Discovery of a Novel Piperidine-Based Inhibitor of Cholesteryl Ester Transfer Protein (CETP) That Retains Activity in Hypertriglyceridemic Plasma.

Herein we describe the discovery and characterization of a novel, piperidine-based inhibitor of cholesteryl ester transfer protein (CETP) with a core structure distinct from other reported CETP inhibitors. A versatile synthesis starting from 4-methoxypyridine enabled an efficient exploration of the SAR, giving a lead molecule with potent CETP inhibition in human plasma. The subsequent optimization focused on improvement of pharmacokinetics and mitigation of off-target liabilities, such as CYP inhibition, whose improvement correlated with increased lipophilic efficiency. The effort led to the identification of an achiral, carboxylic acid-bearing compound 16 (TAP311) with excellent pharmacokinetics in rats and robust efficacy in hamsters. Compared to anacetrapib, the compound showed substantially reduced lipophilicity, had only modest distribution into adipose tissue, and retained potency in hypertriglyceridemic plasma in vitro and in vivo. Furthermore, in contrast to torcetrapib, the compound did not increase aldosterone secretion in human adrenocortical carcinoma cells nor in chronically cannulated rats. On the basis of its preclinical efficacy and safety profile, the compound was advanced into clinical trials.

Genetic association between the dopamine D3 receptor gene polymorphism (Ser9Gly) and tardive dyskinesia in patients with schizophrenia: a reevaluation in East Asian populations.

The dopamine D3 receptor gene (DRD3) is considered being one of the candidate genes contributing to the development of tardive dyskinesia (TD). In a recent meta-analysis with mixed ethnicities, only a barely positive association was found between the functional DRD3 Ser9Gly polymorphism and TD in patients with schizophrenia (OR=1.17; 95% CI: 1.01-1.37; p=0.041). To further evaluate the controversial association between the polymorphism and TD using only Japanese subjects, we tested the association in a case-control design. We also conducted a meta-analysis including 8 studies with 3 East Asian populations (Japanese, Chinese, and Korean). In our Japanese case-control sample (43 with TD/157 without TD), we found no association between the DRD3 Ser9Gly polymorphism in schizophrenia and TD (genotype: p=0.92; allele: p=1.00). Furthermore, no significant difference in the mean AIMS score among the three genotypic groups was observed in our sample. The meta-analysis comprising 1291 East Asian subjects also showed no association between the polymorphism and TD; the Mantel-Haenszel pooled OR for TD among carriers of the DRD3 Ser9Gly of the eight Asian studies was 0.94 (95% CI: 0.78-1.12). Overall, our results suggest that the DRD3 Ser9Gly polymorphism may not confer susceptibility to TD in East Asian populations. Given that the Ser9Gly variant may play a putative role in the DRD3 function, further studies on the DRD3 are warranted.

Lack of association between the leptin receptor gene (LEPR) Gln223Arg polymorphism and late-onset Alzheimer disease.

The principal hypothesis for pathogenesis of Alzheimer disease (AD) is the amyloid cascade hypothesis, which emphasizes an imbalance between production and clearance of beta-amyloid (Abeta) in the brain. Insulin has important effects on the regulation of the Abeta level in the brain, modulating both Abeta production and clearance. An optimal brain insulin level promotes Abeta clearance, which may play protective roles against AD. A functional human leptin receptor gene (LEPR) polymorphism, a glutamine to an arginine substitution at codon 223 (Gln223Arg), has been associated with insulin resistance capacity and an altered leptin-binding activity. The LEPR Gln223Arg polymorphism may thus play an important role in the pathogenesis of AD. In this study, we examined the association between the LEPR Gln223Arg polymorphism and late-onset Alzheimer disease (LOAD) in a Japanese population. Our sample includes 49 patients with LOAD and 134 normal controls. Our preliminary data showed no significant association between the LEPR Gln223Arg polymorphism and LOAD (genotype distribution: chi=0.11, df=2, P=0.945; allele frequency: chi=0.058, df=1, P=0.81, odds ratio=1.08, 95% confidence interval=0.59 to 2.03). Our results suggest that the LEPR polymorphism may not play a major role in the development of LOAD.

Association analysis between the C-1291G polymorphism in the promoter region of the adrenergic alpha2A receptor gene and polydipsia in schizophrenia.

