Suresh K. Balani

Comparison of Intrinsic Clearance in Liver Microsomes and Hepatocytes from Rats and Humans: Evaluation of Free Fraction and Uptake in Hepatocytes

Apparent intrinsic clearance (CLint,app) of 7-ethoxycoumarin, phenacetin, propranolol, and midazolam was measured using rat and human liver microsomes and freshly isolated and cryopreserved hepatocytes to determine factors responsible for differences in rates of metabolism in these systems. The cryopreserved and freshly isolated hepatocytes generally provided similar results, although there was greater variability using the latter system. The CLint,app values in hepatocytes are observed to be lower than that in microsomes, and this difference becomes greater for compounds with high CLint,app. This could partly be attributed to the differences in the free fraction (fu). The fu in hepatocyte incubations (fu,hep-inc) was influenced not only by the free fraction of compounds in the incubation buffer (fu,buffer) but also by the rate constants of uptake (kup) and metabolism (kmet). This report provides a new derivation for fu,hep-inc, which can be expressed as fu,hep-inc = [kup/(kmet + kup)]/[1 + (Chep/Cbuffer) × (Vhep/Vbuffer)], where the Chep, Cbuffer, Vhep, and Vbuffer represent the concentrations of a compound in hepatocytes and buffer and volumes of hepatocytes and buffer, respectively. For midazolam, the fu,hep-inc was calculated, and the maximum metabolism rate in hepatocytes was shown to be limited by the uptake rate.

Strategy of utilizing in vitro and in vivo ADME tools for lead optimization and drug candidate selection.

The high-throughput screening in drug discovery for absorption, distribution, metabolism and excretion (ADME) properties has become the norm in the industry. Only a few years ago it was ADME properties that were attributed to more failure of drugs than efficacy or safety in the clinic trials. With the realization of new techniques and refinement of existing techniques better projections for the pharmacokinetic properties of compounds in humans are being made, shifting the drug failure attributes more to the safety and efficacy properties of drug candidates. There are a tremendous number of tools available to discovery scientists to screen compounds for optimization of ADME properties and selection of better candidates. However, the use of these tools has generally been to characterize these compounds rather than to select among them. This report discusses applications of the available ADME tools to better understand the clinical implication of these properties, and to optimize these properties. It also provides tracts for timing of studies with respect to the stage of the compound during discovery, by means of a discovery assay by stage (DABS) paradigm. The DABS provide the team with a rationale for the types of studies to be done during hit-to-lead, early and late lead optimization stages of discovery, as well as outlining the deliverables (objectives) at those stages. DABS has proven to be optimal for efficient utilization of resources and helped the discovery team to track the progress of compounds and projects.

EVALUATION OF MICRODOSING TO ASSESS PHARMACOKINETIC LINEARITY IN RATS USING LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY

The microdosing strategy allows for early assessment of human pharmacokinetics of new chemical entities using more limited safety assessment requirements than those requisite for a conventional phase I program. The current choice for evaluating microdosing is accelerator mass spectrometry (AMS) due to its ultrasensitivity for detecting radiotracers. However, the AMS technique is still expensive to be used routinely and requires the preparation of radiolabeled compounds. This report describes a feasibility study with conventional liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology for oral microdosing assessment in rats, a commonly used preclinical species. The nonlabeled drugs fluconazole and tolbutamide were studied because of their similar pharmacokinetics characteristics in rats and humans. We demonstrate that pharmacokinetics can be readily characterized by LC-MS/MS at a microdose of 1 μg/kg for these molecules in rats, and, hence, LC-MS/MS should be adequate in human microdosing studies. The studies also exhibit linearity in exposure between the microdose and ≥1000-fold higher doses in rats for these drugs, which are known to show a linear dose-exposure relationship in the clinic, further substantiating the potential utility of LC-MS/MS in defining pharmacokinetics from the microdose of drugs. These data should increase confidence in the use of LC-MS/MS in microdose pharmacokinetics studies of new chemical entities in humans. Application of this approach is also described for an investigational compound, MLNX, in which the pharmacokinetics in rats were determined to be nonlinear, suggesting that MLNX pharmacokinetics at microdoses in humans also might not reflect those at the therapeutic doses. These preclinical studies demonstrate the potential applicability of using traditional LC-MS/MS for microdose pharmacokinetic assessment in humans.

