Validation of a New Methodology to Create Oral Drugs beyond the Rule of 5 for Intracellular Tough Targets.

Establishing a technological platform for creating clinical compounds inhibiting intracellular protein-protein interactions (PPIs) can open the door to many valuable drugs. Although small molecules and antibodies are mainstream modalities, they are not suitable for a target protein that lacks a deep cavity for a small molecule to bind or a protein found in intracellular space out of an antibody's reach. One possible approach to access these targets is to utilize so-called middle-size cyclic peptides (defined here as those with a molecular weight of 1000-2000 g/mol). In this study, we validated a new methodology to create oral drugs beyond the rule of 5 for intracellular tough targets by elucidating structural features and physicochemical properties for drug-like cyclic peptides and developing library technologies to afford highly N-alkylated cyclic peptide hits. We discovered a KRAS inhibitory clinical compound (LUNA18) as the first example of our platform technology.

Selective TRK Inhibitor CH7057288 against TRK Fusion-Driven Cancer.

Members of the tropomyosin receptor kinase (TRK) family are expressed in their constitutively activated forms as a result of a gene fusion that occurs across a wide variety of cancer types. We have identified CH7057288 as a potent and selective TRK inhibitor that belongs to a novel chemical class. CH7057288 showed selective inhibitory activity against TRKA, TRKB, and TRKC in cell-free kinase assays and suppressed proliferation of TRK fusion-positive cell lines, but not that of TRK-negative cell lines. Strong in vivo tumor growth inhibition was observed in subcutaneously implanted xenograft tumor models of TRK fusion-positive cells. Furthermore, in an intracranial implantation model mimicking brain metastasis, CH7057288 significantly induced tumor regression and improved event-free survival. Recently, resistant mutations in the kinase domain of TRK have been reported in patients who show disease progression after treatment with the TRK inhibitors now under clinical development. Our compound maintained similar levels of in vitro and in vivo activity against one of these resistant mutants as it did to wild-type TRK. An X-ray crystal structure of the TRKA and CH7057288 complex supported the activity against the mutant. In addition, gene expression analysis revealed that CH7057288 suppressed MAPK and E2F pathways as downstream signaling of TRK fusion. Therefore, CH7057288 could be a promising therapeutic agent for TRK fusion-positive cancer.

Hepatocellular carcinoma in a mouse model fed a choline-deficient, L-amino acid-defined, high-fat diet.

Hepatocellular carcinoma (HCC) is a common cancer worldwide and represents the outcome of the natural history of chronic liver disease. The growing rates of HCC may be partially attributable to increased numbers of people with non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). However, details of the liver-specific molecular mechanisms responsible for the NAFLD-NASH-HCC progression remain unclear, and mouse models that can be used to explore the exact factors that influence the progression of NAFLD/NASH to the more chronic stages of liver disease and subsequent HCC are not yet fully established. We have previously reported a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) as a dietary NASH model with rapidly progressive liver fibrosis in mice. The current study in C57BL/6J mice fed CDAHFD provided evidence for the chronic persistence of advanced hepatic fibrosis in NASH and disease progression towards HCC in a period of 36 weeks. When mice fed CDAHFD were switched back to a standard diet, hepatic steatosis was normalized and NAFLD activity score improved, but HCC incidence increased and the phenotype of fibrosis-associated HCC development was observed. Moreover, when mice continued to be fed CDAHFD for 60 weeks, HCC further developed without severe body weight loss or carcinogenesis in other organs. The autochthonous tumours showed a variety of histological features and architectural patterns including trabecular, pseudoglandular and solid growth. The CDAHFD mouse model might be a useful tool for studying the development of HCC from NAFLD/NASH, and potentially useful for better understanding pathological changes during hepatocarcinogenesis.

Discovery of novel motilin antagonists: Conversion of tetrapeptide leads to orally available peptidomimetics.

We successfully discovered peptidomimetic motilin antagonists (17c and 17d) through the improvement of physicochemical properties of a tetrapeptide antagonist (2). Furthermore, with oral administration and based on motilin antagonistic activity, both compounds suppressed motilin-induced colonic and gastric motility in conscious dogs.

Prediction of drug-drug interactions based on time-dependent inhibition from high throughput screening of cytochrome P450 3A4 inhibition.

A method of assessing the risk of drug-drug interaction (DDI) caused by mechanism-based inhibition (MBI) was developed for early-stage drug development using cytochrome P450 (CYP) 3A4 inhibition screening data. CYP3A4 inhibition was evaluated using a fluorescent substrate with or without preincubation containing an inhibitor. The results showed that five well-known mechanism-based inhibitors, but not the competitive inhibitor ketoconazole, had lower IC(50) after preincubation, suggesting the utility of the IC(50) shift by preincubation to discern mechanism-based inhibitors. A method to approximately predict the change in the area under the concentration-time curve (AUC) of a co-administered drug by MBI was found using IC(50) shift data and the unbound mean plasma concentration of the inhibitor. From our predictions of change in the AUC for 38 drugs using this method, all mechanism-based inhibitors causing change in the AUC of more than 200% were predicted to be high risk. In conclusion, our method provides a simple assessment of the risk of DDI from mechanism-based inhibitors, especially in the early stages of drug development.

Nonlinear intestinal pharmacokinetics of mitemcinal, the first acid-resistant non-peptide motilin receptor agonist, in rats.

