Toshio Teramura

A Comparison of Pharmacokinetics Between Humans and Monkeys

To verify the availability of pharmacokinetic parameters in cynomolgus monkeys, hepatic availability (Fh) and the fraction absorbed multiplied by intestinal availability (FaFg) were evaluated to determine their contributions to absolute bioavailability (F) after intravenous and oral administrations. These results were compared with those for humans using 13 commercial drugs for which human pharmacokinetic parameters have been reported. In addition, in vitro studies of these drugs, including membrane permeability, intrinsic clearance, and p-glycoprotein affinity, were performed to classify the drugs on the basis of their pharmacokinetic properties. In the present study, monkeys had a markedly lower F than humans for 8 of 13 drugs. Although there were no obvious differences in Fh between humans and monkeys, a remarkable species difference in FaFg was observed. Subsequently, we compared the FaFg values for monkeys with the in vitro pharmacokinetic properties of each drug. No obvious FaFg differences were observed between humans and monkeys for drugs that undergo almost no in vivo metabolism. In contrast, low FaFg were observed in monkeys for drugs that undergo relatively high metabolism in monkeys. These results suggest that first-pass intestinal metabolism is greater in cynomolgus monkeys than in humans, and that bioavailability in cynomolgus monkeys after oral administration is unsuitable for predicting pharmacokinetics in humans. In addition, a rough correlation was also observed between in vitro metabolic stability and Fg in humans, possibly indicating the potential for Fg prediction in humans using only in vitro parameters after slight modification of the evaluation system for in vitro intestinal metabolism.

Detection of cocrystal formation based on binary phase diagrams using thermal analysis.

ABSTRACTPurposeAlthough a number of studies have reported that cocrystals can form by heating a physical mixture of two components, details surrounding heat-induced cocrystal formation remain unclear. Here, we attempted to clarify the thermal behavior of a physical mixture and cocrystal formation in reference to a binary phase diagram.MethodsPhysical mixtures prepared using an agate mortar were heated at rates of 2, 5, 10, and 30°C/min using differential scanning calorimetry (DSC). Some mixtures were further analyzed using X-ray DSC and polarization microscopy.ResultsWhen a physical mixture consisting of two components which was capable of cocrystal formation was heated using DSC, an exothermic peak associated with cocrystal formation was detected immediately after an endothermic peak. In some combinations, several endothermic peaks were detected and associated with metastable eutectic melting, eutectic melting, and cocrystal melting. In contrast, when a physical mixture of two components which is incapable of cocrystal formation was heated using DSC, only a single endothermic peak associated with eutectic melting was detected.ConclusionThese experimental observations demonstrated how the thermal events were attributed to phase transitions occurring in a binary mixture and clarified the relationship between exothermic peaks and cocrystal formation.

Quantitative Prediction of Intestinal Metabolism in Humans from a Simplified Intestinal Availability Model and Empirical Scaling Factor

This study aimed to establish a practical and convenient method of predicting intestinal availability (Fg) in humans for highly permeable compounds at the drug discovery stage, with a focus on CYP3A4-mediated metabolism. We constructed a “simplified Fg model,” described using only metabolic parameters, assuming that passive diffusion is dominant when permeability is high and that the effect of transporters in epithelial cells is negligible. Five substrates for CYP3A4 (alprazolam, amlodipine, clonazepam, midazolam, and nifedipine) and four for both CYP3A4 and P-glycoprotein (P-gp) (nicardipine, quinidine, tacrolimus, and verapamil) were used as model compounds. Observed fraction of drug absorbed (FaFg) values for these compounds were calculated from in vivo pharmacokinetic (PK) parameters, whereas in vitro intestinal intrinsic clearance (CLint,intestine) was determined using human intestinal microsomes. The CLint,intestine for the model compounds corrected with that of midazolam was defined as CLm,index and incorporated into a simplified Fg model with empirical scaling factor. Regardless of whether the compound was a P-gp substrate, the FaFg could be reasonably fitted by the simplified Fg model, and the value of the empirical scaling factor was well estimated. These results suggest that the effects of P-gp on Fa and Fg are substantially minor, at least in the case of highly permeable compounds. Furthermore, liver intrinsic clearance (CLint,liver) can be used as a surrogate index of intestinal metabolism based on the relationship between CLint,liver and CLm,index. Fg can be easily predicted using a simplified Fg model with the empirical scaling factor, enabling more confident selection of drug candidates with desirable PK profiles in humans.

