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R S Obach

Publications and source records attributed to R S Obach.

At least 19 recordsLinked to original sources
PubMed2001

Metabolic characterization of the major human small intestinal cytochrome p450s.

Human small intestine epithelial cells (enterocytes) provide the first site for cytochrome P450 (CYP)-catalyzed metabolism of orally ingested xenobiotics. CYP3A4 is the major form of CYP expressed in enterocytes and CYP2C is also expressed at a significant level. In this study, we further characterized the expression of CYP3A4 and CYP2C in human enterocytes and their interindividual variations by examining the metabolic activities from 10 individuals. CYP3A4 in human jejunum microsomes, as determined by 6beta-testosterone hydroxylase activity, varied from 0.36 to 2.46 nmol/min/mg. The apparent average K(m) and V(max) values from two representative individuals were 54 microM and 3.2 nmol/min/mg, respectively. CYP2C9 and CYP2C19 in human jejunum microsomes, as determined by diclofenac 4'-hydroxylase and mephenytoin 4'-hydroxylase activities, varied over an 18-fold range (7.3-129 pmol/min/mg) and 17-fold range (0.8-13.1 pmol/min/mg), respectively. The mean apparent K(m) for diclofenac 4'-hydroxylase was 9.9 microM , whereas the apparent mean K(m) for S-mephenytoin 4'-hydroxylase was 79.3 microM . The mean intrinsic clearance (V(max)/K(m)) was approximately 130-fold greater for diclofenac 4'-hydroxylase than for mephenytoin 4'-hydroxylase. The metabolic activities of CYP2C9 and CYP2C19 were confirmed by inhibition by sulfaphenazole for CYP2C9 and ticlopidine for CYP2C19. In addition, CYP2C9 activities did not correlate with CYP3A4 activities, while CYP2C19 activities had a significant but poor correlation with those of CYP3A4. Thus the major CYP activities in human enterocytes have large interindividual variabilities that are not strongly related.

Adult↗
PubMed2001

Cytochrome P450-catalyzed metabolism of ezlopitant alkene (CJ-12,458), a pharmacologically active metabolite of ezlopitant: enzyme kinetics and mechanism of an alkene hydration reaction.

Experiments were conducted to characterize the metabolism of ezlopitant alkene (CJ-12,458), an active metabolite of ezlopitant, in human liver microsomes. In incubations with human liver microsomes and cofactors required for cytochrome P450 (CYP) activity, CJ-12,458 was converted to two metabolites: a diol (CP-611,781) and a 1 degrees alcohol (CP-616,762). In human liver microsomes, apparent K(M) values of 5.4 and 8.5 microM were determined for the formation of diol and 1 degrees alcohol metabolites, respectively. High K(M) activities were also observed for formation of these metabolites; however, the aforementioned low K(M) activities accounted for greater than 90% of the total intrinsic clearance. In pooled human liver microsomes, formation of both metabolites was partially inhibited by both quinidine and ketoconazole, suggesting that CYP2D6 and CYP3A enzymes are involved in the metabolism of CJ-12,458. This evidence was corroborated through the use of heterologously expressed CYP enzymes and correlation analysis with a panel of human liver microsomes. The data suggest that CYP2D6 is quantitatively more important than CYP3A in the metabolism of CJ-12,458 by a factor of about 2 to 1. The conversion of an alkene to a 1 degrees alcohol represents a novel biotransformation reaction. Incubations using (18)O(2), (2)H(2)O, [(2)H(5)]CJ-12,458, and [(2)H]NADPH were conducted and the 1 degrees alcohol product was characterized by ion trap mass spectrometry. From these data, a mechanism for this reaction is proposed involving epoxidation, an exocyclic hydride shift, and reduction at the benzylic position.

Benzylamines↗
PubMed2001

Mechanism of cytochrome P4503A4- and 2D6-catalyzed dehydrogenation of ezlopitant as probed with isotope effects using five deuterated analogs.

