Prediction of Oral Drug Absorption in Rats from In Vitro Data.

PURPOSE: In drug discovery, rats are widely used for pharmacological and toxicological studies. We previously reported that a mechanism-based oral absorption model, the gastrointestinal unified theoretical framework (GUT framework), can appropriately predict the fraction of a dose absorbed (Fa) in humans and dogs. However, there are large species differences between humans and rats. The purpose of the present study was to evaluate the predictability of the GUT framework for rat Fa. METHOD: The Fa values of 20 model drugs (a total of 39 Fa data) were predicted in a bottom-up manner. Based on the literature survey, the bile acid concentration (Cbile) and the intestinal fluid volume were set to 15 mM and 4 mL/kg, respectively, five and two times higher than in humans. LogP, pKa, molecular weight, intrinsic solubility, bile micelle partition coefficients, and Caco-2 permeability were used as input data. RESULTS: The Fa values were appropriately predicted for highly soluble drugs (absolute average fold error (AAFE) = 1.65, 18 Fa data) and poorly soluble drugs (AAFE = 1.57, 21 Fa data). When the species difference in Cbile was ignored, Fa was over- and under-predicted for permeability and solubility limited cases, respectively. High Cbile in rats reduces the free fraction of drug molecules available for epithelial membrane permeation while increasing the solubility of poorly soluble drugs. CONCLUSION: The Fa values in rats were appropriately predicted by the GUT framework. This result would be of great help for a better understanding of species differences and model-informed preclinical formulation development.

Bottom-Up Physiologically Based Oral Absorption Modeling of Free Weak Base Drugs.

In this study, we systematically evaluated "bottom-up" physiologically based oral absorption modeling, focusing on free weak base drugs. The gastrointestinal unified theoretical framework (the GUT framework) was employed as a simple and transparent model. The oral absorption of poorly soluble free weak base drugs is affected by gastric pH. Alternation of bulk and solid surface pH by dissolving drug substances was considered in the model. Simple physicochemical properties such as pKa, the intrinsic solubility, and the bile micelle partition coefficient were used as input parameters. The fraction of a dose absorbed (Fa) in vivo was obtained by reanalyzing the pharmacokinetic data in the literature (15 drugs, a total of 85 Fa data). The AUC ratio with/without a gastric acid-reducing agent (AUCr) was collected from the literature (22 data). When gastric dissolution was neglected, Fa was underestimated (absolute average fold error (AAFE) = 1.85, average fold error (AFE) = 0.64). By considering gastric dissolution, predictability was improved (AAFE = 1.40, AFE = 1.04). AUCr was also appropriately predicted (AAFE = 1.54, AFE = 1.04). The Fa values of several drugs were slightly overestimated (less than 1.7-fold), probably due to neglecting particle growth in the small intestine. This modeling strategy will be of great importance for drug discovery and development.

ONO-8430506: A Novel Autotaxin Inhibitor That Enhances the Antitumor Effect of Paclitaxel in a Breast Cancer Model.

Lysophosphatidic acid (LPA) is a bioactive lipid mediator that elicits a number of biological functions, including smooth muscle contraction, cell motility, proliferation, and morphological change. LPA is endogenously produced by autotaxin (ATX) from extracellular lysophosphatidylcholine (LPC) in plasma. Herein, we report our medicinal chemistry effort to identify a novel and highly potent ATX inhibitor, ONO-8430506 (20), with good oral availability. To enhance the enzymatic ATX inhibitory activity, we designed several compounds by structurally comparing our hit compound with the endogenous ligand LPC. Further optimization to improve the pharmacokinetic profile and enhance the ATX inhibitory activity in human plasma resulted in the identification of ONO-8430506 (20), which enhanced the antitumor effect of paclitaxel in a breast cancer model.

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.

Prediction Accuracy of Mechanism-Based Oral Absorption Model for Dogs.

The purpose of the present study was to evaluate the prediction accuracy of a mechanism-based oral absorption model for the fraction of a dose absorbed (Fa) in dogs, focusing on poorly soluble drugs. As an open mechanism-based model, the gastrointestinal unified theoretical framework was used in this study. The prediction accuracy of the gastrointestinal unified theoretical framework was evaluated using Fa data in dogs (63 data sets for marketed drugs and proprietary compounds). For neutral compounds, Fa was accurately predicted, suggesting that the physiological parameters of dogs were appropriate except for gastrointestinal pH. An extensive literature survey on the small intestinal pH of dogs was then conducted. The result suggested that the pH value ranged between 6.5 and 7.5, with the midst value of 7.0, but there was a great variation among the literature. To confirm the appropriateness of this pH value, the Fa of free acid compounds was predicted by setting the small intestinal pH to 6.5, 7.0, and 7.5. The proportions of compounds with <2-fold error were 57%, 90%, and 76%, respectively. The results of the present study would enable an appropriate use of a mechanism-based model for drug discovery and development.

Harmonizing solubility measurement to lower inter-laboratory variance - progress of consortium of biopharmaceutical tools (CoBiTo) in Japan.

