Discovery of 2,4-diamino-5-cyanopyrimidine derivatives as protein kinase C theta inhibitors with mitigated time-dependent drug-drug interactions.

Protein kinase C theta (PKCθ) plays a critical role in T cell signaling and has therapeutic potential for T cell-mediated diseases such as transplant rejection and rheumatoid arthritis. PKCθ inhibitors have emerged as effective immunomodulative agents for the prevention of transplant rejection. We previously reported that the 2,4-diamino-5-cyanopyrimidine derivative 2 was a potent PKCθ inhibitor; however, it exhibited CYP3A4 time-dependent inhibition (TDI). Here, we report the structural modification of compound 2 into 34 focusing on mitigating CYP3A4 TDI. Compound 34 exhibited potent in vitro activity with mitigated CYP3A4 TDI and efficacy in vivo transplant model.

A novel 2,4-diaminopyrimidine derivative as selective inhibitor of protein kinase C theta prevents allograft rejection in a rat heart transplant model.

Protein kinase C theta (PKCθ) plays a critical role in T cell signaling and is an attractive target for the treatment of T cell-mediated diseases such as transplant rejection and autoimmune disease. To identify PKCθ inhibitors, we focused on the 2,6-diamino-3-carbamoyl-5-cyanopyrazine derivative 2, which exhibited moderate PKCθ inhibitory activity. Optimization of 2 identified the 2,4-diamino-5-cyanopyrimidine derivative 16c, which exhibited potent PKCθ inhibitory activity and showed good selectivity against other PKC isozymes. Compound 16c prolonged graft survival in an in vivo rat heterotopic cardiac transplant model.

A novel JAK inhibitor, peficitinib, demonstrates potent efficacy in a rat adjuvant-induced arthritis model.

The Janus kinase (JAK) family of tyrosine kinases is associated with various cytokine receptors. JAK1 and JAK3 play particularly important roles in the immune response, and their inhibition is expected to provide targeted immune modulation. Several oral JAK inhibitors have recently been developed for treating autoimmune diseases, including rheumatoid arthritis (RA). Here, we investigated the pharmacological effects of peficitinib (formerly known as ASP015K), a novel, chemically synthesized JAK inhibitor. We found that peficitinib inhibited JAK1 and JAK3 with 50% inhibitory concentrations of 3.9 and 0.7 nM, respectively. Peficitinib also inhibited IL-2-dependent T cell proliferation in vitro and STAT5 phosphorylation in vitro and ex vivo. Furthermore, peficitinib dose-dependently suppressed bone destruction and paw swelling in an adjuvant-induced arthritis model in rats via prophylactic or therapeutic oral dosing regimens. Peficitinib also showed efficacy in the model by continuous intraperitoneal infusion. Area under the concentration versus time curve (AUC) at 50% inhibition of paw swelling via intraperitoneal infusion was similar to exposure levels of AUC at 50% inhibition via oral administration, implying that AUC might be important for determining the therapeutic efficacy of peficitinib. These data suggest that peficitinib has therapeutic potential for the oral treatment of RA.

Optimization of 2,4-diamino-5-fluoropyrimidine derivatives as protein kinase C theta inhibitors with mitigated time-dependent drug-drug interactions and P-gp liability.

Protein kinase C theta (PKCθ) plays a critical role in T cell signaling and has therapeutic potential for T cell-mediated diseases such as transplant rejection and rheumatoid arthritis. Here, a series of 2,4-diamino-5-fluoropyrimidine derivatives were prepared and evaluated for their inhibition of PKCθ. Of these compounds, 14f was found to exhibit potent PKCθ inhibitory activity and significantly weak CYP3A4 time-dependent inhibition (TDI) and P-glycoprotein (P-gp) liability.

Pharmacological characterization of a novel potent, selective, and orally active phosphodiesterase 2A inhibitor, PDM-631.

Recently, we identified a novel phosphodiesterase 2A (PDE2A) inhibitor, PDM-631 ((S)-3-cyclopropyl-6-methyl-1-(1-(4-(trifluoromethoxy)phenyl)propan-2-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one). PDM-631 showed potent inhibitory activities for human and rat PDE2A with IC50 values of 1.5 and 4.2nM, respectively and more than 2000-fold selectivity against other phosphodiesterases. In rat studies, PDM-631 showed oral bioavailability and good brain penetration, and increased the cGMP levels in the cortex. These data indicate that PDM-631 is a potent, selective, orally active, and brain-penetrable PDE2A inhibitor. In behavioral studies using rat models, PDM-631 (3-30mg/kg) resulted in better discrimination between a novel object and a familiar one 48h after the acquisition phase in the novel object recognition test, thus indicating that PDM-631 increased object recognition memory. In contrast, PDM-631 did not attenuate the conditioned avoidance response at the same dose range (3-30mg/kg) in rats, indicating that PDM-631 did not show an antipsychotic-like effect. In test for extrapyramidal side effect, PDM-631 had no effect on catalepsy at the effective doses (10 and 30mg/kg) in the novel object recognition test, while haloperidol caused catalepsy at a dose of 3mg/kg. Our results suggest that PDM-631 is a good pharmacological tool that can be used to investigate the role of PDE2A and may have therapeutic potential for the treatment of cognitive impairments associated with schizophrenia and neurodegenerative disorders, without any extrapyramidal side effects.