The objective was to investigate the mechanism of nonlinear pharmacokinetics of mitemcinal, the first acid-resistant non-peptide motilin agonist, in rats. Super-proportional increases of the cumulative rates of radioactivity excreted into bile and urine following oral administration of [3H]-mitemcinal suggested nonlinear absorption of mitemcinal in rats. To evaluate the fraction dose absorbed (Fa) and intestinal availability (Fg), [3H]-mitemcinal was orally administrated to rats, and tritium radioactivity and unchanged mitemcinal concentration were determined in the portal blood. Fa values for 0.2 mg/kg1, 0.5 mg/kg, and 5.0 mg/kg dose groups were 0.314, 0.353, and 0.569, respectively. Corresponding Fg values were 0.243, 0.296, and 0.513, respectively. In Caco-2 experiments, the permeation of [3H]-mitemcinal in the secretory direction was larger than in the absorptive direction and was inhibited by P-glycoprotein (P-gp) substrate digoxin. The results indicate that the saturation of P-gp-mediated membrane permeation and intestinal metabolism causes the nonlinear pharmacokinetics of mitemcinal in rats.

Oral absorption of poorly water-soluble drugs: computer simulation of fraction absorbed in humans from a miniscale dissolution test.

PURPOSE: The purpose of this study was to develop a new system for computer simulation to predict fraction absorbed (F(a)) of Biopharmaceutical Classification System (BCS) class II (low solubility-high permeability) drugs after oral administration to humans, from a miniscale dissolution test. METHODS: Human oral absorption of 12 lipophilic drugs was simulated theoretically by using the dissolution and permeation parameters of the drugs. A miniscale dissolution test and a solubility study were carried out in a conventional buffer and a biorelevant medium (pH 6.5). A dissolution parameter, which can simulate in vivo dissolution, was obtained from the in vitro dissolution curve. Human intestinal permeability was estimated assuming that the permeation was limited by diffusion through the unstirred water layer. The F(a) in humans was predicted and then compared with clinical data. RESULTS: The dissolution and solubility of most model drugs were faster and higher in a biorelevant medium than in a conventional buffer. The simulated absorption was limited by the drug dissolution rate and/or solubility. Predicted F(a) was significantly correlated with clinical data (correlation coefficient r2 = 0.82, p < 0.001) when the dissolution profiles in biorelevant medium were used for the simulation. CONCLUSIONS: This new system quantitatively simulated human absorption and would be beneficial for the prediction of human F(a) values for BCS class II drugs.

Prediction of oral drug absorption in humans by theoretical passive absorption model.

The purpose of the present study was to examine the oral drug absorption predictability of the theoretical passive absorption model (TPAM). As chemical descriptors of drugs, the octanol/buffer distribution coefficient at pH 6.0 (D(ow)), intrinsic octanol-water partition coefficient (P(ow)), pK(a), and molecular weight (MW) were calculated from the chemical structure. Total passive intestinal membrane permeation consists of transcellular, paracellular and unstirred water layer (UWL) permeation. Transcellular permeation was modeled based on the pH-partition hypothesis with correction for cationic species permeation, and the independent variables were D(ow), P(ow), and pK(a). Paracellular permeation was modeled as a size-restricted diffusion within a negative electrostatic field-of-force, and the independent variables were MW and pK(a). UWL permeation was modeled as diffusion across a water layer, and the independent variable was MW. Cationic species permeation in the transcellular permeation model and the effect of a negative electric field-of-force in the paracellular permeation model were the extensions to the previous TPAM. The coefficients of the paracellular and UWL permeation models were taken from the literature. A data set of 258 compounds with observed values of Fa% (the fraction of a dose absorbed in humans) taken from the literature was employed to optimize four fitting coefficients in the transcellular permeation model. The TPAM predicted Fa%, with root mean square errors of 15-21% and a correlation coefficient (CC) of 0.78-0.88. In addition, the TPAM predicted the effective human intestinal membrane permeability with a CC of 0.67-0.77, as well as the contribution of paracellular permeation. The TPAM was found to predict oral absorption from the chemical structure of drugs with adequate predictability for usage in drug discovery.

Correction of permeability with pore radius of tight junctions in Caco-2 monolayers improves the prediction of the dose fraction of hydrophilic drugs absorbed by humans.

PURPOSE: To improve predictions of fraction dose absorbed (Fa) for hydrophilic drugs, a correction of paracellular permeability using the pore radius of tight junctions (TJs) in Caco-2 monolayers was performed. METHODS: The apparent permeability coefficient (P9app)) of drugs was measured using the Caco-2 assay and the parallel artificial membrane permeation assay (PAMPA), and values were corrected with the pore radius of TJs. RESULTS: An equation for calculating the pore radius of TJs from the P(app) of lucifer yellow was obtained. The optimal pore radius of TJs in Caco-2 monolayers for predicting human Fa was calculated to be 7 A. The correlation between the actual and predicted Fa was improved by using the P(app) corrected with the pore radius of TJs. Permeability in the PAMPA, which was corrected using the pore radius and membrane potential, was well correlated with that in the Caco-2 assay. Most of the hydrophilic drugs tested in this study were absorbed mainly through the paracellular pathway. CONCLUSIONS: The results suggest the necessity of optimizing paracellular permeation for the prediction of Fa, and also the importance of the paracellular pathway to the absorption of hydrophilic drugs. This method might contribute to the setting of appropriate dosages and the development of hydrophilic drugs.