Marked species differences in the bioavailability of midazolam in cynomolgus monkeys and humans

The bioavailability (F) of midazolam in cynomolgus monkeys (0.02) was markedly lower than that in humans (0.24–0.46) and the reason for this difference in F between the two species was investigated. Based on the area under the plasma concentration–time curve after intravenous and intraportal infusion to cynomolgus monkeys, the hepatic availability (Fh) was estimated as 0.66. The fraction of dose absorbed (Fa) estimated from the single-pass intestinal perfusion method was 1.0 in cynomolgus monkeys. The intestinal availability (Fg = F/Fa/Fh) was calculated as 0.03 in cynomolgus monkeys. Since the Fa of midazolam has been reported to be almost 1.0 in humans, Fh and Fg were calculated as 0.33–0.76 and 0.46–1.00 when the reference values for hepatic blood flow (1026–1530 ml h−1 kg−1) were used. In conclusion, the main reason for low F in cynomolgus monkeys was the markedly higher first-pass intestinal metabolism seen in cynomolgus monkeys compared with humans.

Prediction of Human Metabolism of FK3453 by Aldehyde Oxidase Using Chimeric Mice Transplanted with Human or Rat Hepatocytes

During drug development, it is important to predict the activities of multiple metabolic enzymes, not only cytochrome P450 (P450) but also non-P450 enzymes, such as conjugative enzymes and aldehyde oxidase (AO). In this study, we focused on prediction of AO-mediated human metabolism and pharmacokinetics (PK) of 6-(2-amino-4-phenylpyrimidine-5-yl)-2-isopropylpyridazin-3(2H)-one (FK3453) (Astellas Pharma Inc.), the development of which was suspended due to extremely low exposure in human, despite good oral bioavailability in rat and dog. We examined species difference in oxidative metabolism of the aminopyrimidine moiety of FK3453, catalyzed by AO, using human-chimeric mice with humanized liver (h-PXB mice) and rat-chimeric mice (r-PXB mice) transplanted with rat hepatocytes. AO activity of h-PXB mouse hepatocytes was higher than that of r-PXB mouse hepatocytes. Moreover, higher concentrations of human-specific AO-generated FK3453 metabolite A-M were detected in urine and feces after administration of FK3453 to h-PXB mice versus r-PXB mice. The total clearance of h-PXB mice was 2-fold higher than that of r-PXB mice. These results agreed reasonably well with the metabolism and PK profiles of FK3453 in human and rat. Our results indicated that h-PXB mice should be helpful for predicting the metabolic profile of drugs in humans, and the use of both h-PXB and r-PXB mice should be helpful for evaluation of species differences of AO metabolic activity.

Species differences in intestinal glucuronidation activities between humans, rats, dogs and monkeys

Abstract 1. Glucuronidation via UDP-glucuronosyltransferase (UGT) in the intestine has been reported to influence the pharmacokinetics (PK) of drugs; however, information concerning the differences in activity between species is limited. Here, we investigated the in vitro and in vivo activities of intestinal glucuronidation for 17 UGT substrates in humans, rats, dogs and monkeys. 2. Although in vitro intrinsic clearance (CLint,u,UGT) in intestinal microsomes showed a good correlation between humans and laboratory animals, values tended to be lower in humans than in laboratory animals. The ratio of CLint,u,UGT in the absence and presence of bovine serum albumin differed between species. In vivo, the fraction of drug absorbed (FaFg) in humans correlated with that in dogs and monkeys, but not in rats. 3. While an inverse correlation between CLint,u,UGT and FaFg was observed in each species, the CLint,u,UGT values in the intestinal microsomes corresponding to FaFg values in dogs were three to four times higher than in other animals. 4. These results indicate the need for a degree of caution when extrapolating PK data from laboratory animals to humans.

Pharmacokinetics of barnidipine hydrochloride, a new dihydropyridine calcium channel blocker, in the rat, dog and human

1. The pharmacokinetics of a new calcium antagonist barnidipine hydrochloride, a stereochemically pure enantiomer, was studied after intravenous and oral dosing to the rat and dog, and oral to man. 2. After intravenous dosing, plasma concentrations of barnidipine hydrochloride declined bi-exponentially with the terminal half-lives of 0.6 h in the rat and 4.1 h in the dog. The blood clearance was 5.2 l/h/kg in the rat and 3.3 l/h/kg in the dog, and was comparable with hepatic blood flow in both species. 3. After oral dosing, plasma concentrations of barnidipine hydrochloride peaked rapidly (0.3-0.4 h in the rat and dog, 1.0-1.6 h in man). Cmax and AUC rose non-linearly with increasing doses in all three species. 4. The absolute bioavailability was low (11-18% in the rat and 6-9% in the dog), suggesting a marked first-pass metabolism.

