Utilization of estimated physicochemical properties as an integrated part of predicting hepatic clearance in the early drug-discovery stage: Impact of plasma and microsomal binding.

Rapid prediction of hepatic clearance for drug candidates plays an important role for decision-making in the early drug-discovery stage. Although knowledge of protein binding in both plasma and microsomal components is needed in the prediction of metabolic clearance from metabolic stability studies, the capacity of protein binding assays are generally lower than those of metabolic stability assays. However, many in silico prediction methods for protein binding are now available and software packages such as ACDLabs, ADMET Predictor and SimCYP incorporate various aspects of in silico predictions relevant to estimating binding and clearance. This has facilitated the use of various estimated or measured physicochemical parameters, relevant to binding, to predict clearance. In this study, prediction of protein binding for 33 drugs was evaluated using various combinations of estimated physicochemical properties. Subsequently, the most accurate estimated protein binding values were used to predict hepatic clearance using the SimCYP software. For the drugs used herein, SimCYP provided the most accurate prediction for protein binding in both plasma and microsomes using physiochemical properties estimated with the ACDLabs software. In conclusion, the use of in silico methods as an integrated part of predicting hepatic clearance in early drug-discovery stage is recommended.

Functional characterization of CYP3A4.16: catalytic activities toward midazolam and carbamazepine.

1. To assess the substrate-dependent effects of the low-activity allele of human CYP3A4, CYP3A4*16 (Thr185Ser), a recombinant wild-type (CYP3A4.1) or variant (CYP3A4.16) protein was co-expressed with human NADPH-P450 reductase in Sf21 insect cells using a baculovirus-insect cell system. 2. The holo-CYP3A4 protein level of CYP3A4.16 in insect microsomes was slightly higher than that of CYP3A4.1, while no difference in total (apo- and holo-) CYP3A4 protein levels was observed between them. 3. When midazolam was used as a substrate, K(m) and V(max) for 1'-hydroxylation in CYP3A4.16 were significantly higher and lower, respectively, than those in the wild-type, resulting in a 50% decrease in intrinsic clearance (V(max)/K(m)) of the variant. In contrast, intrinsic clearance for 4-hydroxylation of the variant was decreased by 30% due to a significant increase in K(m) without a difference in V(max). 4. Both the wild-type and variant exhibited sigmoidal kinetic profiles for carbamazepine 10,11-epoxide formation. When the modified two-site equation was applied for the analysis of kinetic parameters, K(m2) and V(max2) of CYP3A4.16 were approximately two times higher and lower than those of the wild-type, resulting in a 74% decrease in intrinsic clearance. 5. These results demonstrated that CYP3A4.16 shows the substrate-dependent altered kinetics compared with CYP3A4.1.

Characterization of Contributing Factors to Variability in Morphine Clearance Through PBPK Modeling Implemented With OCT1 Transporter.

Morphine shows large interindividual variability in its pharmacokinetics; however, the cause of this has not been fully addressed. The variability in morphine disposition is considered to be due to a combination of pharmacogenetic and physiological determinants related to morphine disposition. We previously reported the effect of organic cation transporter (OCT1) genotype on morphine disposition in pediatric patients. To further explore the underlying mechanisms for variability arising from relevant determinants, including OCT1, a physiologically based pharmacokinetic (PBPK) model of morphine was developed. The PBPK model predicted morphine concentration-time profiles well, in both adults and children. Almost all of the observed morphine clearances in pediatric patients fell within a twofold range of median predicted values for each OCT1 genotype in each age group. This PBPK modeling approach quantitatively demonstrates that OCT1 genotype, age-related growth, and changes in blood flow as important contributors to morphine pharmacokinetic (PK) variability.

Characterizing the Developmental Trajectory of Sirolimus Clearance in Neonates and Infants.

Sirolimus is increasingly being used in neonates and infants, but the mechanistic basis of age-dependent changes in sirolimus disposition has not been fully addressed yet. In order to characterize the age-dependent changes, serial sirolimus clearance (CL) estimates in individual young pediatric patients were collected and analyzed by population modeling analysis. In addition, sirolimus metabolite formation was also investigated to further substantiate the corresponding age-dependent change in CYP3A activity. The increasing pattern over time of allometrically size-normalized sirolimus CL estimates vs. age was well described by a sigmoidal Emax model. This age-dependent increase was also observed within each individual patient over a 4-year study period. CYP3A-dependent sirolimus metabolite formation changed in a similar fashion. This study clearly demonstrates the rapid increase of sirolimus CL over time in neonates and infants, indicating the developmental change. This developmental pattern can be explained by a parallel increase in CYP3A metabolic activity.