Ezlopitant is metabolized by cytochrome P450 primarily to two metabolites: a benzyl alcohol and a corresponding alkene. The alkene arises as a direct product of metabolism of ezlopitant rather than through dehydration of the benzyl alcohol. The mechanism of this cytochrome P450 (P450)-catalyzed dehydrogenation reaction was probed with five different deuterium-labeled analogs of ezlopitant. At saturating ezlopitant concentrations, deuterium substitution resulted in small differences in reaction velocity. When deuterium was incorporated into the benzylic position ([d(1)]ezlopitant and [d(7)]ezlopitant), low isotope effects on the formation of both the benzyl alcohol and alkene were observed (1.25-1.55 for CYP3A4 and 1.48-2.61 for CYP2D6), suggesting that abstraction of the benzylic hydrogen is obligatory in the formation of both metabolites. A small amount of metabolic switching occurred because isotope effects were slightly higher for alkene and alcohol formation than for ezlopitant consumption. Intramolecular deuterium isotope effects of the dehydrogenation reaction for tri- and tetradeuterated analogs were very low (1.13-1.15) for both CYP3A4 and CYP2D6, whereas intramolecular isotope effects for the chemical dehydration of correspondingly deuterated ezlopitant benzyl alcohol (CJ-12,764) were 3.8 to 5.9. Thus, dehydrogenation does not appear to occur via enzyme-mediated general acid catalysis of the benzyl alcohol. A mechanism for the dehydrogenation of ezlopitant is proposed in consideration of the data presented.

Benzylamines↗
PubMed2001

The prediction of human clearance from hepatic microsomal metabolism data.

Human liver microsomal intrinsic clearance has become a commonly measured parameter during drug discovery, and such data are used to design compounds predicted to possess optimal drug disposition characteristics. Liver microsomal intrinsic clearance values can be scaled and used to predict hepatic clearance in humans. Clearance, when combined with the volume of distribution, determines the half-life of a drug. Hepatic clearance, when combined with absorption, determines the oral bioavailability of a drug. Half-life and oral bioavailability are key determinants of the dosing regimen, i.e., size of dose and frequency of administration. Thus, the accurate prediction of human clearance is important in the selection of new compounds for progression into development, as new drugs on the market must not only be efficacious and safe, but must also be convenient to use for patients and physicians. Over the past decade, exploring methods whereby human clearance can be predicted from in vitro data has been an area of active research in drug metabolism science. Human liver microsomes have been a key tool in this research. This in vitro system possesses many of the major drug metabolizing enzymes and is thus applicable to a wide variety of compounds. This review describes the theoretical and practical aspects of predicting clearance from human liver microsomal intrinsic clearance data, a summary of advantages and shortcomings of this in vitro system, a synopsis of recent applications of human liver microsomal intrinsic clearance data in clearance predictions, and a discussion of potential future directions for this field.

Animals↗
PubMed2000

Assay of ezlopitant, a substance P receptor antagonist, and metabolites in biological matrices by gas chromatography with mass spectrometric detection: simultaneous analysis of a benzyl alcohol and alkene.

A method for the analysis of the substance P antagonist ezlopitant and two active metabolites in serum using solid-phase extraction followed by GC-MS analysis is described. The linear dynamic range was 1.0 to 100 ng/ml and precision and accuracy over this range were within 15%. Upon injection of reconstituted sample extracts into the hot injector port of the gas chromatograph, the benzyl alcohol metabolite undergoes a small amount of spontaneous dehydration to the alkene metabolite. We have incorporated an additional hexadeuterated internal standard of the benzyl alcohol into the assay to permit measurement of the extent of dehydration in each sample. This novel approach should be generally applicable to the simultaneous determination of benzyl alcohols and corresponding alkenes by GC-MS when the possibility exists that the alcohol can undergo spontaneous dehydration to the alkene in the injector port of GC instrumentation.

Alkenes↗
PubMed2000

Microsomal binding of amitriptyline: effect on estimation of enzyme kinetic parameters in vitro.