Several lines of studies have shown the existence of an important inhibitory mechanism for the control of water intake involving adrenergic alpha2A receptors (ADRA2A). A human study using patients with schizophrenia demonstrated an exacerbation of polydipsia by the administration of clonidine, an ADRA2A-agonist, and a relief of polydipsia by mianserin, an ADRA2A-antagonist, suggesting the involvement of the central adrenergic system in the drinking behavior of patients with schizophrenia. Based on these findings we examined a possible association between the C-1291G polymorphism in the promoter region of the ADRA2A gene and polydipsia in schizophrenia using a Japanese case-control sample. Our sample includes 348 patients with schizophrenia (DSM-IV) (84 with polydipsia and 264 without polydipsia). No significant association between the ADRA2A C-1291G polymorphism and polydipsia was found. Our result suggests that the ADRA2A C-1291G polymorphism may not confer susceptibility to polydipsia in schizophrenia in our sample. Further studies with larger samples are warranted.

Inhibition of focal adhesion kinase as a potential therapeutic strategy for imatinib-resistant gastrointestinal stromal tumor.

Focal adhesion kinase (FAK) is often up-regulated in a variety of malignancies, including gastrointestinal stromal tumor (GIST), and its overexpression seems to be associated with tumor progressiveness and poor prognosis. GIST is well known to have a mutation to c-KIT; thus, a specific c-KIT inhibitor (imatinib) is recognized as the first-line chemotherapy for GIST, although a certain type of c-KIT mutation reveals a resistance to imatinib due to as yet uncertain molecular mechanisms. To assess the c-KIT mutation-related variation of cellular responses to imatinib, murine lymphocyte-derived Ba/F3 cells, which are stably transduced with different types of c-KIT mutation, were treated with either imatinib or a FAK inhibitor (TAE226), and their antitumor effects were determined in vitro and in vivo. A mutation at exon 11 (KITdel559-560) displayed a high sensitivity to imatinib, whereas that at exon 17 (KIT820Tyr) showed a significant resistance to imatinib in vitro and in vivo. KIT820Tyr cells appeared to maintain the activities of FAK and AKT under the imatinib treatment, suggesting that FAK might play a role in cell survival in imatinib-resistant cells. When FAK activity in those cells was inhibited by TAE226, cell growth was equally suppressed and the cells underwent apoptosis regardless of the c-KIT mutation types. Oral administration of TAE226 significantly diminished tumor growth in nude mice bearing KIT(820Tyr) xenografts. In summary, c-KIT mutation at exon 17 displayed a resistance to imatinib with maintained activations of FAK and subsequent survival signals. Targeting FAK could be a potential therapeutic strategy for imatinib-resistant GISTs.

No association between a functional polymorphism in the promoter region of the neuropeptide Y gene (-485C>T) and schizophrenia.

It has been suggested that hypoactivity of neuropeptide Y (NPY) may be involved in the pathophysiology of schizophrenia. A post-mortem study revealed a decreased level of NPY in the brain of patients with schizophrenia. An increased level of NPY after antipsychotic treatment was also reported in animal brain and cerebrospinal fluid of patients. Previously Itokawa et al. reported a positive association between the functional -485C>T polymorphism in the NPY gene and schizophrenia in a Japanese population. The aim of this study is to replicate their positive findings in an independent Japanese case-control sample. Our sample includes 260 patients with schizophrenia (DSM-IV) and 196 control subjects. No significant differences in distribution of genotype or allele frequencies between patients and controls were observed. Our results suggest that the NPY -485C>T polymorphism may not confer susceptibility to schizophrenia, at least in our sample. Further studies in larger samples are warranted.

Genetic association between the dopamine D3 gene polymorphism (Ser9Gly) and schizophrenia in Japanese populations: evidence from a case-control study and meta-analysis.

