Prediction of drug-drug interaction risk of P-glycoprotein substrate in drug discovery.

We aimed at predicting the drug-drug interaction (DDI) risk of P-glycoprotein (P-gp) substrates by using P-gp expressing LLC-PK1 cells and its knockout mice (KO). The area under the curve (AUC) of 16 marketed drugs and plasma concentration (Cplasma) of 207 screening compounds, with corrected efflux ratio (CER) ≥ 2, were compared between P-gp KO mice and wild type mice (WT). At permeability (Papp) ≥ 10 × 10-6 cm/s in parent LLC-PK1 cells, AUC ratios (KO/WT) and Cplasma ratios (KO/WT) of these compounds were within 3-fold. AUC ratios (KO/WT) of clinical P-gp substrates, with human AUC ratios with and without P-gp inhibitor administration ≥2, were higher than 8.7. These observations led us to establish a work-flow of P-gp substrate assessment with the threshold AUC ratio (KO/WT) ≥ 9 leading to a DDI risk of AUC ratio (human) ≥ 2. A screening compound showing high CER (=57.6) was found, but its AUC ratio (KO/WT) was 3.7, had been presumed to be a weak risk and its AUC ratio (human) was 1.2 in a later clinical DDI study. Our proposed workflow should be useful for predicting the DDI risk of P-gp substrates in drug discovery.

Species difference in brain penetration of P-gp and BCRP substrates among monkey, dog and mouse.

The brain penetration of 19 drugs, including P-glycoprotein (P-gp) and/or breast cancer resistance protein (BCRP) substrates, was compared among mice, cynomolgus monkeys and beagle dogs. The brain-to-plasma concentration ratios (Kp,brain) of the tested compounds in monkey and dog showed good correlation, whereas species differences were observed between non-rodents (monkey/dog) and rodents (mouse). In particular, the Kp,brain values of 7 compounds out of 12 P-gp substrates (Kp,brain ratio in P-gp knockout mice versus wild-type mice ≥3) in monkey and dog were more than three-fold higher than those in mice and a similar trend was observed in the brain-to-plasma unbound concentration ratios (Kp,uu,brain). The cerebral spinal fluid (CSF) drug concentrations (CCSF), a surrogate for unbound brain concentration (Cu,brain), were also compared between dog and monkey, and the CSF-to-plasma unbound concentration ratios (Kp,uu,CSF) of BCRP substrates in dog were notably higher than those in monkey, although non-bcrp substrates showed good correlation. Also, the Kp,uu,CSF values of BCRP substrates in dog were clearly higher than the Kp,uu,brain values, indicating that the dog CCSF of BCRP substrates was not suitable as a surrogate of Cu,brain. These observations should be useful when selecting the appropriate animal models for CNS drug discovery.

Suppression of joint pain in transient receptor potential vanilloid 4 knockout rats with monoiodoacetate-induced osteoarthritis.

INTRODUCTION: Transient receptor potential vanilloid 4 (TRPV4) modulates osteoarthritic (OA) pain in animal models. However, the pathophysiological function of TRPV4 in regulating OA pain remains poorly understood. METHODS: We developed TRPV4-knockout (TRPV4-KO) rats and assessed the effects of Trpv4 gene deficiency in a monoiodoacetate (MIA)-induced OA pain model (MIA rats) by examining pain-related behavior, pathological changes, and electrophysiological changes in dorsal root ganglion (DRG) neurons. The changes detected in TRPV4-KO rats were confirmed in wild-type rats using a TRPV4 antagonist. RESULTS: Transient receptor potential vanilloid 4-KO rats showed the same pain threshold as wild-type rats for thermal or pressure stimuli under normal conditions. Trpv4 gene deletion did not suppress the development of osteoarthritis pathologically in MIA rats. However, the OA-related mechanical pain behaviors observed in MIA rats, including decreased grip strength, increased mechanical allodynia, and reduced weight-bearing on the ipsilateral side, were completely suppressed in TRPV4-KO rats. The DRG neurons in wild-type but not TRPV4-KO MIA rats were depolarized with increased action potentials. Transient receptor potential vanilloid 4 antagonist treatments recapitulated the effects of genetic Trpv4 deletion. CONCLUSION: Transient receptor potential vanilloid 4 was sensitized in the DRG neurons of MIA rats and played a critical role in the development of OA pain. These results suggest that the inhibition of TRPV4 might be a novel potent analgesic strategy for treating OA pain.