EFFECTIVE DOSING REGIMEN OF 1-AMINOBENZOTRIAZOLE FOR INHIBITION OF ANTIPYRINE CLEARANCE IN GUINEA PIGS AND MICE USING SERIAL SAMPLING

Single-dose pharmacokinetics of 1-aminobenzotriazole (ABT), a potent nonspecific inhibitor of cytochromes P450 (P450s), were characterized after oral administration to mice and guinea pigs at doses of 50, 100, and 150 mg/kg using serial sampling in both species. Only 30-μl blood samples were drawn from jugular vein-cannulated mice using Microvette capillary tubes containing lithium heparin. A comparison of the pharmacokinetics of antipyrine (AP) administered i.v. at 20 mg/kg to mice followed by serial and terminal sampling techniques yielded similar results. The ABT concentrations in plasma were sustained at high levels (5-100 μM) for at least 12 h in both species. Pretreatment of animals with ABT 2 h prior to AP administration decreased the plasma AP clearance by about 95% in mice at all ABT doses studied and 84, 95, and 95% in guinea pigs at a dose of 50, 100, and 150 mg/kg ABT, respectively. In vitro, the dissociation constants (KI) for ABT as the P450 mechanism-based inactivator were determined to be 45.6 and 193 μM, and the maximal inactivation rate constants (kinact) were determined to be 0.089 and 0.075 min-1 for the mouse and guinea pig liver microsomes, respectively. The projected P450 inactivations at the plasma Cmax of ABT agreed with the inhibitions of P450-mediated AP clearance observed in vivo. For mechanistic studies in vivo overall, a 2-h prior oral pretreatment with ABT at 50 mg/kg in mice and 100 mg/kg in guinea pigs would provide significant systemic concentrations of the inhibitor over 24 h and inhibition of P450-dependent clearance of test compounds.

ESTERASE INHIBITION BY GRAPEFRUIT JUICE FLAVONOIDS LEADING TO A NEW DRUG INTERACTION

Our previous studies described a newly identified potential of grapefruit juice (GFJ) in mediating pharmacokinetic drug interactions due to its capability of esterase inhibition. The current study identifies the active components in GFJ responsible for its esterase-inhibitory effect. The esterase-inhibitory potential of 10 constitutive flavonoids and furanocoumarins toward p-nitrophenylacetate (PNPA) hydrolysis was investigated. The furanocoumarins bergamottin, 6′,7′-dihydroxybergamottin, and bergapten, and the glycoside flavonoids naringin and hesperidin, at concentrations found in GFJ or higher, did not inhibit the hydrolysis of PNPA by purified porcine esterase and human liver microsomes. However, the flavonoid aglycones morin, galangin, kaempferol, quercetin, and naringenin showed appreciable inhibition of PNPA hydrolysis in purified porcine esterase, and human and rat liver systems. In Caco-2 cells, demonstrated to contain minimal CYP3A activity, the permeability coefficient of the prodrugs lovastatin and enalapril was increased in the presence of the active flavonoids kaempferol and naringenin, consistent with inhibition of esterase activity. In rats, oral coadministration of kaempferol and naringenin with these prodrugs led to significant increases in plasma exposure to the active acids. In addition, in portal vein-cannulated rats, coadministration of lovastatin with kaempferol (10 mg/kg) led to a 154% and a 113% increase in the portal plasma exposure to the prodrug and active acid, respectively, compared with coadministration with water. The contribution of CYP3A inhibition was demonstrated to be minimal. Overall, a series of flavonoids present in GFJ are identified as esterase inhibitors, of which kaempferol and naringenin are shown to mediate pharmacokinetic drug interaction with the prodrugs lovastatin and enalapril due to their capability of esterase inhibition.