Dysregulation in the dopaminergic system has been implicated in the pathophysiology of schizophrenia (SCZ). Dopamine D3 receptors (DRD3) concentrated in limbic regions of the brain (important for cognitive, emotional and endocrine function) may be particularly relevant to SCZ. A recent meta-analysis with mixed ethnicities reported a marginal significant association between the Ser9Gly homozygosity in the first exon of the DRD3 gene and SCZ. To further evaluate the controversial association between this polymorphism and SCZ, a case-control study and meta-analysis was conducted using the homogeneous Japanese population. In our Japanese case-control sample (246 cases/198 controls), we found an association between the DRD3 Ser9Gly polymorphism and SCZ (genotype: chi(2) = 9.76, d.f. = 2, p = 0.008; Ser allele versus Gly allele: chi(2) = 7.96, d.f. = 1, p = 0.0048; OR = 0.65; 95% CI = 0.48-0.88). However in a meta-analysis of nine Japanese case-control studies comprising 2056 subjects the association between DRD3 Ser9Gly polymorphism and SCZ did not persisted. The Mantel-Haenszel pooled OR for SCZ among carriers of the DRD3 Ser9Gly homozygosity (Ser/Ser homozygotes and Gly/Gly homozygotes) of the nine Japanese studies was 1.16 (95% CI 0.97-1.39), pointing to a non-significant effect of the DRD3 Ser9Gly homozygosity as a risk factor for SCZ. Overall, our results suggest that the DRD3 Ser9Gly polymorphism may not confer susceptibility to SCZ in the Japanese population. Given that the Ser9Gly variant may play a putative role in DRD3 function, further studies on the DRD3 with linked variants are warranted.

Dual tyrosine kinase inhibitor for focal adhesion kinase and insulin-like growth factor-I receptor exhibits anticancer effect in esophageal adenocarcinoma in vitro and in vivo.

PURPOSE: Focal adhesion kinase (FAK) regulates integrin and growth factor-mediated signaling pathways to enhance cell migration, proliferation, and survival, and its up-regulation correlates malignant grade and poor outcome in several types of cancer. In this study, we aimed to raise a potential therapeutic strategy using a FAK inhibitor for Barrett's esophageal adenocarcinoma. EXPERIMENTAL DESIGN: The expression status of FAK in clinical Barrett's esophageal adenocarcinoma tissues was determined by immunohistochemistry. Cultured esophageal adenocarcinoma cells were treated with TAE226, a specific FAK inhibitor with an additional effect of inhibiting insulin-like growth factor-I receptor (IGF-IR), to assess its anticancer effect in vitro. Western blot was carried out to explore a participating signaling pathway for TAE226-induced cell death. Furthermore, TAE226 was orally administered to s.c. xenograft animals to investigate its anticancer effect in vivo. RESULTS: Strong expression of FAK was found in 94.0% of Barrett's esophageal adenocarcinoma compared with 17.9% of Barrett's epithelia, suggesting that FAK might play a critical role in the progression of Barrett's esophageal adenocarcinoma. When esophageal adenocarcinoma cells were treated with TAE226, cell proliferation and migration were greatly inhibited with an apparent structural change of actin fiber and a loss of cell adhesion. The activities of FAK, IGF-IR, and AKT were suppressed by TAE226 and subsequent dephosphorylation of BAD at Ser(136) occurred, resulting in caspase-mediated apoptosis. In vivo tumor volume was significantly reduced by oral administration of TAE226. CONCLUSIONS: These results suggest that TAE226, a dual tyrosine kinase inhibitor for FAK and IGF-IR, could become a new remedy for Barrett's esophageal adenocarcinoma.

Functional polymorphism of the human multidrug resistance gene (MDR1) and polydipsia-hyponatremia in schizophrenia.

P-glycoprotein (P-gp), which is coded by the MDR1 gene, in the brain capillary endothelial cell limits the entry of many drugs including antipsychotics into the brain. The aim of this study is to examine whether a functional polymorphism, a C to T substitution at position 3435 in exon 26 of the MDR1 gene, is associated with susceptibility to polydipsia-hyponatremia in schizophrenia (SCZ) in a Japanese case-control sample. It has been reported that individuals homozygous for this polymorphism had significantly lower MDR1 expression levels and dysfunction of MDR1 (PNAS 97:3473-3478, 2000). Furthermore, the brain entry of risperidone and 9-hydroxyrisperidone has been shown to be greatly limited by P-gp (Int J Neuropsychopharmacol 7:415-419, 2004). In order to our knowledge, this is the first association study between the MDR1 polymorphism and polydipsia-hyponatremia in SCZ. Our sample includes 331 patients with SCZ (DSM-IV) (84 with polydipsics and 247 non-polydipsic controls). The common C3435T polymorphism of the MDR1 was genotyped for both groups and differences in genotype and allele frequency between cases and controls were evaluated using the chi(2)-test. A significant association between the MDR1 C3435T polymorphism and polydipsia was found (chi(2) = 4.43, d.f. = 1, P = 0.035; OR = 1.46; 95%CI = 1.03-2.07). Our results suggest that the MDR1 C3435T polymorphism may confer susceptibility to polydipsia in SCZ.

