Impact of Plasma Protein Binding in Drug Clearance Prediction: A Data Base Analysis of Published Studies and Implications for In Vitro-In Vivo Extrapolation.

Plasma protein-mediated uptake (PMU) and its effect on clearance (CL) prediction have been studied in various formats; however, a comprehensive analysis of the overall impact of PMU on CL parameters from hepatocyte assays (routinely used for IVIVE) has not previously been performed. The following work collated data reflecting the effect of PMU for 26 compounds with a wide variety of physicochemical, drug, and in vivo CL properties. PMU enhanced the unbound intrinsic clearance in vitro (CLint,u in vitro) beyond that conventionally calculated using fraction unbound and was correlated with the unbound fraction of drug in vitro and in plasma (fup) and absolute unbound intrinsic clearance in vivo (CLint,u in vivo) in both rat and human hepatocytes. PMU appeared to be more important for highly bound (fup < 0.1) and high CLint,u in vivo drugs. These trends were independent of species, assay conditions, ionization, and extended clearance classification system group, although the type of plasma protein used in in vitro assays may require further investigation. Such generalized trends (spanning fup 0.0008-0.99) may suggest a generic mechanism behind PMU; however, multiple drug-dependent mechanisms are also possible. Using the identified relationship between the impact of PMU on CLint,u in vitro and fup, PMU-enhanced predictions of CLint,u in vivo were calculated for both transporter substrates and metabolically cleared drugs. PMU was accurately predicted, and incorporation of predicted PMU improved the IVIVE of hepatic CL, with an average fold error of 1.17 and >50% of compounds predicted within a 2-fold error for both rat and human data sets (n ≥ 100). SIGNIFICANCE STATEMENT: Current strategies for prediction of hepatic clearance from in vitro data are recognized to be inaccurate, but they do not account for PMU. The impact of PMU on CLint,u in vitro is wide ranging and can be predicted based on fraction unbound in plasma and applied to CLint,u in vitro values obtained by standard procedures in the absence of plasma protein. Such PMU-enhanced predictions improved IVIVE, and future studies may easily incorporate this PMU relationship to provide more accurate IVIVE.

Use of Segregated Hepatocyte Scaling Factors and Cross-Species Relationships to Resolve Clearance Dependence in the Prediction of Human Hepatic Clearance.

Human and rat hepatocytes have a strong tendency to underpredict hepatic intrinsic clearance (CLint) and the extent of underprediction increases with increasing observed CLint In this study, application of the log average rat hepatocyte-rat in vivo empirical scaling factor (ESF) of 4.2 to human hepatocyte prediction successfully removed bias but did not improve precision. An analogous method using individual drug rat ESFs only achieved marginal improvement in accuracy but not precision. A novel approach to resolve clearance-dependent prediction, involving rat ESFs calculated for particular (order of magnitude) ranges of observed CLint (log average range, 0.12-2.1) improved human prediction precision but only modestly reduced bias. However, rat in vivo CLint was several-fold greater than human in vivo CLint and this was reflected in greater rat hepatocyte and microsome CLint, suggesting that rat metabolic enzymes are more efficient than their human counterparts, by several-fold. By applying the segregated rat ESFs followed by the human/rat CLint ratio, which was consistent regardless of CLint (log average 3.5), both accuracy and precision were improved, providing both a means of mitigating clearance dependence and reaffirming the potential role of rat hepatocytes for prediction of human metabolic CLint These cross-species observations indicate that underprediction from human in vitro systems may be predominantly consequential of an intrinsic property of the in vitro system rather than individual drug properties.

Clearance Prediction Methodology Needs Fundamental Improvement: Trends Common to Rat and Human Hepatocytes/Microsomes and Implications for Experimental Methodology.

Although prediction of clearance using hepatocytes and liver microsomes has long played a decisive role in drug discovery, it is widely acknowledged that reliably accurate prediction is not yet achievable despite the predominance of hepatically cleared drugs. Physiologically mechanistic methodology tends to underpredict clearance by several fold, and empirical correction of this bias is confounded by imprecision across drugs. Understanding the causes of prediction uncertainty has been slow, possibly reflecting poor resolution of variables associated with donor source and experimental methods, particularly for the human situation. It has been reported that among published human hepatocyte predictions there was a tendency for underprediction to increase with increasing in vivo intrinsic clearance, suggesting an inherent limitation using this particular system. This implied an artifactual rate limitation in vitro, although preparative effects on cell stability and performance were not yet resolved from assay design limitations. Here, to resolve these issues further, we present an up-to-date and comprehensive examination of predictions from published rat as well as human studies (where n = 128 and 101 hepatocytes and n = 71 and 83 microsomes, respectively) to assess system performance more independently. We report a clear trend of increasing underprediction with increasing in vivo intrinsic clearance, which is similar both between species and between in vitro systems. Hence, prior concerns arising specifically from human in vitro systems may be unfounded and the focus of investigation in the future should be to minimize the potential in vitro assay limitations common to whole cells and subcellular fractions.