A novel model for the prediction of drug-drug interactions in humans based on in vitro cytochrome p450 phenotypic data.

Ketoconazole has generally been used as a standard inhibitor for studying clinical pharmacokinetic drug-drug interactions (DDIs) of drugs that are primarily metabolized by CYP3A4/5. However, ketoconazole at therapeutic, high concentrations also inhibits cytochromes P450 (P450) other than CYP3A4/5, which has made the predictions of DDIs less accurate. Determining the in vivo inhibitor concentration at the enzymatic site is critical for predicting the clinical DDI, but it remains a technical challenge. Various approaches have been used in the literature to estimate the human hepatic free concentrations of this inhibitor, and application of those to predict DDIs has shown some success. In the present study, a novel approach using cryopreserved human hepatocytes suspended in human plasma was applied to mimic the in vivo concentration of ketoconazole at the enzymatic site. The involvement of various P450s in the metabolism of compounds of interest was quantitatively determined (reactive phenotyping). Likewise, the effect of ketoconazole on various P450s was quantitated. Using this information, P450-mediated change in the area under the curve has been predicted without the need of estimating the inhibitor concentrations at the enzyme active site or the Ki. This approach successfully estimated the magnitude of the clinical DDI of an investigational compound, MLX, which is cleared by multiple P450-mediated metabolism. It also successfully predicted the pharmacokinetic DDIs for several marketed drugs (theophylline, tolbutamide, omeprazole, desipramine, midazolam, alprazolam, cyclosporine, and loratadine) with a correlation coefficient (r2) of 0.992. Thus, this approach provides a simple method to more precisely predict the DDIs for P450 substrates when coadministered with ketoconazole or any other competitive P450 inhibitors in humans.

P-Glycoprotein and Breast Cancer Resistance Protein Affect Disposition of Tandutinib, A Tyrosine Kinase Inhibitor

Tandutinib is a tyrosine kinase inhibitor under investigation for the treatment of solid and hematological tumors. We evaluated efflux transporter substrate specificity of tandutinib in Caco-2 cells, and the role of efflux transporters in the disposition of tandutinib in rats and efflux transporter knock-out mice. These studies demonstrated that tandutinib is a substrate of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) in Caco-2 cells. In rats, administration of GF120918, before treatment with tandutinib orally resulted in approximately a seven-fold increase in the mean plasma area under the concentration-versus-time curve (AUC) compared to the vehicle control group. In mice, after intravenous administration of tandutinib, the mean plasma AUC values in the Bcrp1(-/-) mice and Mdr1a/b(-/-) mice was 1.53- and 1.20-fold greater than that of the wild type (WT) mice, respectively. After oral administration, the drug exposure in Mdr1a/b(-/-), Bcrp1(-/-), and Mdr1a/b(-/-)/Bcrp1(-/-) mice was higher than in the WT mice. The brain to plasma exposure ratio (B/P) of tandutinib in Mdr1a/b(-/-) mice increased by 2- to 3-fold over that in the WT mice. There was a 13-fold increase in B/P in Mdr1a/b(-/-)/Bcrp1(-/-) mice. This finding illustrates that P-gp and Bcrp play a role in oral absorption, systemic clearance, and brain penetration of tandutinib in the rodents. P-gp affected oral absorption and brain penetration of tandutinib to a greater extent than Bcrp, but Bcrp contribution to systemic clearance of tandutinib was greater than P-gp. Thus, co-administration of efflux pump inhibitors may be a useful strategy to enhance tandutinib absorption and brain penetration clinically.

