Structure-Guided In Vitro to In Vivo Pharmacokinetic Optimization of Propargyl-Linked Antifolates.

Pharmacokinetic/pharmacodynamic properties are strongly correlated with the in vivo efficacy of antibiotics. Propargyl-linked antifolates, a novel class of antibiotics, demonstrate potent antibacterial activity against both Gram-positive and Gram-negative pathogenic bacteria, including multidrug-resistant Staphylococcus aureus Here, we report our efforts to optimize the pharmacokinetic profile of this class to best match the established pharmacodynamic properties. High-resolution crystal structures were used in combination with in vitro pharmacokinetic models to design compounds that not only are metabolically stable in vivo but also retain potent antibacterial activity. The initial lead compound was prone to both N-oxidation and demethylation, which resulted in an abbreviated in vivo half-life (∼20 minutes) in mice. Stability of leads toward mouse liver microsomes was primarily used to guide medicinal chemistry efforts so robust efficacy could be demonstrated in a mouse disease model. Structure-based drug design guided mitigation of N-oxide formation through substitutions of sterically demanding groups adjacent to the pyridyl nitrogen. Additionally, deuterium and fluorine substitutions were evaluated for their effect on the rate of oxidative demethylation. The resulting compound was characterized and demonstrated to have a low projected clearance in humans with limited potential for drug-drug interactions as predicted by cytochrome P450 inhibition as well as an in vivo exposure profile that optimizes the potential for bactericidal activity, highlighting how structural data, merged with substitutions to introduce metabolic stability, are a powerful approach to drug design.

Metabolism, pharmacokinetics and excretion of the GABA(A) receptor partial agonist [(14)C]CP-409,092 in rats.

The metabolism and excretion of a GABA(A) partial agonist developed for the treatment of anxiety, CP-409,092; 4-oxo-4,5,6,7-tetrahydro-1H-indole-3-carboxylic acid (4-methylaminomethyl-phenyl)-amide, were studied in rats following intravenous and oral administration of a single doses of [(14)C]CP-409,092. The pharmacokinetics of CP-409,092 following single intravenous and oral doses of 4 and 15 mg kg(-1), respectively, were characterized by high clearance of 169 + or - 18 ml min(-1) kg(-1), a volume of distribution of 8.99 + or - 1.46 l kg(-1), and an oral bioavailability of 2.9% + or - 3%. Following oral administration of 100 mg kg(-1) [(14)C]CP-409,092, the total recovery was 89.1% + or - 3.2% for male rats and 89.3% + or - 0.58% for female rats. Approximately 87% of the radioactivity recovered in urine and faeces were excreted in the first 48 h. A substantial portion of the radioactivity was measured in the faeces as unchanged drug, suggesting poor absorption and/or biliary excretion. There were no significant gender-related quantitative/qualitative differences in the excretion of metabolites in urine or faeces. The major metabolic pathways of CP-409,092 were hydroxylation(s) at the oxo-tetrahydro-indole moiety and oxidative deamination to form an aldehyde intermediate and subsequent oxidation to form the benzoic acid. The minor metabolic pathways included N-demethylation and subsequent N-acetylation and oxidation. The present work demonstrates that oxidative deamination at the benzylic amine of CP-409,092 and subsequent oxidation to form the acid metabolite seem to play an important role in the metabolism of the drug, and they contribute to its oral clearance and low exposure.

Understanding the clinical pharmacokinetics of a GABAA partial agonist by application of in vitro tools.