Selective blockade of transient receptor potential vanilloid 4 reduces cyclophosphamide-induced bladder pain in mice.

Transient receptor potential vanilloid 4 (TRPV4) is a non-selective cation channel activated by various physical stimuli such as cell swelling and shear stress. TRPV4 is expressed in bladder sensory nerves and epithelium, and its activation produces urinary dysfunction in rodents. However, there have been few reports regarding its involvement in bladder pain. Therefore, we investigated whether TRPV4 is involved in bladder pain in mouse cystitis model. Intraperitoneal injection of cyclophosphamide (CYP; 300 mg/kg) produced mechanical hypersensitivity in the lower abdomen associated with a severe inflammatory bladder in mice. The mechanical threshold was reversed significantly in Trpv4-knockout (KO) mice. Repeated injections of CYP (150 mg/kg) daily for 4 days provoked mild bladder inflammation and persistent mechanical hypersensitivity in mice. Trpv4-KO mice prevented a reduction of the mechanical threshold without an alteration in bladder inflammation. A selective TRPV4 antagonist also reversed the mechanical threshold in chronic cystitis mice. Although expression of Trpv4 was unchanged in the bladders of chronic cystitis mice, the level of phosphorylated TRPV4 was increased significantly. These results suggest involvement of TRPV4 in bladder pain of cystitis mice. A TRPV4 antagonist might be useful for patients with irritable bladder pain such as those with interstitial cystitis/painful bladder syndrome.

ACC2 Deletion Enhances IMCL Reduction Along With Acetyl-CoA Metabolism and Improves Insulin Sensitivity in Male Mice.

Intramyocellular lipid (IMCL) accumulation in skeletal muscle greatly contributes to lipid-induced insulin resistance. Because acetyl-coenzyme A (CoA) carboxylase (ACC) 2 negatively modulates mitochondrial fatty acid oxidation (FAO) in skeletal muscle, ACC2 inhibition is expected to reduce IMCL via elevation of FAO and to attenuate insulin resistance. However, the concept of substrate competition suggests that enhanced FAO results in reduced glucose use because of an excessive acetyl-CoA pool in mitochondria. To identify how ACC2-regulated FAO affects IMCL accumulation and glucose metabolism, we generated ACC2 knockout (ACC2-/-) mice and investigated skeletal muscle metabolites associated with fatty acid and glucose metabolism, as well as whole-body glucose metabolism. ACC2-/- mice displayed higher capacity of glucose disposal at the whole-body levels. In skeletal muscle, ACC2-/- mice exhibited enhanced acylcarnitine formation and reduced IMCL levels without alteration in glycolytic intermediate levels. Notably, these changes were accompanied by decreased acetyl-CoA content and enhanced mitochondrial pathways related to acetyl-CoA metabolism, such as the acetylcarnitine production and tricarboxylic acid cycle. Furthermore, ACC2-/- mice exhibited lower levels of IMCL and acetyl-CoA even under HFD conditions and showed protection against HFD-induced insulin resistance. Our findings suggest that ACC2 deletion leads to IMCL reduction without suppressing glucose use via an elevation in acetyl-CoA metabolism even under HFD conditions and offer new mechanistic insight into the therapeutic potential of ACC2 inhibition on insulin resistance.

The role of acylated-ghrelin in the regulation of sucrose intake.