Prediction of Pharmacokinetic Drug-Drug Interactions Using Human Hepatocyte Suspension in Plasma and Cytochrome P450 Phenotypic Data. III. In Vitro-in Vivo Correlation with Fluconazole

Traditional cytochrome P450 (P450) based drug-drug interaction (DDI) predictions are based on the ratio of an inhibitors physiological concentration [I] and its inhibition constant Ki. Determining [I] at the enzymatic site, although critical for predicting clinical DDIs, remains a technical challenge. In our previous study, a novel approach using cryopreserved human hepatocytes suspended in human plasma was investigated to mimic the in vivo concentration of ketoconazole at the enzymatic site (Lu et al., 2007), effectively eliminating the estimation of the elusive [I] value. P450 inhibition in this system appears to model that in vivo. Using the ketoconazole inhibition information in a human hepatocyte-plasma suspension together with quantitative P450 phenotypic information, we successfully predicted the pharmacokinetic DDIs for a small set of drugs, such as theophylline, tolbutamide, omeprazole, desipramine, midazolam, loratadine, cyclosporine, and alprazolam, as well as an investigational compound. For the applicability of this model on a wider scale the in vitro-in vivo correlation data set needed to be expanded. However, for most drugs in the literature there is not enough quantitative information on the involvement of individual P450s to predict DDIs retrospectively. To facilitate that, in this study we determined quantitative P450 phenotyping for seven marketed drugs: budesonide, buprenorphine, loratadine, sirolimus, tacrolimus, docetaxel, and methylprednisolone. Augmentation of the new data set with the one generated previously produced broader a database that provided further support for the wider applicability of this approach using ketoconazole as a potent CYP3A inhibitor. This application is predicted to be equally effective with other P450 inhibitors that are not substrates of efflux pumps.

Disposition of Caspofungin, a Novel Antifungal Agent, in Mice, Rats, Rabbits, and Monkeys

ABSTRACT The metabolism, excretion, and pharmacokinetics of caspofungin (Cancidas; Merck & Co., Inc.) were investigated after administration of a single intravenous dose to mice, rats, rabbits, and monkeys. Caspofungin had a low plasma clearance (0.29 to 1.05 ml/min/kg) and a long terminal elimination half-life (11.7 h to 59.7 h) in all preclinical species. The elimination kinetics of caspofungin were multiphasic and displayed an initial distribution phase followed by a dominant β-elimination phase. The presence of low levels of prolonged radioactivity in plasma was observed and was partially attributable to the chemical degradation product M0. Excretion studies with [3H]caspofungin indicated that the hepatic and renal routes play an important role in the elimination of caspofungin, as a large percentage of the radiolabeled dose was recovered in urine and feces. Excretion of radioactivity in all species studied was slow, and low levels of radioactivity were detected in daily urine and fecal samples throughout a prolonged collection period. Although urinary profiles indicated the presence of several metabolites (M0, M1, M2, M3, M4, M5, and M6), the majority of the total radioactivity was associated with the polar metabolites M1 [4(S)-hydroxy-4-(4-hydroxyphenyl)-l-threonine] and M2 [N-acetyl-4(S)-hydroxy-4-(4-hydroxyphenyl)-l-threonine]. Caspofungin was thus primarily eliminated by metabolic transformation; however, the rate of metabolism was slow. These results suggest that distribution plays a prominent role in determining the plasma pharmacokinetics and disposition of caspofungin, as very little excretion or biotransformation occurred during the early days after dose administration, a period during which concentrations in plasma fell substantially. The disposition of caspofungin in preclinical species was similar to that reported previously in humans.

MLN8054 and Alisertib (MLN8237): Discovery of Selective Oral Aurora A Inhibitors

The Aurora kinases are essential for cell mitosis, and the dysregulation of Aurora A and B have been linked to the etiology of human cancers. Investigational agents MLN8054 (8) and alisertib (MLN8237, 10) have been identified as high affinity, selective, orally bioavailable inhibitors of Aurora A that have advanced into human clinical trials. Alisertib (10) is currently being evaluated in multiple Phase II and III clinical trials in hematological malignancies and solid tumors.