Modeling and simulation in pediatric drug therapy: Application of pharmacometrics to define the right dose for children.

During the past decades significant progress has been made in our understanding of the importance of age-appropriate development of new drug therapies in children. Importantly, several regulatory initiatives in Europe and the US have provided a framework for a rationale. In the US, most notably the enactment of the Best Pharmaceuticals for Children Act (BPCA) and Product Research and Equity Act (PREA) has facilitated the studying of on-patent and off-patent drugs in children. The biggest challenge in pediatric studies is defining a safe and effective dose or dose range in a patient population that can span from premature neonates to adolescents. From a mechanism-based perspective, advances in the science of quantitative pharmacology and pharmacometrics have resulted in the development of model-based approaches to better describe and understand important age-related factors influencing drug disposition and response in pediatric patients. The application of modeling and simulation has been shown to result in better estimates of pediatric doses as evidenced by several studies, although the optimal approach is still being debated. The extrapolation of efficacy findings from adults to the pediatric population has streamlined the development process especially for studies in older children. However, a focus on developmental changes in neonates and infants as well as further developing a paradigm for conducting pharmacodynamic studies in neonates, infants, and children remain important unmet needs. In this overview we will review current approaches for age-appropriate dose selection and highlight ongoing efforts to define exposure-response and clinical outcome relationships across the pediatric age spectrum.

Development of a Pediatric Physiologically Based Pharmacokinetic Model for Sirolimus: Applying Principles of Growth and Maturation in Neonates and Infants.

This study describes the maturation of sirolimus clearance in a cohort of very young pediatric patients with vascular anomalies. The relationship between allometrically scaled in vivo clearance and age was described by the Emax model in patients aged 1 month to 2 years. Consistent with the observed increase, in vitro intrinsic clearance of sirolimus using pediatric liver microsomes showed a similar age-dependent increase. In children older than 2 years, allometrically scaled sirolimus clearance did not show further maturation. Simulated clearance estimates with a sirolimus physiologically based pharmacokinetic model that included CYP3A4/5/7 and CYP2C8 maturation profiles were in close agreement with observed in vivo clearance values. In addition, physiologically based pharmacokinetic model-simulated sirolimus pharmacokinetic profiles predicted the actual observations well. These results demonstrate the utility of a physiologically based pharmacokinetic modeling approach for the prediction of the developmental trajectory of sirolimus metabolic activity and its effects on total body clearance in neonates and infants.

Development of a Physiologically-Based Pharmacokinetic Model for Sirolimus: Predicting Bioavailability Based on Intestinal CYP3A Content.

Sirolimus is an inhibitor of mammalian target of rapamycin (mTOR) and is increasingly being used in transplantation and cancer therapies. Sirolimus has low oral bioavailability and exhibits large pharmacokinetic variability. The underlying mechanisms for this variability have not been explored to a large extent. Sirolimus metabolism was characterized by in vitro intrinsic clearance estimation. Pathway contribution ranked from CYP3A4 > CYP3A5 > CYP2C8. With the well stirred and Qgut models sirolimus bioavailability was predicted at 15%. Interindividual differences in bioavailability could be attributed to variable intestinal CYP3A expression. The physiologically-based pharmacokinetics (PBPK) model developed in Simcyp predicted a high distribution of sirolimus into adipose tissue and another elimination pathway in addition to CYP-mediated metabolism. PBPK model predictive performance was acceptable with Cmax and area under the curve (AUC) estimates within 20% of observed data in a dose escalation study. The model also showed potential to assess the impact of hepatic impairment and drug-drug interaction (DDI) on sirolimus pharmacokinetics.CPT: Pharmacometrics & Systems Pharmacology (2013) 2, e59; doi:10.1038/psp.2013.33; published online 24 July 2013.

Non-invasive method to detect induction of CYP3A4 in chimeric mice with a humanized liver.