The octanoyl modification of ghrelin by ghrelin O-acyltransferase (GOAT) is essential for exerting its physiologic actions. Since exogenous acylated-ghrelin has shown to stimulate food intake in humans and rodents, GOAT has been regarded as a promising target for modulating appetite, thereby treating obesity and diabetes. However, GOAT-knockout (KO) mice have been reported to show no meaningful body weight reduction, when fed a high-fat diet. In this study, we sought to determine whether GOAT has a role in the regulation of body weight and food intake when fed a dietary sucrose. We found that GOAT KO mice showed significantly reduced food intake and marked resistance to obesity, when fed a high-fat + high-sucrose diet. In addition, GOAT KO mice fed a medium-chain triglyceride (MCT) + high-sucrose diet showed a marked resistance to obesity and reduced feed efficiency. These results suggest that blockade of acylated-ghrelin production offers therapeutic potential for obesity caused by overconsumption of palatable food.

Reduced intake of carbohydrate prevents the development of obesity and impaired glucose metabolism in ghrelin O-acyltransferase knockout mice.

A close relationship between acylated-ghrelin and sucrose intake has been reported. However, little has been examined about the physiological action of ghrelin on preference for different types of carbohydrate such as glucose, fructose, and starch. The current study was aimed to investigate the role of acylated-ghrelin in the determinants of the choice of carbohydrates, and pathogenesis of chronic disorders, including obesity and insulin resistance. In a two-bottle-drinking test, ghrelin O-acyltransferase (GOAT) knockout (KO) mice consumed a less amount of glucose and maltodextrin, and almost the same amount of fructose and saccharin solution compared to WT littermates. The increased consumption of glucose and maltodextrin was observed when acylated-ghrelin, but not unacylated-ghrelin, was exogeneously administered in normal C57BL/6J mice, suggesting an association of acylated-ghrelin with glucose-containing carbohydrate intake. When fed a diet rich in maltodextrin, starch and fat for 12 weeks, GOAT KO mice showed less food intake and weight gain, as well as improved glucose tolerance and insulin sensitivity than WT mice. Our data suggests that blockade of GOAT activity may offer a therapeutic option for treatment of obesity and its associated metabolic syndrome by preventing from overconsumption of carbohydrate-rich food.

Ghrelin O-acyltransferase knockout mice show resistance to obesity when fed high-sucrose diet.

Ghrelin is an appetite-stimulating hormone secreted from stomach. Since the discovery that acylation of the serine-3 residue by ghrelin O-acyltransferase (GOAT) is essential for exerting its functions, GOAT has been regarded as an therapeutic target for attenuating appetite, and thus for the treatment of obesity and diabetes. However, contrary to the expectations, GOAT-knockout (KO) mice have not shown meaningful body weight reduction, under high-fat diet. Here, in this study, we sought to determine whether GOAT has a role in body weight regulation and glucose metabolism with a focus on dietary sucrose, because macronutrient composition of diet is important for appetite regulation. We found that peripherally administered acylated-ghrelin, but not unacylated one, stimulated sucrose consumption in a two-bottle-drinking test. The role of acylated-ghrelin in sucrose preference was further supported by the finding that GOAT KO mice consumed less sucrose solution compared with WT littermates. Then, we investigated the effect of dietary composition of sucrose on food intake and body weight in GOAT KO and WT mice. As a result, when fed on high-fat diet, food intake and body weight were similar between GOAT KO and WT mice. However, when fed on high-fat, high-sucrose diet, GOAT KO mice showed significantly reduced food intake and marked resistance to obesity, leading to amelioration of glucose metabolism. These results suggest that blockade of acylated-ghrelin production offers therapeutic potential for obesity and metabolic disorders caused by overeating of palatable food.

Exploring new gene integration sites for gene knock-in by gene-trapping strategy.

The knock-in mouse is a powerful tool for biological research, but the stability of expression of an integrated gene strongly depends on where it is integrated in the mouse genome. At present, there are an insufficient number of loci suitable for gene knock-in, such as the Rosa26 locus. Therefore, in this study, we developed an efficient strategy for identifying genome loci suitable for gene knock-in and characterized the properties of such loci for gene integration. For efficient discovery and characterization, we constructed a new gene-trapping vector that enables monitoring of the expression of both trapped and integrated genes using fluorescence. We successfully obtained fluorescent-positive mouse embryonic stem cell (mESC) clones with the vector. Thorough analysis of the expression of fluorescent proteins in chimera embryos generated with the obtained mESC clones, some of the gene-trapped chimera embryos showed stable and ubiquitous expression of the integrated gene. Furthermore, adult mice derived from one of the gene-trapped mESC clones showed ubiquitous expression of the integrated gene in various tissues without any unusual phenotype. This indicated that the identified locus possesses high potential for foreign gene integration. Our strategy allows for efficient discovery and characterization of mouse genome loci for gene integration.

