Prediction Characteristics of Oral Absorption Simulation Software Evaluated Using Structurally Diverse Low-Solubility Drugs.

The purpose of the present study was to characterize current biopharmaceutics modeling and simulation software regarding the prediction of the fraction of a dose absorbed (Fa) in humans. As commercial software products, GastroPlus™ and Simcyp® were used. In addition, the gastrointestinal unified theoretical framework, a simple and publicly accessible model, was used as a benchmark. The Fa prediction characteristics for a total of 96 clinical Fa data of 27 model drugs were systematically evaluated using the default settings of each software product. The molecular weight, dissociation constant, octanol-water partition coefficient, solubility in biorelevant media, dose, and particle size of model drugs were used as input data. Although the same input parameters were used, GastroPlus™, Simcyp®, and the gastrointestinal unified theoretical framework showed different Fa prediction characteristics depending on the rate-limiting steps of oral drug absorption. The results of the present study would be of great help for the overall progression of physiologically based absorption models.

In vivo hepatic clearance of lipophilic drugs predicted by in vitro uptake data into cryopreserved hepatocytes suspended in sera of rats, guinea pigs, monkeys and humans.

Evaluation of uptake of lipophilic acid compounds into hepatocytes was an unresolved drug development issue because of their adsorption to cells and materials and low analytical sensitivity and accuracy in assessment of protein bindings. Uptake assays of compounds using hepatocytes suspended in serum were expected to solve these problems for prediction of in vivo hepatic clearance. Here, for compounds with high protein binding (>99%), diflunisal, montelukast, cerivastatin, telmisartan, fluvastatin and six new drug candidates, in vivo hepatic clearance predicted based on hepatic depletion and uptake (CLh, uptake, predicted) data using hepatocytes in the absence and presence of sera was investigated. In vitro hepatic uptake results with hepatocytes suspended in serum improved prediction of human hepatic clearance values for highly lipophilic montelukast and telmisartan. In vivo CLh, uptake, predicted values of six new highly lipophilic acid drug candidates (protein binding >99.97%) and diflunisal, montelukast and cerivastatin predicted based on hepatocytes suspended in serum were within threefold differences of their total clearance in vivo in rats, guinea pigs or monkeys, except for montelukast in monkeys (5.8-fold). These results suggest that the human hepatic uptake in hepatocytes suspended in serum is useful for prediction of CLh, uptake, predicted, especially for highly lipophilic/protein binding acid compounds.

Evaluation of Using Dogs to Predict Fraction of Oral Dose Absorbed in Humans for Poorly Water-Soluble Drugs.

Dogs have been widely used to study the oral absorption of a drug in drug discovery. However, there has been no quantitative validation of using dogs to predict the fraction of oral dose absorbed (Fa) in humans (Fah) for poorly water-soluble drugs. Here, we report the results of using dogs for quantitative Fah prediction, focusing on poorly water-soluble free acid and neutral drugs. The Fa values of 4 acidic and 1 neutral proprietary compounds were measured in humans and dogs. Extensive literature survey was also performed to increase the number of Fa data. Fah and Fa in dogs (Fad) were then compared at equivalent body weight-normalized doses. In the case of neutral compounds, Fad was found to be similar to Fah. In the case of acidic compounds, Fad significantly overestimated Fah in most cases. A difference in intestinal pH was suggested as the main reason for this discrepancy. In conclusion, the use of dogs would not be appropriate to predict Fah for acidic compounds, but more work is required to know about neutral compounds.

Using zebrafish in systems toxicology for developmental toxicity testing.

With the high cost and the long-term assessment of developmental toxicity testing in mammals, the vertebrate zebrafish has become a useful alternative model organism for high-throughput developmental toxicity testing. Zebrafish is also very favorable for the 3R perspective in toxicology; however, the methodologies used by research groups vary greatly, posing considerable challenges to integrative analysis. In this review, we discuss zebrafish developmental toxicity testing, focusing on the methods of chemical exposure, the assessment of morphological abnormalities, housing conditions and their effects on the production of healthy embryos, and future directions. Zebrafish as a systems toxicology model has the potential to elucidate developmental toxicity pathways, and to provide a sound basis for human health risk assessments.

Age-related changes of hepatic clearances of cytochrome P450 probes, midazolam and R-/S-warfarin in combination with caffeine, omeprazole and metoprolol in cynomolgus monkeys using in vitro-in vivo correlation.

1. Pharmacokinetics of human cytochrome P450 probes (caffeine, racemic warfarin, omeprazole, metoprolol and midazolam) were investigated after single intravenous and oral administrations at doses of 0.20 and 1.0 mg kg(-1), respectively, in combination to three young (3-year-old) and three aged (16-year-old) cynomolgus monkeys. 2. The plasma concentrations of caffeine and R-/S-warfarin decreased slowly in a monophasic manner, but those of omeprazole, metoprolol and midazolam decreased rapidly, in a similar manner to those as reported for pharmacokinetics in humans. 3. The mean maximum concentrations of R- and S-warfarin (4.6 and 3.7 µg/mL, respectively) in aged monkeys after oral administration were significantly higher than those in young monkeys (3.3 and 2.7 µg/mL). The mean clearance (CL) values of midazolam in aged monkeys (9.5 mL/min/kg) were significantly lower than those in young monkeys (13 mL/min/kg). 4. Individual intrinsic CL values for omeprazole (r = 0.29) and metoprolol (r = 0.30) of individual monkey livers were inversely correlated with their ages significantly (p < 0.05) in liver microsomes prepared from 55 cynomolgus monkeys. 5. These results suggest that cynomolgus monkeys could be a good model for humans, especially with particular characteristics in reduced CLs of some human P450 substrates by aging.