Methodological uncertainty in quantitative prediction of human hepatic clearance from in vitro experimental systems.

Mechanistic prediction of unbound drug clearance from human hepatic microsomes and hepatocytes correlates with in vivo clearance but is both systematically low (10 - 20 % of in vivo clearance) and highly variable, based on detailed assessments of published studies. Metabolic capacity (Vmax) of commercially available human hepatic microsomes and cryopreserved hepatocytes is log-normally distributed within wide (30 - 150-fold) ranges; Km is also log-normally distributed and effectively independent of Vmax, implying considerable variability in intrinsic clearance. Despite wide overlap, average capacity is 2 - 20-fold (dependent on P450 enzyme) greater in microsomes than hepatocytes, when both are normalised (scaled to whole liver). The in vitro ranges contrast with relatively narrow ranges of clearance among clinical studies. The high in vitro variation probably reflects unresolved phenotypical variability among liver donors and practicalities in processing of human liver into in vitro systems. A significant contribution from the latter is supported by evidence of low reproducibility (several fold) of activity in cryopreserved hepatocytes and microsomes prepared from the same cells, between separate occasions of thawing of cells from the same liver. The large uncertainty which exists in human hepatic in vitro systems appears to dominate the overall uncertainty of in vitro-in vivo extrapolation, including uncertainties within scaling, modelling and drug dependent effects. As such, any notion of quantitative prediction of clearance appears severely challenged.

Reliability of human cryopreserved hepatocytes and liver microsomes as in vitro systems to predict metabolic clearance.

A total of 110 drugs, selected to cover a range of physicochemical and pharmacokinetic properties, were used to explore standard approaches to the prediction of in vivo metabolic clearance using drug-depletion profiles from human liver microsomes (HLMs) and cyropreserved hepatocytes. A total of 41 drugs (37% of the compounds tested) showed measurable depletion rates using HLMs (depletion by 20% or more over the time course). The most reliable correlations in terms of bias (average fold error (AFE) = 2.32) and precision (root mean square error (RMSE) = 3501) were observed by comparing in vivo intrinsic clearance (CL(int)), calculated using the parallel-tube model and incorporating the fraction unbound in blood, with in vitro CL(int) adjusted for microsomal binding. For these reference drugs, 29% of predictions were within two-fold of the observed values and 66% were within five-fold. Compared with HLMs, clearance predictions with cryopreserved hepatocytes (57 drugs) were of similar precision (RMSE = 3608) but showed more bias (AFE = 5.21) with 18% of predictions within two-fold of the observed values and 46% within five-fold. However, with a broad complement of drug-metabolizing enzymes, hepatocytes catalysed measurable CL(int) values for a greater proportion (52%) of the reference compounds and were particularly proficient at defining metabolic rates for drugs with predominantly phase 2 metabolic routes.

Effect of activated charcoal on acetaminophen absorption.

Acetaminophen intoxication can cause hepatic, renal, and myocardial necrosis which is often fatal. These lesions develop very rapidly, perhaps during the first pass of the drug through the liver. In case of acute ingestion of an overdose it is therefore essential to employ measures for reducing the absorption of acetaminophen. The effect of activated charcoal on acetaminophen absorption by normal volunteers was determined as a function of the dose of charcoal, the dosage form of acetaminophen, and the charcoal-to-acetaminophen dose ratio. The results indicate that activated charcoal can be an effective antidote for acute acetaminophen intoxication, if administered promptly and in sufficient quantity.

Drug metabolizing capacity in vitro and in vivo--II. Correlations between hepatic microsomal monooxygenase markers in phenobarbital-induced rats.

Pretreatment with various doses of phenobarbital (PB) has been used to create a pool of rats with a wide range of hepatic microsomal monooxygenase activity to systematically examine relationships between and within in vivo and in vitro markers. The in vivo clearance of tolbutamide (TOL), theophylline (TH), antipyrine (AP) and its metabolites were determined in the same rats used for hepatic microsome preparation and assessment of P450 content and activities (via 7-ethoxycoumarin O-deethylase (ECOD), 7 ethoxyresorufin O-deethylase, 7-methoxycoumarin O-demethylase (MCOD) and aldrin epoxidase determinations). A graded dose-response relationship was found between PB treatment and most but not all parameters. The need for careful selection of in vivo and as well as in vitro markers is apparent from these studies. The most responsive parameters--TOL and AP clearances, MCOD and ECOD activities--were also those producing the strongest in vivo-in vitro correlations. Despite the diffuse nature of the PB induced response in P450 complement, good predictive relationships were apparent between ECOD and TOL clearance (r2 = 0.88).