1. 4-Oxo-4,5,6,7-tetrahydro-1H-indole-3-carboxylic acid (4-methylaminomethyl-phenyl)-amide (1), developed for general anxiety disorder, was discontinued from clinical development due to unsuitable oral pharmacokinetics. 2. In humans, (1) demonstrated an unacceptable high apparent oral clearance (Cl(p)/F) that also demonstrated a supraproportional dose-exposure relationship. Secondary peaks in the plasma concentration-time profile suggested possible enterohepatic recirculation of (1). A combination of in vitro mechanistic tools was applied to better understand the processes underlying these complex clinical pharmacokinetic profiles of (1). 3. In metabolism experiments, (1) was shown to be a substrate of monoamine oxidase A (MAO-A) as well as being metabolized by cytochrome P450. The former appeared to be a high K(M) process with a high capacity, while the latter showed saturation between 1 and 10 microM, consistent with the supraproportional dose-exposure relationship. 4. In a sandwich-cultured hepatocyte model, (1) was shown to be a substrate for both uptake and efflux into the canicular space, which is consistent with the observation of pharmacokinetics suggestive of enterohepatic recirculation. Finally, in human epithelial colon adenocarcinoma cell line (Caco-2) and Madin-Darby canine kidney cells transwell flux experiments, (1) was shown to have relatively low permeability and a basolateral-to-apical flux ratio consistent with the activity of P-glycoprotein. 5. In combination, a compounding of the contributions of MAO-A, hepatic uptake and efflux transporters, and P-glycoprotein to the disposition of (1) may underlie the low oral exposure, saturable clearance, and aberrant concentration versus time profiles observed for this compound in humans.

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.

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.

Evaluation of various static and dynamic modeling methods to predict clinical CYP3A induction using in vitro CYP3A4 mRNA induction data.

Several drug-drug interaction (DDI) prediction models were evaluated for their ability to identify drugs with cytochrome P450 (CYP)3A induction liability based on in vitro mRNA data. The drug interaction magnitudes of CYP3A substrates from 28 clinical trials were predicted using (i) correlation approaches (ratio of the in vivo peak plasma concentration (Cmax) to in vitro half-maximal effective concentration (EC50); and relative induction score), (ii) a basic static model (calculated R3 value), (iii) a mechanistic static model (net effect), and (iv) mechanistic dynamic (physiologically based pharmacokinetic) modeling. All models performed with high fidelity and predicted few false negatives or false positives. The correlation approaches and basic static model resulted in no false negatives when total Cmax was incorporated; these models may be sufficient to conservatively identify clinical CYP3A induction liability. Mechanistic models that include CYP inactivation in addition to induction resulted in DDI predictions with less accuracy, likely due to an overprediction of the inactivation effect.

Evaluation of various static in vitro-in vivo extrapolation models for risk assessment of the CYP3A inhibition potential of an investigational drug.

Nine static models (seven basic and two mechanistic) and their respective cutoff values used for predicting cytochrome P450 3A (CYP3A) inhibition, as recommended by the US Food and Drug Administration and the European Medicines Agency, were evaluated using data from 119 clinical studies with orally administered midazolam as a substrate. Positive predictive error (PPE) and negative predictive error (NPE) rates were used to assess model performance, based on a cutoff of 1.25-fold change in midazolam area under the curve (AUC) by inhibitor. For reversible inhibition, basic models using total or unbound systemic inhibitor concentration [I] had high NPE rates (46-47%), whereas those using intestinal luminal ([I]gut) values had no NPE but a higher PPE. All basic models for time-dependent inhibition had no NPE and reasonable PPE rates (15-18%). Mechanistic static models that incorporate all interaction mechanisms and organ specific [I] values (enterocyte and hepatic inlet) provided a higher predictive precision, a slightly increased NPE, and a reasonable PPE. Various cutoffs for predicting the likelihood of CYP3A inhibition were evaluated for mechanistic models, and a cutoff of 1.25-fold change in midazolam AUC appears appropriate.

Heterotropic effects on drug-metabolizing enzyme activities: in vitro curiosity emerges as a clinically meaningful phenomenon (perhaps?).

Alterations in cytochrome P4503A4 are the most frequent underlying cause of drug-drug interactions (DDIs). This enzyme exhibits some unusual behaviors; for example, it has been observed that certain inhibitors can affect some CYP3A4-catalyzed reactions more than others, even for the same substrate. This has been proposed to be due to the simultaneous binding of more than one ligand to the enzyme. This behavior has been frequently observed in vitro, but seldom are analogous effects evident in vivo.