The effect of binding of amitriptyline to human liver microsomes and to microsomes from human B-lymphoblastoid cells on the estimation of enzyme kinetic parameters describing N-demethylation to nortriptyline was investigated using a combination of microsomal binding and in vitro enzyme kinetic studies. Quantitative binding in both matrices increased with higher microsomal protein concentrations (free fractions 0.88-0.32 at 100-500 microg protein/ml in human liver microsomes and 0.82-0.26 at 250-1000 microg protein/ml in microsomes from B-lymphoblastoid cells) and was independent of amitriptyline concentration over a concentration range of 0.2 to 200 microM. Addition of heat-inactivated microsomal protein (50-450 microg/ml) to native human liver microsomes (50 microg/ml) reduced the amitriptyline N-demethylation rate in a protein concentration dependent manner. This effect was greater at lower substrate concentrations and was overcome by saturating concentrations of substrate, thereby decreasing the apparent affinities of the high- and low-affinity components of the N-demethylation process, with minimal effect on the net V(max). Addition of metabolically inactive microsomes from untransfected human lymphoblastoid cells (750 microg/ml) to CYP2C19 (250 microg/ml protein) increased the apparent K(m) value for amitriptyline N-demethylation by 3.5-fold and increased the uncompetitive substrate inhibition constant (K(s)) by 2.2-fold, making substrate inhibition essentially undetectable. A similar effect was seen with CYP3A4, with a 1.8-fold increase in the S(50) (substrate concentration at which half-maximal velocity of a Hill enzyme is achieved). Microsomal binding did not alter the V(max) of either CYP isoform to any appreciable extent. These findings emphasize the importance of incorporating microsomal binding in the estimation of enzyme kinetic parameters in vitro and making appropriate corrections for unbound drug concentrations.

Algorithms↗
PubMed2000

Inhibition of human cytochrome P450 enzymes by constituents of St. John's Wort, an herbal preparation used in the treatment of depression.

Commercially available St. John's wort (Hypericum perforatum) extracts, preparations that are used in the treatment of depression, were examined for the potential to inhibit human cytochrome P450 (CYP) enzyme activities, specifically CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4. Crude extracts demonstrated inhibition of each of these five enzymes, with CYP2D6, CYP2C9, and CYP3A4 being more sensitive than CYP1A2 and CYP2C19. Extracts were fractionated by HPLC, and each of the fractions was tested for inhibition of these five CYPs to identify individual constituents with inhibitory activity. Several fractions were shown to possess inhibitory activity, including the fractions containing hyperforin (the putative active antidepressant constituent), I3,II8-biapigenin, and hypericin. Hyperforin and I3,II8-biapigenin were isolated from the extract, and inhibition constants for the five CYP activities were measured. In addition, three other constituents, hypericin, quercetin, and chlorogenic acid, were tested for inhibitory activity toward the CYP enzymes. The flavonoid compound I3,II8-biapigenin was shown to be a potent, competitive inhibitor of CYP3A4, CYP2C9, and CYP1A2 activities with K(i) values of 0.038, 0.32, and 0.95 microM, respectively. Hyperforin was a potent noncompetitive inhibitor of CYP2D6 activity (K(i) = 1.5 microM) and competitive inhibitor of CYP2C9 and CYP3A4 activities (K(i) = 1.8 and 0.48 microM, respectively). Hypericin also demonstrated potent inhibition of several CYP activities. These in vitro data indicate that St. John's wort preparations contain constituents that can potently inhibit the activities of major human drug-metabolizing enzymes and suggest that these preparations should be examined for potential pharmacokinetic drug interactions in vivo.

Anthracenes↗
PubMed2000

Metabolism of ezlopitant, a nonpeptidic substance P receptor antagonist, in liver microsomes: enzyme kinetics, cytochrome P450 isoform identity, and in vitro-in vivo correlation.

The enzyme kinetics of the metabolism of ezlopitant in liver microsomes from various species have been determined. The rank order of the species with regard to the in vitro intrinsic clearance of ezlopitant was monkey >> guinea pig > rat >> dog > human. CJ-12,764, a benzyl alcohol analog, was observed as a major metabolite, and a dehydrogenated metabolite (CJ-12,458) was equally important in human liver microsomes. Scale-up of the liver microsomal intrinsic clearance data and correcting for both serum protein binding and nonspecific microsomal binding yielded predicted hepatic clearance values that showed a good correlation with in vivo systemic blood clearance values. Including microsomal binding was necessary to achieve agreement between hepatic clearance values predicted from in vitro data and systemic clearance values measured in vivo. Cytochrome P450 (CYP) 3A4, 3A5, and 2D6 demonstrated the ability to metabolize ezlopitant to CJ-12,458 and CJ-12,764. However, in liver microsomes, the CYP3A isoforms appear to play a substantially more important role in the metabolism of ezlopitant than CYP2D6, as assessed through the use of CYP-specific inhibitors, correlation to isoform-specific marker substrate activities, and appropriate scale-up of enzyme kinetic data generated in microsomes containing individual heterologously expressed recombinant CYP isoforms. The apparent predominance of CYP3A over CYP2D6 is consistent with observations of the pharmacokinetics of ezlopitant in humans in vivo.