The orexin 1 receptor (HCRTR1) gene as a susceptibility gene contributing to polydipsia-hyponatremia in schizophrenia.

The underlying pathophysiology of primary polydipsia in schizophrenia (SCZ) is poorly understood. Our previous study, however, suggested that this condition may have a genetic component [Shinkai et al 2003 Am J Med Genet 119B 7-12]. Orexins, also called hypocretins, play an important role in feeding and drinking behavior. Administration of orexin in rats has been shown to induce increased water intake with a longer-lasting effect than angiotensin II, which is also known as a potent dipsogen. Meerabux et al. [2005 Biol Psychiatry 58 401-407] reported an association between the 408Val allele of the orexin 1 receptor (HCRTR1) gene and polydipsia-hyponatremia in a sample of Japanese patients with SCZ. In the present study, we attempted to replicate the findings of Meerabux et al. in an independent Japanese case-control sample. Our sample included 312 patients with SCZ (DSM-IV) (65 with polydipsia and 247 without polydipsia). We also observed an association between the HCRTR1 Ile408Val polymorphism and polydipsia (genotype distribution: chi2 = 9.85, df = 2, P = 0.007). Meerabux et al. (2005) previously demonstrated an association between the 408Val allele of the HCRTR1 gene and polydipsia. In contrast with Meerabux et al. study, we found that the 408Ile allele was associated with polydipsia in our sample (chi2 = 8.00, df = 1, P = 0.0047; OR = 0.53; 95%CI = 0.34-0.83). How either allele contributes to the development of polydipsia in SCZ is unclear at this stage. It is possible that Ile408Val polymorphism is a non-functional marker that lies in linkage disequilibrium with an as-yet undetected functional variant. In any case, our results support the hypothesis that the HCRTR1 Ile408Val polymorphism may confer susceptibility to polydipsia in SCZ. Further studies examining the association between the orexin system and polydipsia in SCZ are warranted.

Novel scaffold for cathepsin K inhibitors.

Pyrrolopyrimidine, a novel scaffold, allows to adjust interactions within the S3 subsite of cathepsin K. The core intermediate 10 facilitated the P3 optimization and identified highly potent and selective cathepsin K inhibitors 11-20.

Association analyses of the DAOA/G30 and D-amino-acid oxidase genes in schizophrenia: further evidence for a role in schizophrenia.

A number of linkage studies have previously implicated the region of chromosome 13q34 in schizophrenia. Chumakov and colleagues (2002) identified a gene complex called G72 (now termed D-amino acid oxidase activator: DAOA)/G30 in this region and performed association analyses of the DAOA/G30 as well as the D-amino-acid oxidase (DAAO) gene with schizophrenia. DAAO oxidizes D-serine, a potent activator of the N-methyl-D-aspartate (NMDA) type glutamate receptor in the human brain whereas the DAOA protein is considered an activator of DAAO. The interaction of these two genes has thus been implicated in the NMDA receptor regulation pathway in schizophrenia. To date, several studies have shown a relatively consistent positive association between schizophrenia and DAOA/G30, but not with DAAO. The aim of our study was to further evaluate the contributions of these genes to the susceptibility to schizophrenia using two different sample sets. Our sample consisted of 168 matched case-control pairs as well as a family-based sample (n=113) for transmission disequilibrium test. Significant associations between the DAOA/G30 M-7 and M-18 polymorphisms and schizophrenia were observed in our case-control sample whereas no associations were observed for DAAO markers. We also observed significant or suggestive transmission disequilibrium for DAOA/G30 M-7, M-23, and M-24 to probands with schizophrenia in our family-based sample. Subsequent analysis of haplotypes made up of four DAOA/G30 markers, one marker selected from each of two linkage disequilibrium blocks that were observed in our sample as well as both ends (M-7 and M-25), were also associated with schizophrenia. Our data provide further evidence that the DAOA/G30 locus may play a role in the pathophysiology of schizophrenia. Although no direct link to genetic polymorphism in these genes and NMDA receptor function has been revealed, the present findings support previous reports implicating DAOA/G30 as susceptibility genes for schizophrenia. Further research is warranted to determine the functional variation underlying these findings and to relate this to the pathophysiology of schizophrenia.