The purpose of the present study was to harmonize the protocol of equilibrium solubility measurements for poorly water-soluble drugs to lower inter-laboratory variance. The "mandatory" and "recommended" procedures for the shake-flask method were harmonized based on the knowledge and experiences of each company and information from the literature. The solubility of model drugs was measured by the harmonized protocol (HP) and the non-harmonized proprietary protocol of each company (nonHP). Albendazole, griseofulvin, dipyridamole, and glibenclamide were used as model drugs. When using the nonHP, the solubility values showed large inter-laboratory variance. In contrast, inter-laboratory variance was markedly reduced when using the HP.

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.

A new CysLT1 and CysLT2 receptors-mediated anaphylaxis guinea pig model.

Although the effectiveness of CysLT1 receptor antagonists on asthma has been clinically established, the effects of CysLT2 receptor antagonists are still unclear. The purpose of this study was to develop a new CysLT1 and CysLT2 receptors-mediated anaphylaxis guinea pig model using S-hexyl GSH, a γ-glutamyl transpeptidase (GTP) inhibitor, to suppress conversion of LTC4 to LTD4. Actively sensitized guinea pigs were challenged with OVA in the absence or presence of S-hexyl GSH, and survival rate following anaphylactic response was monitored. OVA-induced fatal anaphylaxis in the absence of S-hexyl GSH was almost completely inhibited by montelukast, a CysLT1 receptor antagonist, but not by the CysLT2 receptor antagonist BayCysLT2RA. However, under treatment with S-hexyl-GSH, the inhibitory effect of motelukast was dramatically diminished, whereas that of BayCysLT2RA was markedly increased. The dual CysLT1/2 receptor antagonist ONO-6950 effectively inhibited anaphylactic response in both S-hexyl GSH-treated and non-treated animals. LC/MS/MS analysis revealed that S-hexyl GSH treatment actually inhibited LTC4 metabolism in the blood and lung tissues. Using S-hexyl GSH, we developed a novel CysLT1 and CysLT2 receptors-mediated anaphylaxis guinea pig model that can be useful for not only screening both CysLT2 and CysLT1/2 receptors antagonists, but also for functional analysis of CysLT2 receptors.

Discovery of Gemilukast (ONO-6950), a Dual CysLT1 and CysLT2 Antagonist As a Therapeutic Agent for Asthma.

An orally active dual CysLT1 and CysLT2 antagonist possessing a distinctive structure which consists of triple bond and dicarboxylic acid moieties is described. Gemilukast (ONO-6950) was generated via isomerization of the core indole and the incorporation of a triple bond into a lead compound. Gemilukast exhibited antagonist activities with IC50 values of 1.7 and 25 nM against human CysLT1 and human CysLT2, respectively, and potent efficacy at an oral dose of 0.1 mg/kg given 24 h before LTD4 challenge in a CysLT1-dependent guinea pig asthmatic model. In addition, gemilukast dose-dependently reduced LTC4-induced bronchoconstriction in both CysLT1- and CysLT2-dependent guinea pig asthmatic models, and it reduced antigen-induced constriction of isolated human bronchi. Gemilukast is currently being evaluated in phase II trials for the treatment of asthma.

Discovery of a potent, orally available dual CysLT1 and CysLT2 antagonist with dicarboxylic acid.

A potent, orally available dual CysLT1 and CysLT2 receptor antagonist with a dicarboxylic acid is described. 4-(3-(Carboxymethyl)-4-{(E)-2-[4-(4-phenoxybutoxy)phenyl]vinyl}-1H-indol-1-yl)butanoic acid (15: ONO-4310321, IC50: CysLT1=13nM, CysLT2=25 nM) showed excellent pharmacokinetic profiles (%Frat=100) compared with our previously reported compound 1 (%Frat=1.5). In addition, we describe a new rule for dicarboxylic acid derivatives to show good oral bioavailability (%Frat⩾40) in rats (HBDs: ⩽2, ClogP: >6.5 and TPSA: <100). Especially, reduction of only one hydrogen-bond donor (HBDs) showed dramatically improved oral bioavailability. This small change of HBDs in dicarboxylic acid derivatives is generally a very effective modification.

Leukotriene C4 induces bronchoconstriction and airway vascular hyperpermeability via the cysteinyl leukotriene receptor 2 in S-hexyl glutathione-treated guinea pigs.

