1), [14C]-urea (6.8 ml hr-1), [14C]-inulin (1.0 ml hr-1) and [125I]-albumin (0.6 ml hr-1). Coadministration of equipotent anti-inflammatory doses of the NSAIDs did not affect local blood flow to the air pouch (as assessed by urea kinetics) but did reduced vascular permeability (as assessed by albumin flux into the pouch). CONCLUSIONS: Comparison of the NSAIDs with the permeabilities of the molecular weight markers indicates that a perfusion rate limitation probably exists. Systemic absorption is complete over the first two hours following intrapouch administration of the NSAIDs, therefore albumin flux into the pouch is insufficient to materially affect the permeability of the NSAIDs. However, subsequently (post 5hr) albumin concentration in the pouch rises sufficiently to lower the effective flux of the NSAIDs.
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Effect of norfloxacin on theophylline disposition: a comparison with other fluoroquinolones.
The effects of norfloxacin (NOR), at steady-state plasma concentrations of 0-32 mg.l-1, on the plasma clearance of a 6 mg.kg-1 i.v. bolus dose of theophylline (THEO) in the male Sprague-Dawley rat have been studied. The effects were characterised by a Ki value (Ki = 12 microM), which was comparable with Ki values obtained previously under identical conditions for ciprofloxacin, but higher than that obtained for enoxacin. The distributional characteristics, volume of distribution and liver to plasma concentration ratio, were very similar for the three compounds. The only marked pharmacokinetic differences were in hepatic clearance, where there was a rank order NOR > ciprofloxacin > enoxacin, a reverse of the order in the reduction of THEO clearance seen in clinical studies. The advantages of using the steady-state experimental design described here are that equivalent concentrations are utilised to compare related drugs and differences in pharmacokinetics are accounted for, to allow a direct comparison of potency. This information, together with additional pharmacokinetic considerations, suggests that the different effects on THEO clearance seen in the clinic for NOR, ciprofloxacin and enoxacin are not solely due to differences in inhibitory potency, but also involve differences in hepatic clearance and hence systemic availability of the fluoroquinolones.
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Pharmacodynamic comparison of regional drug delivery for non-steroidal anti-inflammatory drugs, using the rat air-pouch model of inflammation.
The inhibition of prostaglandin E2 (PGE2) synthesis by S-(+)-ibuprofen and piroxicam have been assessed following intravenous and regional (intrapouch) drug delivery using the rat air-pouch model of inflammation. Anti-inflammatory response was defined as the decrease in the area under the exudate PGE2 concentration-time curve between 3 and 10 h, following regional administration of the irritant carrageenan. Dose-response studies indicated that bolus regional administration of S-(+)-ibuprofen increased potency 30-fold compared with systemic administration and could be further improved 10-fold by regional infusion, whereas regional administration of piroxicam showed no therapeutic advantage. Examination of the concentration-response using AUC revealed that for a given response, average pouch concentrations for S-(+)-ibuprofen during the PGE2 inflammatory response (3 to 10 h) was similar, irrespective of route or mode of administration. In contrast, an advantage following systemic rather than regional administration was revealed for piroxicam, based on plasma concentration-response data, indicating a major systemic anti-inflammatory component for piroxicam but not for S-(+)-ibuprofen. These observations stress the need to take account of both pharmacodynamics and pharmacokinetics when considering the potential advantage of regional administration.
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Concentration-response relationships for three nonsteroidal anti-inflammatory drugs in the rat intestine.
1. The effect of diclofenac, piroxicam and (S+)-ibuprofen upon the rat intestine has been measured at constant drug plasma concentrations in the rat, using (51Cr)-EDTA intestinal permeation as a measure of damage. Initially disposition studies after sc administration of the three NSAIDs were carried out. From these studies it was found that constant-rate iv infusions were necessary to maintain plasma concentrations of diclofenac and (S+)-ibuprofen. Administration of piroxicam by sc bolus gave relatively constant plasma concentrations, thus iv infusions were not necessary to obtain concentration-response data for this drug. Relative potency was found by comparing the concentration-response profiles of the three NSAIDs and the rank order of potency obtained was: diclofenac > piroxicam > (S+)-ibuprofen. 2. The effect of mode of administration upon intestinal damage was also investigated using diclofenac. Intestinal permeability was measured in rats given diclofenac either by sc bolus or iv infusion and dose-response data compared. It was found that for the same dose, administration by sc bolus gave a higher degree of damage than by iv infusion.
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Kinetics of diazepam metabolism in rat hepatic microsomes and hepatocytes and their use in predicting in vivo hepatic clearance.