Chimeric mice with a humanized liver have been previously established by the transplantation of human hepatocytes to urokinase-type plasminogen activator/severe combined immunodeficiency mice. A non-invasive method to detect the induction of cytochrome P450 (CYP) 3A4 was evaluated in chimeric mice with a humanized liver. Dexamethasone (DEX) was used as a probe drug to detect induction; and rifampicin was used as a model drug to induce CYP3A4. Before and after rifampicin treatment (50 mg kg(-1), intraperitoneal injection once a day for 4 days) in the chimeric mice, DEX was subcutaneously injected and the urinary excretion of 6beta-hydroxydexamethason (6betaOHD) and DEX was determined. The metabolic ratio (6betaOHD/DEX) significantly increased after rifampicin treatment. Livers from the control and rifampicin-treated chimeric mice were stained immunohistolochemically with antibodies against CYP3A4 and CYP3A5. CYP3A4 and CYP3A5 were detected in the area of humanized liver, but staining was intense for CYP3A4 and very weak for CYP3A5. Only the staining of CYP3A4 was increased after rifampicin treatment. Formation of 6betaOHD by human liver microsomes was higher than that formed by mouse liver microsomes. Metabolite formation was catalysed by both CYP3A4 and CYP3A5 and the intrinsic clearance (V(max)/K(m)) by CYP3A4 was found to be 50-fold higher than that of CYP3A5. The results of the present study indicate that estimation of the changes of the urinary metabolic ratio (6betaOHD/DEX) in the chimeric mice with a humanized liver is a very useful tool for detecting the induction of CYP3A4 by a non-invasive method.

Approach to predict the contribution of cytochrome P450 enzymes to drug metabolism in the early drug-discovery stage: the effect of the expression of cytochrome b(5) with recombinant P450 enzymes.

In order to evaluate the potential adverse effects due to genetic polymorphism and/or inter-individual variation, it is necessary to calculate the cytochrome P450 (CYP) contribution to the metabolism of new drugs. In the current study, the in vitro intrinsic clearance (CL(int)) values of marker substrates and drugs were determined by measuring metabolite formation and substrate depletion, respectively. Recombinant CYP microsomes expressing CYP2C9, CYP2C19 and CYP3A4 with co-expressed cytochrome b(5) were used, but those expressing CYP1A2 and CYP2D6 did not have co-expressed cytochrome b(5). The following prediction methods were compared to determine the CL(int) value using data from recombinant CYP enzymes: (1) relative CYP enzyme content in human liver microsomes; (2) relative activity factor (RAF) estimated from the V(max) value; and (3) RAF estimated from the CL(int) value. Estimating RAF from CL(int) proved the most accurate prediction method among the three tested, and differences in the CYP3A4 marker reactions did not affect its accuracy. The substrate depletion method will be useful in the early drug-discovery stage when the main metabolite and/or metabolic pathway has not been identified. In addition, recombinant CYP microsomes co-expressed with cytochrome b(5) might be suitable for the prediction of the CL(int) value.

Relative roles of CYP2C19 and CYP3A4/5 in midazolam 1'-hydroxylation.

1. During the characterization of recombinant CYP2C19, it was observed that this enzyme metabolized midazolam, which is generally regarded as CYP3A4/5 substrate, and we therefore decided to pursue this observation further. 2. CYP2C19 showed a Michaelis-Menten pattern for midazolam 1'-hydroxylation and was inhibited by (+)-N-3-benzylnirvanol and S-mephenytoin, which are a standard potent inhibitor and a substrate of CYP2C19, respectively. 3. The inhibitory potency by CYP3A4/5 inhibitor on the midazolam 1'-hydroxylation in human liver microsomes (HLM) was correlated with the CYP3A4/5 specific catalytic activity, but such correlation was not observed in CYP2C19 enzyme. The in vitro intrinsic clearance value for midazolam 1'-hydroxylation was not changed by the addition of (+)-N-3-benzylnirvanol in four individual HLM preparations. 4. These results indicated that although CYP2C19 is capable of catalyzing midazolam 1'-hydroxylation, CYP3A4/5 play a more important role.

Metabolism of CJ-036878, N-(3-phenethoxybenzyl)-4-hydroxybenzamide, in liver microsomes and recombinant cytochrome P450 enzymes: metabolite identification by LC-UV/MS(n) and (1)H-NMR.

The identification of metabolites in the early stages of drug discovery is important not only for guiding structure-activity relationships (SAR) and structure-metabolism relationships (SMR) strategies, but also for predicting the potential for adverse events. The present study investigated the phase I metabolism of CJ-036878 (N-(3-phenethoxybenzyl)-4-hydroxybenzamide), a potent antagonist of the N-methyl-D-asparatate (NMDA) receptor, using liver microsomes and representative recombinant cytochrome P450 enzymes. The structures of the oxidative metabolites M1-M11 were confirmed by LC-UV/MS(n) and/or (1)H-nuclear magnetic resonance (NMR). It was found that CJ-036878 is metabolized through three routes: (1) aliphatic hydroxylation that generates M1 and M2; (2) aromatic hydroxylation that produces M3-M5, M7 and M8; and (3) dimerization through an oxidative phenol coupling reaction that yields M10 and M11. The use of recombinant human cytochrome P450 enzymes suggested that CYP3A4 is the major enzyme involved in the oxidative metabolism of CJ-036878, with minor contributions from CYP1A2, CYP2C19, and CYP2D6.