The translation elongation factor eEF2 is a novel tumor­associated antigen overexpressed in various types of cancers.

Recent studies have shown that cancer immunotherapy could be a promising therapeutic approach for the treatment of cancer. In the present study, to identify novel tumor-associated antigens (TAAs), the proteins expressed in a panel of cancer cells were serologically screened by immunoblot analysis and the eukaryotic elongation factor 2 (eEF2) was identified as an antigen that was recognized by IgG autoantibody in sera from a group of patients with head and neck squamous cell carcinoma (HNSCC) or colon cancer. Enzyme-linked immunosorbent assay showed that serum eEF2 IgG Ab levels were significantly higher in colorectal and gastric cancer patients compared to healthy individuals. Immunohistochemistry experiments showed that the eEF2 protein was overexpressed in the majority of lung, esophageal, pancreatic, breast and prostate cancers, HNSCC, glioblastoma multiforme and non-Hodgkin's lymphoma (NHL). Knockdown of eEF2 by short hairpin RNA (shRNA) significantly inhibited the growth in four eEF2-expressing cell lines, PC14 lung cancer, PCI6 pancreatic cancer, HT1080 fibrosarcoma and A172 glioblastoma cells, but not in eEF2-undetectable MCF7 cells. Furthermore, eEF2-derived 9-mer peptides, EF786 (eEF2 786-794 aa) and EF292 (eEF2 292-300 aa), elicited cytotoxic T lymphocyte (CTL) responses in peripheral blood mononuclear cells (PBMCs) from an HLA-A*24:02- and an HLA-A*02:01-positive healthy donor, respectively, in an HLA-A-restricted manner. These results indicated that the eEF2 gene is overexpressed in the majority of several types of cancers and plays an oncogenic role in cancer cell growth. Moreover, the eEF2 gene product is immunogenic and a promising target molecule of cancer immunotherapy for several types of cancers.

Overexpression of eukaryotic elongation factor eEF2 in gastrointestinal cancers and its involvement in G2/M progression in the cell cycle.

A high level protein synthesis is one of the characteristics of cancer cells. The aim of this study is to show the contribution of eukaryotic elongation factor 2 (eEF2), which plays an essential role in the polypeptide chain elongation step, in the tumorigenesis of gastrointestinal cancers. In the present study, we demonstrated by using immunohistochemistry that eEF2 protein was overexpressed in 92.9% (13 of 14) of gastric and 91.7% (22 of 24) of colorectal cancers. No mutations were found in any of the exons of the eEF2 gene in six gastric and six colorectal cancers. Knockdown of eEF2 by eEF2-specific short-hairpin RNA (shEF2) inhibited cancer cell growth in two gastric cancer cell lines, AZ-521 and MKN28, and one colon cancer cell line, SW620. Flow cytometric analysis showed that knockdown of eEF2 induced G2/M arrest and resulted in inactivation of Akt and cdc2 (a G2/M regulator) and activation of eEF2 kinase (a negative regulator of eEF2) in these cancer cells. Conversely, forced expression of eEF2 in AZ-521 cells significantly enhanced the cell growth through promotion of G2/M progression in cell cycle, activated Akt and cdc2, and inactivated eEF2 kinase. Furthermore, forced expression of eEF2 in these cancer cells enhanced in vivo tumorigenicity in a mouse xenograft model. These results showed that overexpressed eEF2 in gastrointestinal cancers promoted G2/M progression and enhanced their cell growth in vitro and in vivo. These results also suggested a novel linkage between translational elongation and cell cycle mechanisms, implying that the linkage might play an important role to orchestrate the deregulated translation and cell cycle mechanisms for promotion of the development of gastrointestinal cancers.