Animals↗
PubMed2000

(R)-, (S)-, and racemic fluoxetine N-demethylation by human cytochrome P450 enzymes.

Fluoxetine is one of the most widely prescribed selective serotonin reuptake inhibitors (SSRIs) that is marketed worldwide. However, details of its human hepatic metabolism have been speculative and incomplete, possibly due to the sensitivity of analytical techniques and selectivity of specific in vitro probes and reagents used. Studies with (R)-, (S)-, and racemic fluoxetine were undertaken to determine the stereospecific nature of its metabolism and estimate intrinsic clearance contributions of each CYP for fluoxetine N-demethylation. Measurable fluoxetine N-demethylase activity was catalyzed by CYP1A2, -2B6, -2C9, -2C19, -2D6, -3A4, and -3A5. All enzymes catalyzed this reaction for both enantiomers and the racemate, and intrinsic clearance values were similar for the enantiomers for all CYP enzymes except CYP2C9, which demonstrated stereoselectivity for R- over the S-enantiomer. Scaling the intrinsic clearance values for the individual CYP enzymes to estimate contributions of each in human liver microsomes suggested that CYP2D6, CYP2C9, and CYP3A4 contribute the greatest amount of fluoxetine N-demethylation in human liver microsomes. These data were corroborated with the examination of the effects of CYP-specific inhibitors quinidine (CYP2D6), sulfaphenazole (CYP2C9), and ketoconazole (CYP3A4) on fluoxetine N-demethylation in pooled human liver microsomes. Together, these findings suggest a significant role for the polymorphically expressed CYP2D6 in fluoxetine clearance and are consistent with reports on the clinical pharmacokinetics of fluoxetine.

Cytochrome P-450 Enzyme System↗
PubMed2000

Three-dimensional quantitative structure activity relationship computational approaches for prediction of human in vitro intrinsic clearance.

Future alternatives to the presently accepted in vitro paradigm of prediction of intrinsic clearance, which could be used earlier in the drug discovery process, would potentially accelerate efforts to identify better drug candidates with more favorable metabolic profiles and less likelihood of failure with regard to human pharmacokinetic attributes. In this study we describe two computational methods for modeling human microsomal and hepatocyte intrinsic clearance data derived from our laboratory and the literature, which utilize pharmacophore features or descriptors derived from molecular structure. Human microsomal intrinsic clearance data generated for 26 known therapeutic drugs were used to build computational models using commercially available software (Catalyst and Cerius(2)), after first converting the data to hepatocyte intrinsic clearance. The best Catalyst pharmacophore model gave an r of 0.77 for the observed versus predicted clearance. This pharmacophore was described by one hydrogen bond acceptor, two hydrophobic features, and one ring aromatic feature essential to discriminate between high and low intrinsic clearance. The Cerius(2) quantitative structure activity relationship (QSAR) model gave an r(2) = 0.68 for the observed versus predicted clearance and a cross-validated r(2) (q(2)) of 0.42. Similarly, literature data for human hepatocyte intrinsic clearance for 18 therapeutic drugs were also used to generate two separate models using the same computational approaches. The best Catalyst pharmacophore model gave an improved r of 0.87 and was described by two hydrogen bond acceptors, one hydrophobe, and 1 positive ionizable feature. The Cerius(2) QSAR gave an r(2) of 0.88 and a q(2) of 0.79. Each of these models was then used as a test set for prediction of the intrinsic clearance data in the other data set, with variable successes. These present models represent a preliminary application of QSAR software to modeling and prediction of human in vitro intrinsic clearance.