CYP3A4 and CYP3A5 catalyse the conversion of the N-methyl-D-aspartate (NMDA) antagonist CJ-036878 to two novel dimers.

CJ-036878, N-(3-phenethoxybenzyl)-4-hydroxybenzamide, was developed as an antagonist of the N-methyl-D-aspartate receptor NR2B subunit. Two dimeric metabolites, CJ-047710 and CJ-047713, were identified from the incubation mixture with CJ-036878 in human liver microsomes (HLM). The identification of the enzymes involved in the formation of these dimeric metabolites was investigated in the current study. Inhibition of the formation of CJ-047710 and CJ-047713 in pooled HLM by 1-aminobenztriazole, SKF-525A, and ketoconazole were observed. Ketoconazole played a significant role in inhibiting formation of these two metabolites in a concentration-dependent manner. Recombinant CYP3A4 and CYP3A5 exhibited a markedly high activity toward the formation of CJ-047710 and CJ-047713 from CJ-036878, but the contribution of other CYP enzymes to these formations was at a very low level or negligible. The formation of CJ-047710 and CJ-047713 in pooled HLM, CYP3A4, and CYP3A5 showed sigmoid characteristics. S50 values for CJ-047710 and CJ-047713 formation in HLM were almost equivalent with those for CYP3A4 and CYP3A5. For the CYP3A enzymes, maximal clearance due to auto-activation values for CJ-047710 and CJ-047713 formation catalysed by CYP3A5 were 3.6- and 3.1-fold higher than those catalysed by CYP3A4. This is the first report that shows both CYP3A4 and CYP3A5 simultaneously contribute to dimerization through oxidative C-C and C-O coupling reactions.

Enzymatic characteristics of CYP3A5 and CYP3A4: a comparison of in vitro kinetic and drug-drug interaction patterns.

CYP3A4 and CYP3A5 exhibit significant overlap in substrate specificity, but can differ in catalytic activity and regioselectivity. To investigate their characteristics further, the enzymatic reactions of the two CYP3A enzymes were compared using midazolam, nifedipine, testosterone and terfenadine as substrates. Both CYP3A5 and CYP3A4 showed sigmoid and substrate inhibition patterns for testosterone 6beta-hydroxylation and terfenadine t-butylhydroxylation (TFDOH), respectively. In the other reactions, the kinetic model for CYP3A5 was not similar to that for CYP3A4. An inhibition study demonstrated that the interactions between alpha-naphthoflavone (alphaNF) and CYP3A substrates were different for the two CYP3A enzymes. alphaNF stimulated nifedipine oxidation catalysed by CYP3A5, but did not stimulate that catalysed by CYP3A4. alphaNF at less than 32 microM inhibited TFDOH catalysed by CYP3A5, but did not inhibit that catalysed by CYP3A4. These results indicate that CYP3A5 has different enzymatic characteristics from CYP3A4 in some CYP3A catalysed reactions.

Approach to the prediction of the contribution of major cytochrome P450 enzymes to drug metabolism in the early drug-discovery stage.

It is important to determine the cytochrome P450 (CYP) contribution of certain drugs by taking into consideration the attrition due to issues such as genetic polymorphism and inter-individual variation. In many cases in the early discovery stage, the metabolites of a new chemical have not been identified. Therefore, the present paper devised an approach in which the in vitro intrinsic clearance (CLint) value for new chemicals was determined by measuring substrate depletion. The following prediction methods were compared to calculate CLint using data from recombinant CYP enzymes: (1) the relative CYP content in human liver microsomes; (2) the relative activity factor (RAF) based on the Vmax value; and (3) the RAF value based on the CLint value. The most accurate prediction method was RAF based on CLint. This method would be useful in the early drug-discovery process in cases in which the main metabolite is not identified.

Characterization of cytochrome P450 enzymes involved in drug oxidations in mouse intestinal microsomes.