Comparison of intestinal metabolism of CYP3A substrates between rats and humans: application of portal–systemic concentration difference method

Abstract 1. Rats are frequently used in pharmacokinetic studies during drug discovery. However, there is limited information regarding species differences in intestinal availability (Fg) between rats and humans. 2. Here, we directly estimated the fraction of dose absorbed in the portal vein (FaFg) of rats for nine CYP3A substrates using portal–systemic concentration difference method and compared them with human FaFg. No distinct difference in FaFg between the two species was observed, and seven of the nine compounds were within a two-fold difference. Given that their net fraction of dose absorbed (Fa) are expected to be high, this result indicates a moderate correlation in Fg between the two species. 3. In contrast, the in vitro intrinsic clearance (CLint,u) in rat intestinal microsomes tended to be lower than that in humans, and the correlation between intestinal CLint,u and FaFg in rats was poor compared with that in humans. 4. Our finding indicates that rats are appropriate animals for evaluation of the intestinal absorption and metabolism of CYP3A substrates. However, a degree of caution is required when estimating rat Fg from rat intestinal microsomes due to the low metabolic activity and the poor correlation between in vitro and in vivo intestinal metabolism.

An evaluation tool for FKBP12-dependent and -independent mTOR inhibitors using a combination of FKBP-mTOR fusion protein, DSC and NMR

Mammalian target of rapamycin (mTOR), a large multidomain protein kinase, regulates cell growth and metabolism in response to environmental signals. The FKBP rapamycin-binding (FRB) domain of mTOR is a validated therapeutic target for the development of immunosuppressant and anticancer drugs but is labile and insoluble. Here we designed a fusion protein between FKBP12 and the FRB domain of mTOR. The fusion protein was successfully expressed in Escherichia coli as a soluble form, and was purified by a simple two-step chromatographic procedure. The fusion protein exhibited increased solubility and stability compared with the isolated FRB domain, and facilitated the analysis of rapamycin and FK506 binding using differential scanning calorimetry (DSC) and solution nuclear magnetic resonance (NMR). DSC enabled the rapid observation of protein–drug interactions at the domain level, while NMR gave insights into the protein–drug interactions at the residue level. The use of the FKBP12–FRB fusion protein combined with DSC and NMR provides a useful tool for the efficient screening of FKBP12-dependent as well as -independent inhibitors of the mTOR FRB domain.

Exploratory population pharmacokinetics (e-PPK) analysis for predicting human PK using exploratory ADME data during early drug discovery research.

SummaryWe have proposed a novel method by population pharmacokinetics analysis for forecasting the drug concentration time-course in humans. This method is based on the non-linear mixed effect model (NONMEM) combined within vitro-in vivo extrapolation (IVIVE). Eleven clinically tested compounds were selected for retrospective analysis. Thein vivo pharmacokinetic (pk) profiles (rats, dogs, monkeys, and humans) andin vitro ADME data [intrinsic clearance (CLint), plasma unbound fraction (fp), and blood-plasma partition ratio (Rb)] for each compound was routinely tested via a screening system to account for inter-compound differences in pk properties. When evaluating the pk parameters, the hepatic plasma flow (Qph) and plasma volume (Vp) were taken into account to compensate for differences in body size among species. All these data were used to conduct population pk (PPK) analyses under the hypothesis that all species constituted one population. The two-compartment model (ADVAN4 TRANS3) and NONMEM software were used for this analysis. The fixed effect model for total body clearance (CL) and central distribution volume (Vd) were constructed as ϑCLQph·Eh and ϑVd·Vp, respectively, where the hepatic extraction ratio Eh was calculated using the traditional dispersion model. NONMEM generates both fixed and random effects (η). The key point of this concept was to substitute the η values of each species (rats, dogs, and monkeys) into the human PPK model to simulate three kinds of pk profiles, compound by compound, for use as a general scaling factor. The NONMEM post hoc option was used to perform the simulation, after which the concentration vs. time courses were compared with actual clinical pk data. The true values were almost within the dynamic range. Thus, the advantage of this concept is that it can generate time-courses without the detail of drug-specific parameters, from which the elimination half time can be determined. This proposed exploratory population pharmacokinetic (e-PPK) approach is a useful and progressive tool that can be applied during the early stages of drug discovery research.