Catalysis↗
PubMed1999

Pharmacokinetics of ezlopitant, a novel non-peptidic neurokinin-1 receptor antagonist in preclinical species and metabolite kinetics of the pharmacologically active metabolites.

The pharmacokinetics of ezlopitant were determined in the rat, gerbil, guinea pig, ferret, dog and monkey after intravenous and oral administration. In general, ezlopitant is marked by high clearance values that approach or exceed hepatic blood flow values, moderate to high values for steady-state volume of distribution (3. 9-28 L/kg), and terminal phase half-life values ranging from 0.6 h in the guinea pig to 7.7 h in the rat. Oral bioavailability ranged from <0.2% (guinea pig) to 28% (dog). Data from portal vein cannulated dogs suggested that 37% of an oral dose of ezlopitant enters the portal vein as an unchanged drug in this species. Ezlopitant is metabolized to two pharmacologically active metabolites, an alkene (CJ-12 458) and a benzyl alcohol (CJ-12 764). After administration of the parent compound, CJ-12 764 was found in greater abundance than CJ-12 458 in all species examined. Ezlopitant and CJ-12 458 were highly protein bound in plasma (or serum), whereas the protein binding of CJ-12 764 was somewhat lower. Measurement of the kinetics of ezlopitant, CJ-12 458 and CJ-12 764 in the cerebrospinal fluid (CSF) of dogs demonstrated that all three compounds can partition into the CSF, and thereby, be capable of contributing to centrally mediated pharmacological effects. Thus, these data suggest that the pharmacological activity exhibited by ezlopitant in preclinical species in vivo is likely a result of the parent compound plus the active metabolites. Furthermore, the contributions of ezlopitant and the active metabolites to pharmacological activity probably varies with the identity of the model species, as well as the dose and route of ezlopitant administration.

Animals↗
PubMed1999

Prediction of human clearance of twenty-nine drugs from hepatic microsomal intrinsic clearance data: An examination of in vitro half-life approach and nonspecific binding to microsomes.

Twenty-nine drugs of disparate structures and physicochemical properties were used in an examination of the capability of human liver microsomal lability data ("in vitro T(1/2)" approach) to be useful in the prediction of human clearance. Additionally, the potential importance of nonspecific binding to microsomes in the in vitro incubation milieu for the accurate prediction of human clearance was investigated. The compounds examined demonstrated a wide range of microsomal metabolic labilities with scaled intrinsic clearance values ranging from less than 0.5 ml/min/kg to 189 ml/min/kg. Microsomal binding was determined at microsomal protein concentrations used in the lability incubations. For the 29 compounds studied, unbound fractions in microsomes ranged from 0.11 to 1.0. Generally, basic compounds demonstrated the greatest extent of binding and neutral and acidic compounds the least extent of binding. In the projection of human clearance values, basic and neutral compounds were well predicted when all binding considerations (blood and microsome) were disregarded, however, including both binding considerations also yielded reasonable predictions. Including only blood binding yielded very poor projections of human clearance for these two types of compounds. However, for acidic compounds, disregarding all binding considerations yielded poor predictions of human clearance. It was generally most difficult to accurately predict clearance for this class of compounds; however the accuracy was best when all binding considerations were included. Overall, inclusion of both blood and microsome binding values gave the best agreement between in vivo clearance values and clearance values projected from in vitro intrinsic clearance data.

Atmospheric Pressure↗
PubMed1998

Expression and characterization of canine cytochrome P450 2D15.