Effect of human renal cationic transporter inhibition on the pharmacokinetics of varenicline, a new therapy for smoking cessation: an in vitro-in vivo study.

Varenicline is predominantly eliminated unchanged in urine, and active tubular secretion partially contributes to its renal elimination. Transporter inhibition assays using human embryonic kidney 293 cells transfected with human renal transporters demonstrated that high concentrations of varenicline inhibited substrate uptake by hOCT2 (IC(50)=890 microM), with very weak or no measurable interactions with the other transporters hOAT1, hOAT3, hOCTN1, and hOCTN2. Varenicline was characterized as a moderate-affinity substrate for hOCT2 (K(m)=370 microM) and its hOCT2-mediated uptake was partially inhibited by cimetidine. Co-administration of cimetidine (1,200 mg/day) reduced the renal clearance of varenicline in 12 smokers, resulting in a 29.0% (90% CI: 21.5%-36.9%) increase in systemic exposure. This increase is not considered clinically relevant, as it should not give rise to safety concerns. Consequently, it can be reasonably expected that other inhibitors of hOCT2 would not cause greater renal interactions with varenicline than that seen with the efficient hOCT2 inhibitor cimetidine.

Mechanism-based inactivation of human cytochrome P450 enzymes: strategies for diagnosis and drug-drug interaction risk assessment.

Among drugs that cause pharmacokinetic drug-drug interactions, mechanism-based inactivators of cytochrome P450 represent several of those agents that cause interactions of the greatest magnitude. In vitro inactivation kinetic data can be used to predict the potential for new drugs to cause drug interactions in the clinic. However, several factors exist, each with its own uncertainty, that must be taken into account in order to predict the magnitude of interactions reliably. These include aspects of in vitro experimental design, an understanding of relevant in vivo concentrations of the inactivator, and the extent to which the inactivated enzyme is involved in the clearance of the affected drug. Additionally, the rate of enzyme degradation in vivo is also an important factor that needs to be considered in the prediction of the drug interaction magnitudes. To address mechanism-based inactivation for new drugs, various in vitro experimental approaches have been employed. The selection of approaches for in vitro kinetic characterization of inactivation as well as in vitro-in vivo extrapolation should be guided by the purpose of the exercise and the stage of drug discovery and development, with an increase in the level of sophistication throughout the research and development process.

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.

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.

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.

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.

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.

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.

Radioimmunoassay of nicotine-delta 1'(5')-iminium ion, an intermediate formed during the metabolism of nicotine to cotinine.

Cotinine, one of the major metabolites of nicotine, is formed by two sequential enzyme reactions: [formula: see text] Chemical and immunological methods of analysis are available to quantify these two compounds (I and III). Study of the intermediate (III) is hampered because of its complex chemistry and lack of simple methods for its assay. Generally, (II) is trapped by addition of cyanide and analyzed as the 5'-cyanonicotine adduct. In order to develop an immunoassay for (II), rabbits were immunized with a 3'-succinylmethyl-5'-cyanonicotine-protein conjugate with the expectation that (II) would, after treatment with cyanide, be quantified as 5'-cyanonicotine. Unexpectedly, however, the antibodies recognized the cyano adduct and (II) to the same extent (limit of deletion = 1.2 pmol). Nicotine, cotinine, and several other metabolites do not significantly inhibit the antigen-antibody reaction. Inhibition studies with 5' substituted nicotine analogs indicate that the 5'-hydroxynicotine tautomer (IIb) is the species recognized by the antibodies. Inin vitro metabolic studies, (II) accumulates in the absence of aldehyde oxidase, but in its presence, is converted to cotinine. As judged by serological activity, dilute solutions (under 15 microM) of (II) are stable when allowed to stand for 6 days at pH 7 and 37 degrees C. However, at high concentrations of iminium ion (where NMR studies showed instability and formation of multiple products) serological activity is also lost. Iminium ions are generated during the metabolism of some tertiary amine xenobiotics. The use of alpha-cyanoamine haptens to elicit antibodies specific for iminium ions and/or their cyano adducts may permit development of immunoassays for these compounds.