1. The rates of diazepam (DZ) metabolism to the primary metabolites 3-hydroxydiazepam, 4'-hydroxydiazepam and nordiazepam were studied in vitro using rat hepatic microsomes and hepatocytes. 4'-hydroxydiazepam had the largest intrinsic clearance (Vmax/Km ratio, CL(int)) in both microsomes and hepatocytes representing 49 and 70% of total metabolism respectively. Whereas the contribution of 3-hydroxydiazepam was similar in both systems (21-24%), the N-demethylation pathway was greater in microsomes (27%) than hepatocytes (9%). 2. The pharmacokinetics of DZ were determined in vivo using the intraportal route to avoid blood flow limitations due to the high clearance of DZ. No dose dependency was observed in either clearance or steady state volume of distribution, which were estimated to be 38 ml/min/SRW (where SRW is a standard rat weight of 250 g) and 1.3 L/SRW respectively. Blood binding of DZ was concentration independent, the unbound fraction being 0.22. 3. Scaling factors were used to relate the in vitro CL(int) to the in vivo unbound clearance. Hepatocytes (123 ml/min/SRW) produced a more realistic prediction for the in vivo value (174 ml/min/SRW) than microsomes (41 ml/min/SRW). This situation is believed to arise from the quantitative differences in the three metabolic pathways in the two in vitro systems. It is speculated that end product inhibition is responsible for reduced total metabolism in microsomes whereas hepatocytes operate kinetically in a manner close to in vivo.
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Metabolism of theophylline and its inhibition by fluoroquinolones in rat hepatic microsomes.
1. The effects of beta-naphthoflavone, dexamethasone, phenobarbitone and isosafrole on the metabolism of theophylline by rat liver microsomes have been studied. Only beta-naphthoflavone, a known P4501A inducer, increased the rate of 1-methylxanthine formation (3-fold), whereas all the inducers studied increased the rate of 1,3-dimethyluric acid production (2.5-3-fold). 2. To study the effects of a range of fluoroquinolones on theophylline metabolism, beta-naphthoflavone-induced microsomes were used, as the ratio for metabolite production rates was similar to that of untreated microsomes (4:1,3-dimethyluric acid: 1-methylxanthine at 2 mM theophylline). High concentrations of fluoroquinolones (0.5-1.5 mM) were required to affect microsomal theophylline metabolism. 1-Methylxanthine was more sensitive to fluoroquinolone inhibition by enoxacin, ciprofloxacin, norfloxacin and pipemidic acid than 1,3-dimethyluric acid; CP67015, had a significant effect on 1,3-dimethyluric acid production only; binfloxacin had no effect on either pathway. 3. Ethoxycoumarin, a rapidly metabolized substrate, was also investigated as a surrogate for theophylline in in vitro experiments. Fluoroquinolone inhibition of ethoxycoumarin O-de-ethylation in beta-naphthoflavone-induced microsomes was quantitatively greater but qualitatively similar to theophylline metabolism (IC50s 440-870 microM at 2 microM 7-ethoxycoumarin). 4. These data are comparable with previous rat experiments in vivo, indicating that enoxacin, ciprofloxacin and norfloxacin have similar intrinsic activity in the inhibition of theophylline metabolism.
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In vivo disposition of caffeine predicted from hepatic microsomal and hepatocyte data.
The kinetics of caffeine metabolism has been investigated in freshly isolated hepatocytes, hepatic microsomes, and in vivo in male Sprague-Dawley rats. A simple Michaelis-Menten model provides an adequate description of each of the three sets of data. There is reasonable agreement between the KM values for the three systems (56-200 microM). Vmax values for hepatocytes and microsomes show good agreement when expressed in the same units using scaling factors for hepatic cellularity and microsomal protein yield [315 and 420 nmol/min/standard rat weight (SRW), respectively]. Both values slightly exceed the in vivo-determined Vmax (190 nmol/min/SRW). Taking the Vmax/KM ratio (intrinsic clearance) as the basis for scaling, the in vitro data from both the hepatocyte (2.6 ml/min/SRW) and microsomal (2.7 ml/min/SRW) studies provide a good prediction of the in vivo total body clearance (3.4 ml/min/SRW).
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Kinetics of drug metabolism in rat liver slices. Rates of oxidation of ethoxycoumarin and tolbutamide, examples of high- and low-clearance compounds.
Ethoxycoumarin (EC) and tolbutamide (TOL) were selected as examples of high- and low-clearance drugs, respectively, to investigate suitable methodologies for obtaining kinetic data on metabolism by precision-cut rat liver slices. A number of characteristics of the slice incubation were compound-dependent. TOL showed linear rates of metabolism over a longer time period than EC and was insensitive to the method of incubation (rotating vials and gyrating culture plates). Also, the need for complete tissue disruption (e.g. via sonication) before analysis was essential for EC but unimportant for TOL. This may suggest that conjugates do not freely diffuse out of the liver slice unlike oxidative metabolites. Both drugs showed rates of metabolism that were dependent on slice thickness (150-530 microns). A high turnover (intrinsic clearance 7.9 microliters/min) and low KM (1.3 microM) for EC, and a low turnover (intrinsic clearance 0.8 microliter/min) and high KM (707 microM) for TOL were determined in slices. These differences in kinetic behavior are comparable with those seen in hepatic microsomes, freshly isolated hepatocytes, and in vivo.
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