CYP2D15 is the canine ortholog of human CYP2D6, the human CYP2D isoform involved in the metabolism of drugs such as antiarhythmics, adrenoceptor antagonists, and tricyclic antidepressants. Similar to human, canine CYP2D15 is expressed in the liver, with detectable levels in several other tissues. Three different CYP2D15 cDNA clones were obtained by RT-PCR from dog liver RNA. Two clones corresponded to variant full-length CYP2D15 cDNAs (termed CYP2D15 WT2 and CYP2D15 V1); the third was identified as a splicing variant missing exon 3 (termed CYP2D15 V2). Recombinant baculoviruses were constructed containing full-length cDNAs and used to express CYP2D15 WT2 and CYP2D15 V1 in Spodoptera frugiperda (Sf9) cells with expression levels of up to 0.14 nmol/mg cell protein. As with human CYP2D6, the recombinant CYP2D15 enzymes exhibited bufuralol 1'-hydroxylaseand dextromethorphan O-demethylase activities whencoexpressed with rabbit NADPH:P450 oxidoreductase. For bufuralol 1'-hydroxylase, apparent Km values were 4.9, 3.7, and 2.5 microM and the Vmax values were 0.14, 0.034, and 0.60 nmol/min/mg protein for dog liver microsomes, CYP2D15 WT2, and the variant CYP2D15 V1, respectively. For dextromethorphan O-demethylase, apparent Km values were 0.6, 0.6, and 2.0 microM and the Vmax values were 0.18, 0.034, and 0.057 nmol/min/mg protein for dog liver microsomes, CYP2D15 WT2, and the variant CYP2D15 V1, respectively. The human CYP2D6-specific inhibitor quinidine and the rat CYP2D1-specific inhibitor quinine were both shown to be inhibitors of bufuralol 1'-hydroxylase activity for dog liver microsomes, CYP2D15 WT2, and the CYP2D15 V1 variant with nearly equal potency. Thus, the dog expresses a CYP2D ortholog possessing enzymatic activities similar to human CYP2D6, but is affected by the inhibitors quinine and quinidine in a manner closer to that of rat CYP2D1.

Animals↗
PubMed1998

Cytochrome P4502D6 catalyzes the O-demethylation of the psychoactive alkaloid ibogaine to 12-hydroxyibogamine.

Ibogaine is a psychoactive alkaloid that possesses potential as an agent to treat opiate and cocaine addiction. The primary metabolite arises via O-demethylation at the 12-position to yield 12-hydroxyibogamine. In this report, evidence is presented that the O-demethylation of ibogaine observed in human hepatic microsomes is catalyzed primarily by the polymorphically expressed cytochrome P-4502D6 (CYP2D6). An enzyme kinetic examination of ibogaine O-demethylase activity in pooled human liver microsomes suggested that two (or more) enzymes are involved in this reaction: one with a low KMapp (1.1 microM) and the other with a high KMapp (>200 microM). The low KMapp activity comprised >95% of total intrinsic clearance. Human liver microsomes from three individual donors demonstrated similar enzyme kinetic parameters (mean KMapp = 0.55 +/- 0.09 microM and 310 +/- 10 microM for low and high KM activities, respectively). However, a fourth human microsome sample that appeared to be a phenotypic CYP2D6 poor metabolizer possessed only the high KMapp activity. In hepatic microsomes from a panel of human donors, the low KMapp ibogaine O-demethylase activity correlated with CYP2D6-catalyzed bufuralol 1'-hydroxylase activity but not with other P450 isoform-specific activities. Quinidine, a CYP2D6-specific inhibitor, inhibited ibogaine O-demethylase (IC50 = 0.2 microM), whereas other P450 isoform-specific inhibitors did not inhibit this activity. Also, of a battery of recombinant heterologously expressed human P450 isoforms, only rCYP2D6 possessed significant ibogaine O-demethylase activity. Thus, it is concluded that ibogaine O-demethylase is catalyzed by CYP2D6 and that this isoform is the predominant enzyme of ibogaine O-demethylation in humans. The potential pharmacological implications of these findings are discussed.

Cytochrome P-450 CYP2D6↗
PubMed1997

The effect of CP-99994 on the responses to provocative motion in the cat.

1. The NK1 receptor antagonist CP-99994 has been shown to prevent vomiting elicited by both peripherally and centrally acting emetogens in ferrets and dogs. These results have now been extended to another stimulus, provocative motion, and another species, the cat. 2. CP-99994 displaced [3H]-substance P from cat cortex with IC50 of 0.52 +/- 0.08 nM. Following s.c. administration, peak plasma drug levels were achieved at 30 min. The plasma drug half life was 1.4 h. 3. Subcutaneous administration of CP-99994 inhibited motion-induced vomiting in the cat with an ED50 of 144 micrograms kg-1 but did not change the epiphenomena associated with provocative motion in the cat over the dose range of 30 to 300 micrograms kg-1. The antiemetic effect of CP-99994 can be attributed to antagonism of the NK1 receptor because its enantiomer, CP-100,263, which is 900 fold weaker as an NK1 antagonist, had no effects on any response to provocative motion. 4. The inhibitory effect of CP-99994 on motion-induced retching and vomiting is consistent with a central site of antiemetic action, potentially at the level of the motor nuclei responsible for these behaviours. 5. An investigation into whether the failure of CP-99994 to alter the epiphenomena will also predict a lack of anti-nausea effects in man will provide critical information on the neural organization of the emetic reflex.