[Impact of microdose clinical trials in the preclinical stage].

A microdose clinical trial may be useful as a safe early-phase exploratory study using doses as low as 100 µg or less for determination of the disposition of a candidate compound in humans in a short period of time. This may increase confidence in candidate compounds, especially those for which it is difficult to predict disposition based on the results of in vitro or preclinical studies. In this study, we examined microdose trials performed in the preclinical stage for two first-in-class compounds with a new mechanism of action. These compounds showed species difference in first pass metabolism in the digestive tract and liver, causing uncertainty in prediction of disposition in humans. For this reason, first-in-human microdose clinical trials were performed. The results showed that the two compounds had effective blood concentrations after oral administration at a dose of 100 mg qd. Administration of an extremely small dose of one (14)C-labeled compound permitted identification of major metabolites. No toxic metabolites were detected. The preclinical toxic dose was determined based on prediction of blood exposure at the estimated maximum clinical dose. For the other candidate compound, the findings of the microdose trial indicated a high bioavailability after oral administration and low hepatic clearance after intravenous administration. These results suggested only a small risk of a change in disposition in patients with hepatic disorder. The data obtained for the two compounds suggest that microdose clinical trials can be useful for improving the process of candidate selection in the preclinical stage.

Age-related pharmacokinetic changes of acetaminophen, antipyrine, diazepam, diphenhydramine, and ofloxacin in male cynomolgus monkeys and beagle dogs.

1. The pharmacokinetics of acetaminophen (marker of gastric emptying), antipyrine (marker of hepatic metabolic activity and total body water), diazepam (lipophilic and highly distributed), diphenhydramine (hepatic blood flow-limited and alpha-1 acid glycoprotein bound) and ofloxacin (renally eliminated) were evaluated in cynomolgus monkeys (3-18 years old) and beagle dogs (2-11 years old) as models in elderly persons. 2. Gastric pH fluctuated with aging in monkeys and dogs. The concentration of alpha-1 acid glycoprotein appeared to be increased by aging. There were no age-related differences in the absorption rates of the drugs under the conditions used in the study. Total body fat increased and water decreased in monkeys, but these parameters did not change in dogs. 3. Hepatic blood flow decreased in both species, but a significant decrease of hepatic clearance was only seen in monkeys. Renal clearance decreased significantly with age in monkeys and showed a tendency to decrease in dogs. 4. Age-related alterations of physiological parameters in monkeys are in agreement with clinical observations in humans, except for the lack of a change in the plasma albumin concentration. Therefore, this study suggests that monkey might be a suitable animal model for prediction of age-related changes in pharmacokinetics in humans.

Development of mice exhibiting hepatic microsomal activity of human CYP3A4 comparable to that in human liver microsomes by intravenous administration of an adenovirus vector expressing human CYP3A4.

Cytochrome P450 3A4 (CYP3A4) plays a crucial role in the pharmacokinetic and safety profiles of drugs. However, it is difficult to properly predict the pharmacokinetics and hepatotoxicity of drugs in humans using data from experimental animals, because the catalytic activities of CYP3A4 and other drug-metabolizing enzymes differ between human and animal organs. In order to easily generate an animal model for proper evaluation of human CYP3A4-mediated drug metabolism, we developed a human CYP3A4-expressing adenovirus (Ad) vector based on our novel Ad vector exhibiting significantly lower hepatotoxicity (Ad-E4-122aT-hCYP3A4). Intravenous administration of Ad-E4-122aT-hCYP3A4 at a dose of 2 × 10(11) virus particles/mouse produced a mouse exhibiting human CYP3A4 activity at a level similar to that in the human liver, as shown in the dexamethasone metabolic experiment using liver microsomes. The area under the curve (AUC) of 6ßOHD was 2.7-fold higher in the Ad-E4-122aT-hCYP3A4-administered mice, compared with the mice receiving a control Ad vector. This Ad vector-expressing human CYP3A4 would thus be a powerful tool for evaluating human CYP3A4-mediated drug metabolism in the livers of experimental animals.

Comparative study of skin phototoxicity with three drugs by an in vivo mouse model.

Photosafety evaluation is becoming important during the drug development process in pharmaceutical companies. Both in vitro and in vivo test systems have been developed for the evaluation of phototoxic potential of chemicals. In the present study, we conducted an in vivo phototoxicity test using BALB/c mice. The mice were treated with sparfloxacin, lomefloxacin, or a quinoline derivative orally followed by the irradiation of simulated sunlight, and resulting phototoxic reactions of the ears were assessed. Sparfloxacin and lomefloxacin, but not the quinoline derivative, are well known to cause photoirritation in humans. All three drugs exhibited positive reaction in the 3T3 neutral red uptake phototoxicity test (3T3 NRU PT). In the in vivo test, sparfloxacin and lomefloxacin exhibited positive skin reaction in mice, but the quinoline derivative did not. The results of in vivo phototoxicity test in the mice coincided with phototoxic potential of these drugs in humans. The exposure levels of sparfloxacin or lomefloxacin at the minimum effective dose that exhibited phototoxic reaction in the mice were comparable with those in humans treated with the recommended therapeutic dose.