Drug metabolizing capacity in vitro and in vivo--I. Correlations between hepatic microsomal monooxygenase markers in beta-naphthoflavone-induced rats.

Relationships between and within in vivo and in vitro markers of drug oxidative metabolism have been investigated in rats displaying a wide range of hepatic microsomal monooxygenase activity due to prior treatment with various doses of the inducing agent beta-naphthoflavone (BNF). BNF induction produced large dose-related changes in the in vivo clearance (CL) of theophylline (TH), antipyrine (AP) and the individual AP metabolite formation clearances, 4-hydroxyantipyrine (4H) and norantipyrine, and the in vitro parameters, 7-ethoxycoumarin O-deethylase, 7-ethoxyresorufin and P450. No trends were observed with the formation clearance of 3-hydroxymethylantipyrine and 7-methoxycoumarin O-demethylase whilst a negative response was observed with aldrin epoxidase. The selectivity of the markers towards BNF induction was coincident with the degree of covariance observed between these parameters. Strong correlations were observed in particular between CL(TH) and CL(4H) and ECOD and EROD indicating the high predictive value of these parameters. These studies demonstrate that under the well controlled conditions which may be imposed in animal environments predictively useful relationships (r2 greater than 0.8) can be established between in vitro and in vivo markers of hepatic microsomal monooxygenase activity.

Comparison of two azole antifungal drugs, ketoconazole, and fluconazole, as modifiers of rat hepatic monooxygenase activity.

The mechanism of action of azole antifungal agents is believed to involve inhibition of fungal cytochrome P-450, and, therefore, an investigation of the interaction of these drugs with mammalian cytochrome P-450 systems should provide some indication of their selectivity as antifungal agents. The ability of ketoconazole and fluconazole, the latter representing a new generation of triazole antifungal agents, to modify rat mixed function oxidase activity has been investigated in vitro with hepatic microsomes and in vivo using a N-methyl-[14C] antipyrine breath test. As a measure of selectivity the results have been compared with antifungal potency. Ketoconazole is more potent than fluconazole by an order of magnitude in inhibiting metabolism by O-dealkylation of ethoxycoumarin, methoxycoumarin and ethoxyresorufin (IC50 values of 6, 5 and 130 microM for ketoconazole respectively). The effects on the regio- and stereospecific hydroxylation of [14C] testosterone were also measured; the IC50 values for inhibition of total testosterone metabolism were 0.1 mM and greater than 3 mM for ketoconazole and fluconazole respectively. Marked selectivity differences were observed for the two drugs as indicated by ketoconazole being a potent inhibitor of 7 alpha-hydroxylation of testosterone (IC50 20 microM) while fluconazole did not inhibit this activity at 3 mM. In vivo investigations using a range of doses confirmed their ranking for inhibitory potency; the ED50 values for maximum demethylation rate were 17 mumol/kg and greater than 60 mumol/kg for ketoconazole and fluconazole respectively. Thus fluconazole has a lower propensity to interact with rat hepatic cytochrome P-450 and can be considered a more selective antifungal agent as its in vivo antifungal potency is an order of magnitude greater than ketoconazole.

In vitro human tissue models in risk assessment: report of a consensus-building workshop.

Advances in the technology of human cell and tissue culture and the increasing availability of human tissue for laboratory studies have led to the increased use of in vitro human tissue models in toxicology and pharmacodynamics studies and in quantitative modeling of metabolism, pharmacokinetic behavior, and transport. In recognition of the potential importance of such models in toxicological risk assessment, the Society of Toxicology sponsored a workshop to evaluate the current status of human cell and tissue models and to develop consensus recommendations on the use of such models to improve the scientific basis of risk assessment. This report summarizes the evaluation by invited experts and workshop attendees of the current status of such models for prediction of human metabolism and identification of drug-drug interactions, prediction of human toxicities, and quantitative modeling of pharmacokinetic and pharmaco-toxicodynamic behavior. Consensus recommendations for the application and improvement of current models are presented.

Multiple regression of predictors and criteria of dental school performance.

This study reports normative data for 370 students admitted to the UCLA School of Dentistry, data on commonly used performance predictors, and the regression equation best fitted for forecasting performance in this school. Most variance was associated with denatl school grade point average, National Board Dental Examination averages, and clinical performance. Best predictors were achievement level and trend in preadmission academic performance. The selection interview and high school preformance were also evaluated as predictor criteria.