1. Cytochrome P450 (P450, CYP) enzymes involved in drug oxidations in mouse intestines were characterized for their role in the first-pass metabolism of xenobiotics. 2. Preparation of mouse intestinal microsomes using a buffer containing glycerol and protease inhibitors including (p-amidinophenyl) methanesulphonyl fluoride, EDTA, soybean trypsin inhibitor, aprotinin, bestatin and leupeptine gave the highest testosterone 6beta-hydroxylase activity among several preparation buffers tested in this study. Testosterone 6beta-hydroxylase activity catalysed by mouse intestinal microsomes subjected to freezing and thawing was lower than that catalysed by unfrozen intestinal microsomes. 3. Low but significant catalytic activities of nifedipine oxidation, midazolam 1'- and 4-hydroxylation, chlorzoxazone 6-hydroxylation, bufuralol 1'- and 6-hydroxylations and tolbutamide methylhydroxylation were observed in mouse intestinal microsomes. Testosterone 6beta-hydroxylation, chlorzoxazone 6-hydroxylation, and bufuralol 1'- and 6-hydroxylations were inhibited by ketoconazole, diethyldithiocarbamate and quinine respectively. 4. Immunoblot analysis using anti-rat CYP3A antibodies demonstrated two immunoreactive bands showing similar migration in mouse intestinal and hepatic microsomes, although studies using anti-CYP1A, anti-CYP2C, anti-CYP2D and anti-CYP2E1 antibodies did not detect any band in mouse intestinal microsomes. 5. The results suggest that mouse intestinal microsomes should be prepared with glycerol and several protease inhibitors and that Cyp3a enzymes probably play an important role in drug oxidations catalysed by mouse intestine.

Use of everted sacs of mouse small intestine as enzyme sources for the study of drug oxidation activities in vitro.

1. The use of everted sacs of the small intestine as an enzyme source for the study of the first-pass metabolism of xenobiotics by cytochrome P450s (P450, CYP) is described. Several drug oxidation activities for testosterone, chlorzoxazone, tolbutamide, bufuralol and warfarin were observed when everted sacs (1-cm segment) from different parts of mouse small intestine were incubated with an NADPH-generating system and each substrate. 2. Most of the drug hydroxylase activities resided in the upper part of mouse small intestine and these activities were much higher than those of intestinal microsomes. Drug oxidation activities decreased along the distance from the upper part of the small intestine except for warfarin hydroxylation. 3. Testosterone 6beta-hydroxylation in the everted sacs exhibited the highest catalytic activities among the drug oxidations tested here. In the upper part of the small intestine, the testosterone 6beta-hydroxylase activities of everted sacs subjected once to freezing and thawing were substantially decreased compared with the untreated everted sacs. 4. Testosterone 6beta-hydroxylase activities in the everted sacs of the small intestine were significantly inhibited by ketoconazole. Immunoreactive proteins using anti-CYP3A antibodies were detected in the upper and middle parts of the small intestine. 5. The results demonstrated that the upper part of the mouse small intestine serves as the major site for intestinal P450 mediated first-pass metabolism. Everted sacs of the small intestine are therefore useful for the study of drug metabolism as well as of transport and absorption.

Cooperativity of alpha-naphthoflavone in cytochrome P450 3A-dependent drug oxidation activities in hepatic and intestinal microsomes from mouse and human.

1. The effects of several CYP3A substrates (alpha-naphthoflavone (alphaNF), terfenadine, midazolam, erythromycin) on nifedipine oxidation and testosterone 6beta-hydroxylation activities were investigated in hepatic and intestinal microsomes from mouse and human. 2. alphaNF (10 microM) and terfenadine (100 microM) inhibited nifedipine oxidation activities (at substrate concentration of 100 microM) in mouse hepatic microsomes to approximately 50%, but not in mouse intestinal microsomes. alphaNF (30 microM) stimulated nifedipine oxidation activities in mouse and human intestinal microsomes and in human hepatic microsomes to approximately 1.3-1.8-fold. Inhibitory potencies (50% inhibition concentration, IC50) of midazolam and erythromycin for nifedipine oxidations were calculated to be approximately 90 microM in human intestinal microsomes. In contrast, testosterone (100 microM) stimulated the nifedipine oxidation activities approximately 1.5-fold in hepatic and intestinal microsomes from mouse and human. 3. alphaNF showed different effects on the kinetic parameters including the Hill coefficients of nifedipine oxidation and testosterone 6beta-hydroxylation catalysed by hepatic and intestinal microsomes from mouse and human. Cooperativity in nifedipine oxidation was increased by the addition of alphaNF to pooled human hepatic microsomes, but little effects of alphaNF could be observed in individual human intestinal microsomes. 4. These results suggest that CYP3A enzymes in liver and intestine might have different characteristics and that observations from hepatic microsomes should not be directly applicable to intestine metabolism in some cases. Studies of drug-drug interactions of CYP3A substrates are recommended to be performed using intestinal samples.