Animals↗
PubMed1997

The prediction of human pharmacokinetic parameters from preclinical and in vitro metabolism data.

We describe a comprehensive retrospective analysis in which the abilities of several methods by which human pharmacokinetic parameters are predicted from preclinical pharmacokinetic data and/or in vitro metabolism data were assessed. The prediction methods examined included both methods from the scientific literature as well as some described in this report for the first time. Four methods were examined for their ability to predict human volume of distribution. Three were highly predictive, yielding, on average, predictions that were within 60% to 90% of actual values. Twelve methods were assessed for their utility in predicting clearance. The most successful allometric scaling method yielded clearance predictions that were, on average, within 80% of actual values. The best methods in which in vitro metabolism data from human liver microsomes were scaled to in vivo clearance values yielded predicted clearance values that were, on average, within 70% to 80% of actual values. Human t1/2 was predicted by combining predictions of human volume of distribution and clearance. The best t1/2 prediction methods successfully assigned compounds to appropriate dosing regimen categories (e.g., once daily, twice daily and so forth) 70% to 80% of the time. In addition, correlations between human t1/2 and t1/2 values from preclinical species were also generally successful (72-87%) when used to predict human dosing regimens. In summary, this retrospective analysis has identified several approaches by which human pharmacokinetic data can be predicted from preclinical data. Such approaches should find utility in the drug discovery and development processes in the identification and selection of compounds that will possess appropriate pharmacokinetic characteristics in humans for progression to clinical trials.

Animals↗
PubMed1997

Nonspecific binding to microsomes: impact on scale-up of in vitro intrinsic clearance to hepatic clearance as assessed through examination of warfarin, imipramine, and propranolol.

The nonspecific, noncovalent binding of three drugs, imipramine, warfarin, and propranolol, to pooled human and animal liver microsomes has been determined using equilibrium dialysis in conditions where no cofactor (NADPH) was included in the incubation. The binding of warfarin was dependent upon both protein and drug concentration, whereas the binding of propranolol and imipramine was also dependent upon protein concentration but generally independent of drug concentration. At a microsomal protein concentration of 1.0 mg/ml and a warfarin concentration of 10 microM, the free fraction (fu(mic)) was 0.85. The corresponding values for propranolol and imipramine were 0.41 and 0.16, respectively. Thus, although all three drugs exhibit high binding in plasma (fu<0.1) the acidic drug warfarin differs from the basic drugs propranolol and imipramine in the extent to which each binds to microsomal protein. The binding of all three drugs to liver microsomes obtained from commonly studied animal species (rat, dog, and monkey) was almost identical to that observed in human. Additionally, the binding of warfarin and propranolol to microsomes obtained from insect cells used in baculovirus cytochrome P450 expression systems was similar to that exhibited in liver microsomes, when equal protein concentrations were compared. The enzyme kinetics of propranolol, imipramine, and warfarin oxidative metabolism were determined in pooled human liver microsomes, and the intrinsic clearance values obtained were used in scaling up to project human in vivo clearance. The values obtained by incorporating microsomal binding were compared with those in which this factor is ignored. The findings suggest that the parameter fu(mic) is important to obtain when attempting to relate in vitro intrinsic clearance to in vivo clearance. Also, this value is important to consider when comparing substrates with respect to enzyme specificity, since measured apparent KM values should be converted to true "free KM" values by correcting for the free fraction in the in vitro incubation. Furthermore, the extent of nonspecific binding to microsomes is likely an important parameter to consider when attempting to relate Ki values measured in vitro to observations of drug-drug interactions (or the lack thereof) in vivo.

Animals↗