Inhibition of both focal adhesion kinase and insulin-like growth factor-I receptor kinase suppresses glioma proliferation in vitro and in vivo.

Multiple genetic aberrations in human gliomas contribute to their highly infiltrative and rapid growth characteristics. Focal adhesion kinase (FAK) regulates tumor migration and invasion. Insulin-like growth factor-I receptor (IGF-IR), whose expression correlates with tumor grade, is involved in proliferation and survival. We hypothesized that inhibiting the phosphorylation of FAK and IGF-IR by NVP-TAE226 (hereafter called TAE226), a novel dual tyrosine kinase inhibitor of FAK and IGF-IR, would suppress the growth and invasion of glioma cells. In culture, TAE226 inhibited extracellular matrix-induced autophosphorylation of FAK (Tyr(397)). TAE226 also inhibited IGF-I-induced phosphorylation of IGF-IR and activity of its downstream target genes such as MAPK and Akt. TAE226 retarded tumor cell growth as assessed by a cell viability assay and attenuated G(2)-M cell cycle progression associated with a decrease in cyclin B1 and phosphorylated cdc2 (Tyr(15)) protein expression. TAE226 treatment inhibited tumor cell invasion by at least 50% compared with the control in an in vitro Matrigel invasion assay. Interestingly, TAE226 treatment of tumor cells containing wild-type p53 mainly exhibited G(2)-M arrest, whereas tumor cells bearing mutant p53 underwent apoptosis. Induction of apoptosis by TAE226 was substantiated by detection of caspase-3/7 activation and poly(ADP-ribose) polymerase cleavage and by an Annexin V apoptosis assay. More importantly, TAE226 treatment significantly increased the survival rate of animals in an intracranial glioma xenograft model. Collectively, these data show that blocking the signaling pathways of FAK and IGF-IR with TAE226 has the potential to be an efficacious treatment for human gliomas.

No association between the brain-derived neurotrophic factor (BDNF) Val66Met polymorphism and schizophrenia in Asian populations: Evidence from a case-control study and meta-analysis.

Brain-derived neurotrophic factor (BDNF) is a nerve growth factor that plays an important role in the development and maintenance of adult neurons and is important regulator of synaptic plasticity in human brain. It has been reported that there are alterations in BDNF levels in the brains of patients with schizophrenia. It has also been reported that transneuronal transfer of BDNF is dependent on neuronal activity, suggesting that BDNF plays an important role in neurotransmission. A single nucleotide polymorphism (SNP) in the BDNF gene that causes a valine to methionine substitution at codon 66 (Val66Met) has been demonstrated to affect human memory and hippocampal function. A possible positive association between the BDNF Val66Met polymorphism and schizophrenia has also been shown in Scottish and Spanish populations. Furthermore, the BDNF Val66Met polymorphism has been implicated in the age of onset of schizophrenia. In the present study, we attempted to replicate these findings in a Japanese case-control sample (211 patients with schizophrenia and 205 controls). We did not find an association between the BDNF Val66Met polymorphism and schizophrenia. An association between the Val66Met polymorphism and age of onset was not observed either. Furthermore, a meta-analysis including the present and previous Asian studies comparing 2059 patients with schizophrenia and 2765 controls also revealed no significant association between the BDNF Val66Met polymorphism and schizophrenia. Our results do not support a significant role for the BDNF Val66Met polymorphism in the development of schizophrenia in Asian populations.

A novel low-molecular weight inhibitor of focal adhesion kinase, TAE226, inhibits glioma growth.