Cysteinyl leukotrienes act through G-protein-coupled receptors termed cysteinyl leukotriene 1 (CysLT1) and cysteinyl leukotriene 2 (CysLT2) receptors. However, little is known about the pathophysiological role of CysLT2 receptors in asthma. To elucidate the possible involvement of CysLT2 receptors in bronchoconstriction and airway vascular hyperpermeability, we have established a novel guinea pig model of asthma. In vitro study confirmed that CHO-K1 cells, expressing guinea pig CysLT2 and CysLT1 receptors are selectively stimulated by LTC4 and LTD4, respectively. However, when LTC4 was intravenously injected to guinea pigs, the resulting bronchoconstriction was fully abrogated by montelukast, a CysLT1 receptor antagonist, indicating rapid metabolism of LTC4 to LTD4 in the lung. We found that treatment with S-hexyl glutathione (S-hexyl GSH), an inhibitor of gamma-glutamyl transpeptidase, significantly increased LTC4 content and LTC4/(LTD4 plus LTE4) ratio in the lung. Under these circumstances, LTC4-induced bronchoconstriction became resistant to montelukast, but sensitive to Compound A, a CysLT2 receptor antagonist, depending on the dose of S-hexyl GSH. Combination with montelukast and Compound A completely abrogated this spasmogenic response. Additionally, we confirmed that LTC4 elicits airway vascular hyperpermeability via CysLT2 receptors in the presence of high dose of S-hexyl GSH as evidenced by complete inhibition of LTC4-induced hyperpermeability by Compound A, but not montelukast. These results suggest that CysLT2 receptors mediate bronchoconstriction and airway vascular hyperpermeability in guinea pigs and that the animal model used in this study may be useful to elucidate the functional role of CysLT2 receptors in various diseases, including asthma.

A novel highly potent autotaxin/ENPP2 inhibitor produces prolonged decreases in plasma lysophosphatidic acid formation in vivo and regulates urethral tension.

Autotaxin, also known as ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2), is a secreted enzyme that has lysophospholipase D activity, which converts lysophosphatidylcholine to bioactive lysophosphatidic acid. Lysophosphatidic acid activates at least six G-protein coupled recpetors, which promote cell proliferation, survival, migration and muscle contraction. These physiological effects become dysfunctional in the pathology of cancer, fibrosis, and pain. To date, several autotaxin/ENPP2 inhibitors have been reported; however, none were able to completely and continuously inhibit autotaxin/ENPP2 in vivo. In this study, we report the discovery of a highly potent autotaxin/ENPP2 inhibitor, ONO-8430506, which decreased plasma lysophosphatidic acid formation. The IC50 values of ONO-8540506 for lysophospholipase D activity were 6.4-19 nM for recombinant autotaxin/ENPP2 proteins and 4.7-11.6 nM for plasma from various animal species. Plasma lysophosphatidic acid formation during 1-h incubation was almost completely inhibited by the addition of >300 nM of the compound to human plasma. In addition, when administered orally to rats at a dose of 30 mg/kg, the compound demonstrated good pharmacokinetics in rats and persistently inhibited plasma lysophosphatidic acid formation even at 24 h after administration. Smooth muscle contraction is a known to be promoted by lysophosphatidic acid. In this study, we showed that dosing rats with ONO-8430506 decreased intraurethral pressure accompanied by urethral relaxation. These findings demonstrate the potential of this autotaxin/ENPP2 inhibitor for the treatment of various diseases caused by lysophosphatidic acid, including urethral obstructive disease such as benign prostatic hyperplasia.

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.

Pyrrolo[1,2-b]pyridazines, pyrrolo[2,1-f]triazin-4(3H)-ones, and related compounds as novel corticotropin-releasing factor 1 (CRF1) receptor antagonists.

To identify structurally novel corticotropin-releasing factor 1 (CRF(1)) receptor antagonists, a series of bicyclic core analogs pyrrolo[1,2-b]pyridazines and pyrrolo[2,1-f]triazin-4(3H)-ones, which were designed based on a monocyclic core antagonist, was synthesized and evaluated. Among the compounds tested, 2-difluoromethoxy-4-methylpyridin-5-yl analog 27 was found to show efficacy in a dose-dependent manner in an elevated plus maze test in rats. The discovery process and structure-activity relationship is presented.

Pyrazolo[1,5-a]pyrimidines, triazolo[1,5-a]pyrimidines and their tricyclic derivatives as corticotropin-releasing factor 1 (CRF1) receptor antagonists.

To identify structurally novel CRF1 receptor antagonists, a series of bicyclic core antagonists, pyrazolo[1,5-a]pyrimidines, triazolo[1,5-a]pyrimidines, imidazo[1,2-a]pyrimidines and pyrazolo[1,5-a][1,3,5]triazines were designed, synthesized and evaluated as CRF1 receptor antagonists. Compounds 2-27 showed binding affinity (IC(50)=4.2-418 nM) and antagonist activity (EC(50)=4.0-889 nM). Compound 5 was found to show oral efficacy in an Elevated Plus Maze test in rats. Further chemical modification of them led us to discovery of the tricyclic core antagonists pyrazolo[1,5-a]pyrrolo[3,2-e]pyrimidines. The discovery process of these compounds is presented, as is the study of the structure-activity relationship.

6,7-Dihydro-5H-cyclopenta[d]pyrazolo[1,5-a]pyrimidines and their derivatives as novel corticotropin-releasing factor 1 receptor antagonists.

To identify an orally active corticotropin-releasing factor 1 receptor antagonist, a series of 6,7-dihydro-5H-cyclopenta[d]pyrazolo[1,5-a]pyrimidines and their derivatives were designed, synthesized and evaluated. An in vitro study followed by in vivo and pharmacokinetic studies of these heterotricyclic compounds led us to the discovery of an orally active CRF1 receptor antagonist. The results of a structure-activity relationship study are presented.