Induction of cytochromes P-450 in pancreatic disease: consequence, coincidence or cause?

We have examined the pharmacokinetics of antipyrine and of theophylline--validated probes for cytochromes P-450 activities--in a series of patients with pancreatic disease. The half-life of each drug was significantly lower, and its clearance faster, in patients than in controls and this pattern was detected in the subgroups with acute pancreatitis (6), chronic pancreatitis (22), or pancreatic cancer (4). These data suggest induction of cytochromes P-450 in all forms of exocrine pancreatic disease. Enzyme induction is unlikely to be secondary to pancreatic malfunction since there was no correlation between prevailing exocrine status, as assessed by secretin-pancreozymin tests, and the half-life or clearance of either drug. The corollary is that induction of the mono-oxygenases by environmental agents, both recognised and unidentified, is a primary event in pancreatic disease. The possible relevance of this finding is discussed.

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.

The six-day-old rat air pouch model of inflammation: characterization of the inflammatory response to carrageenan.

Inflammation was induced in the 6-day-old rat air pouch by injection of carrageenan. The model was characterized in terms of exudate volume, leucocyte influx, cell free protein, prostaglandin E2 levels, and granuloma formation. The time course of all these inflammatory markers, except prostaglandin E2, showed a 3-hr lag followed by a rapid increase to 8 hr. Thereafter, the rate of increase was much slower to 48 hr. Differential cell counts indicated a predominantly polymorphonuclear cell response (75%) during the first 48 hr. Prostaglandin E2 levels increased rapidly after a 3-hr lag, to a maximum of 440 +/- 140 ng/mL at 15 hr and thereafter quickly declined to 140 +/- 60 ng/mL at 21 hr. Prostaglandin E2 levels were the most sensitive inflammatory marker to (S+)-ibuprofen and were reduced dose dependently in the range 0.05 to 1 mg/kg. We have demonstrated the time course for duration of NSAID-induced reduction of prostaglandin E2 levels during inflammation in an individual animal. Rac-ibuprofen (0.1-1 mg/kg) reduced leucocyte influx at 3 and 5 hr, after which drug effects gradually diminished by 24 hr. Rac-ibuprofen at 1 mg/kg significantly reduced the volume of air pouch exudate recovered at 24 hr but had no effect on protein levels.

Experimental determination of a drug targeting index for S(+)ibuprofen using the rat air pouch model of inflammation.

We have used the rat air pouch model of inflammation and S(+)ibuprofen as an experimental model system to enable the quantitative assessment of the pharmacokinetic determinants of site specific drug delivery. S(+)ibuprofen (50 & 1mg/kg) was administered directly into six day old air pouches immediately following the injection of the irritant carrageenan. Serial exudate and plasma samples were collected and analysed for ibuprofen by HPLC. The procedure was repeated following administration of S(+)ibuprofen (20 & 5mg/kg) intravenously. The parameters describing events in the air pouch and plasma indicated linear kinetics over the doses employed. The dose normalised AUCs were then used to formulate a quantitative measure of benefit for S(+)ibuprofen delivered directly to the air pouch. A Drug Targeting Index (DTI) was calculated from the ratio of AUC in the air pouch and plasma following direct intrapouch administration divided by the same ratio following intravenous administration and gave a value of 130. This pharmacokinetic measure of benefit represents the maximum advantage afforded by the site specific delivery of S(+)ibuprofen as the whole of the administered dose is delivered directly to the site of action.

Drug biotransformation interactions in man VI: acetaminophen and ascorbic acid.

Oral administration of 3 g of ascorbic acid 1.5 hr after an oral dose of 1 g of acetaminophen caused a rapid and pronounced decrease in the excretion rate of acetaminophen sulfate in five healthy adult volunteers. There was a statistically significant increase in the fractions of the dose of acetaminophen excreted as such as as acetaminophen glucuronide but a decrease in the fraction excreted as acetaminophen sulfate. The apparent biological half-life of acetaminophen increased from 2.3 +/- 0.2 (mean +/- SD) to 3.1 +/- 0.5 hr. Concomitant administration of sodium sulfate prevented these effects. Ascorbic acid, which itself is metabolized in part to the sulfate, inhibits the conjugation of acetaminophen with sulfate by competing for available sulfate in the body.

Effect of carbonated beverages and of an antiemetic containing carbohydrate and phosphoric acid on riboflavin bioavailability and salicylamide biotransformation in humans.