Glioblastomas are highly lethal cancers that resist current therapies. Novel therapies under development target molecular mechanisms that promote glioblastoma growth. In glioblastoma patient specimens, the non-receptor tyrosine kinase focal adhesion kinase (FAK) is overexpressed. Upon growth factor receptor stimulation or integrin engagement, FAK is activated by phosphorylation on critical tyrosine residues. Activated FAK initiates a signal transduction cascade which promotes glioma growth and invasion by increasing cellular adhesion, migration, invasion, and proliferation. We find that human glioma cell lines express different levels of total FAK protein and activating phosphorylation of tyrosine residues Tyr397, Tyr861, and Tyr925. As all glioma cell lines examined expressed phosphorylated FAK, we examined the efficacy of a novel low-molecular weight inhibitor of FAK, TAE226, against human glioma cell lines. TAE226 inhibited the phosphorylation of FAK as well as the downstream effectors AKT, extracellular signal-related kinase, and S6 ribosomal protein in multiple glioma cell lines. TAE226 induced a concentration-dependent decrease in cellular proliferation with an associated G(2) cell cycle arrest in every cell line and an increase in apoptosis in a cell-line-specific manner. TAE226 also decreased glioma cell adhesion, migration, and invasion through an artificial extracellular matrix. Together, these data demonstrate the potential benefit of TAE226 for glioma therapy.

No association between a functional NAD(P)H: quinone oxidoreductase gene polymorphism (Pro187Ser) and tardive dyskinesia.

Several lines of evidence have indicated that free radicals may play a role in the pathophysiology of tardive dyskinesia (TD) (reviewed in Andreassen and Jorgensen, 2000). NAD(P)H: quinone oxidoreductase (NQO1) is an important enzyme in the human body that counteracts the oxidative stress-induced neuronal injury caused by the toxic free radicals such as dopamine-semiquinones. Taking the possible genetic predisposition to TD into account (Yassa and Ananth, 1981), the NQO1 gene is a good candidate gene that may confer increased susceptibility to TD. Based on this hypothesis, Pae et al. (2004) reported a significant association between the Pro187Ser polymorphism in the NQO1 gene and TD. In the present study, we attempted to replicate the findings of Pae et al. (2004) with the same polymorphism in 222 Japanese patients with schizophrenia. No significant difference was detected between patients with and without TD in the allelic distribution (chi2 = 0.070, d.f. = 1, p = 0.795) and in the genotypic distribution (chi2 = 0.910, d.f. = 2, p = 0.657). In addition, there was no significant difference in terms of total Abnormal Involuntary Movement Scale scores among the three genotype groups (p = 0.49). Our results suggest that the NQO1 gene polymorphism does not confer an increased risk of TD.

Association study between functional polymorphisms in the cytochrome P450 1A2 and 2D6 genes and polydipsia in schizophrenia.

The underlying pathophysiology of polydipsia in schizophrenia is poorly understood. Several studies, however, have suggested that there might be a genetic predisposition to polydipsia. In the present study, using a case-control sample that is independent from the previous family sample, we examined a possible association between polydipsia and functional polymorphisms in the genes of cytochrome P450 (CYP) 1A2 and 2D6, primarily important enzymes to the pharmacokinetics of antipsychotic drugs. Japanese patients with schizophrenia (63 polydipsics and 78 nonpolydipsics) were genotyped for two functional polymorphisms, the 734C/A polymorphism in the CYP1A2 gene and the 2D6*10 allele of the CYP2D6 gene. Neither of the polymorphisms was found to be associated with polydipsia nor was any evidence found that the two polymorphisms have an additive effect on the liability to polydipsia. Our results suggest that the CYP1A2 and CYP2D6 polymorphisms are not likely to play a major role in the development of polydipsia in schizophrenia, although further studies testing other alleles of CYP1A2 and CYP2D6 using different ethnic populations are warranted.

Design and synthesis of 7H-pyrrolo[2,3-d]pyrimidines as focal adhesion kinase inhibitors. Part 2.

A series of 2-amino-9-aryl-7H-pyrrolo[2,3-d]pyrimidines were designed and synthesized as focal adhesion kinase (FAK) inhibitors using molecular modeling in conjunction with a co-crystal structure. Chemistry was developed to introduce functionality onto the 9-aryl ring, which resulted in the identification of potent FAK inhibitors. In particular, compound 32 possessed single-digit nanomolar IC(50) and represents one of the most potent FAK inhibitors discovered to date.