Two carbohydrate-phosphoric acid solutions, one a widely used beverage (Solution C) and the other a pharmaceutical product used as an antiemetic (Solution E), administered together with riboflavin-5'-phosphate or salicylamide to healthy human adults, significantly increased the bioavailability of riboflavin and appreciably altered the metabolic fate of salicylamide (increased conversion to the sulfate and decreased formation of the glucuronide). A beverage containing phosphoric acid but no carbohydrates (Solution T) also increased the bioavailability of riboflavin but not as much as Solution C. These effects are attributed to a decrease of the gastric emptying rate caused by carbohydrates and phosphoric acid, consistent with the empirical use of Solution C syrup and Solution E as antinauseants and antiemetics. The results demonstrate also that the choice of beverage to be taken with medication can affect the bioavailability and/or metabolic fate of medicinals with saturable absorption and/or biotransformation characteristics.

Effect of various alcohols on intestinal net water flux and theophylline absorption in rats.

Previous studies in this laboratory demonstrated that the rate of intestinal absorption of theophylline in rats is increased significantly by ethanol in low concentrations and that this absorption enhancing effect is associated with an increased net water flux from the intestine. It is now shown that other alcohols, namely methanol, n-propanol, n-butanol, glycerin, propylene glycol, and, to a lesser extent, mannitol and sorbitol, can also increase net water flux from the small intestine of anesthetized rats. Polyethylene glycol 200 and 400 had no such effect, suggesting that these compounds do not penetrate to a site of action that elicits the increased net water flux. At initial concentrations of 0.1 and 1.0 M, glycerin and propylene glycol increase significantly the intestinal absorption rate of theophylline from the small intestine of anesthetized rats. The results show that the theophylline absorption enhancing effect of ethanol is not limited to that particular alcohol.

Relevance of in vitro kinetic parameters to in vivo metabolism of xenobiotics.

Numerous in vitro systems are available for the study of pathways and kinetics of drug metabolism and the question is often posed of their utility as predictors of in vivo disposition behaviour. Of particular interest is the feasibility of predicting in vivo metabolic clearance of noxious chemicals in humans from data obtained in vitro using human tissue/gene expression systems. In theory the easily defined in vitro parameters V(max) and K(m) can be scaled to provide in vivo metabolic clearance. The cornerstone of this procedure is the parameter intrinsic clearance which may be defined biochemically as the V(max)/K(m) ratio. Extrapolation from the in vitro data requires scaling factors from either microsomal protein recovery, cellularity and/or organ weight and the use of a liver model (e.g. well-stirred liver) to express the kinetic data in terms of circulating drug concentrations (blood or plasma) rather than concentration at the enzyme site. The feasibility of this strategy and the relative merits of using hepatic microsomal and hepatocyte data will be discussed using a body of information on 25 drugs.

Incorporation of in vitro drug metabolism data into physiologically-based pharmacokinetic models.

The liver poses particular problems in constructing physiologically-based pharmacokinetic models since this organ is not only a distribution site for drugs/chemicals but frequently the major site of metabolism. The impact of hepatic drug metabolism in modelling is substantial and it is crucial to the success of the model that in vitro data on biotransformation be incorporated in a judicious manner. The value of in vitro/in vivo extrapolation is clearly demonstrated by considering kinetic data from incubations with freshly isolated hepatocytes. The determination of easily measurable in vitro parameters, such as V(max) and K(m), from initial rate studies and scaling to the in vivo situation by accounting for hepatocellularity provides intrinsic clearance estimates. A scaling factor of 1200 x 10(6) cells per 250 g rat has proved to be a robust parameter independent of laboratory technique and insensitive to animal pretreatment. Similar procedures can also be adopted for other in vitro systems such as hepatic microsomes and liver slices. An appropriate scaling factor for microsomal studies is the microsomal recovery index which allows for the incomplete recovery of cytochrome P-450 with standard differential centrifugation of liver homogenates. The hepatocellularity of a liver slice has been unsatisfactory in scaling kinetic parameters from liver slices. The level of success varies from drug to drug and substrate diffusion is a competing process to metabolism within the slice incubation system; hence, low clearance drugs are better predicted than high clearance drugs. The use of three liver models (venous-equilibration, undistributed sinusoidal and dispersion models) have been compared to predict hepatic clearance from in vitro intrinsic clearance values. As no consistent advantage of one model over the others could be demonstrated, the simplest, the venous-equilibration model, is adequate for the currently available data in hepatocytes. While these successes are encouraging as they establish the fidelity of in vitro systems for in vivo prediction, the level of success varies from drug to drug. It is important to address the reasons for failure of prediction by each in vitro system and it is noteworthy that the current approach simplifies several key issues.