Effect of a novel inducible nitric oxide synthase inhibitor, FR260330, in prevention of renal ischemia/reperfusion injury in vervet monkeys.

Cytotoxic nitric oxide (NO) is produced during ischemia/reperfusion (I/R) injury by the expression of inducible NO synthase (iNOS). Therefore, continuous iNOS inhibition might prevent early graft dysfunction. FR260330, a potent and selective inhibitor of iNOS activity, impedes the dimmerization of iNOS monomer. In this study, the effect of FR260330 in the prevention of renal I/R injury was evaluated in the model of one kidney ischemia in Vervet monkeys. A total of 18 male Vervet monkeys were randomly assigned to two equal groups (n=9). Transient (60 min) left renal ischemia was produced by simultaneous contralateral nephrectomy in treated (FR260330 20 mg/kg/day) and placebo control groups. Renal function and other biochemical parameters as well as FR260330 concentrations were studies until day 15 after I/R injury. All monkeys survived after 60 min I/R injury until sacrifice on day 15. Serum creatinine in the untreated controls increased significantly in comparison to the FR260330-treated group on days 2, 3, 4, and 7 (P<0.05). Plasma FR260330 concentration after oral administration showed that C(max) was 3.251+/-2.526 microg/ml, and T(max) was 4 hr. This study thus finds that FR260330, as a selective iNOS inhibitor, effectively prevents renal I/R injury in Vervet monkeys.

Effect of a novel inducible nitric oxide synthase inhibitor in prevention of rat chronic aortic rejections.

BACKGROUND: Cytotoxic nitric oxide (NO) is produced during ischemia-reperfusion injury and acute and chronic rejection in allografts by expression of inducible (i) NO synthase (NOS). Therefore, continuous inhibition of iNOS may prevent early graft dysfunction and immune injury (rejection) and consequently improve graft survival. FR260330 is a potent and selective inhibitor of iNOS activity that works by preventing iNOS monomers from dimerization. In this study, the authors evaluated the effect of FR260330 in prevention of chronic rejection in a model of rat aortic allografts. METHODS: Male Lewis (LEW, RT1l) rats received male ACI (RT1a) aorta allografts or LEW aorta isografts. Fourteen groups (n > or = 6) were involved in this study. FR260330, tacrolimus, or both were administered orally for 14 or 90 days, according to protocol. The degree of intimal proliferation of graft aorta was determined by a computerized image system. RESULTS: Both low and high doses of FR260330- or tacrolimus-treated grafts showed significantly decreased intima/(intima+media) ratios at day 90 compared with placebo controls. Combination therapy of low-dose FR260330 with low-dose tacrolimus produced a significant decrease of intima/(intima+media) ratios with intact endothelium compared with placebo controls. Anti-alpha-actin immunohistochemical staining demonstrated that one of the mechanisms of intimal proliferation is related to migration of vascular smooth muscle cells. CONCLUSIONS: A selective inhibitor of NOS, FR260330 plays a protective role in chronic aortic allograft rejection in the rat. Combination therapy of low-dose FR260330 with tacrolimus produces significant protection of immune injury and may serve to improve long-term graft survival and function.

Pharmacological profile of FR260330, a novel orally active inducible nitric oxide synthase inhibitor.

In this study, we examined effects of a newly synthesized chemical compound, FR260330, (2E)-3-(4-chlorophenyl)-N-[(1S)-2-oxo-2-{[2-oxo-2-(4-{[6-(trifluoromethyl)-4-pyrimidinyl]oxy}-1-piperidinyl)ethyl]amino}-1-(2-pyridinylmethyl)ethyl]acrylamide on nitric oxide (NO) production in rat splenocytes and human colon cancer cell line, DLD-1 cells. FR260330 inhibited NOx production dose dependently in both cells. In lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) treated murine macrophage cell line, RAW264.7, Western blot analysis with gel filtration chromatography revealed FR260330 might prevent dimerization of inducible nitric oxide synthase (iNOS), but had no effect on the expression of iNOS protein. Furthermore, oral administration of FR260330 reduced NOx production dose dependently in plasma from rats exposed to LPS (IC50=1.6 mg/kg). Meanwhile, higher dose (100 mg/kg) of oral administration of FR260330 did not change mean arterial blood pressure in rats. These results suggest that FR260330 might be a useful therapeutical approach to various inflammatory diseases, in which superoxide or peroxynitrite formed from iNOS-derived NO are involved.

A novel inhibitor of vacuolar ATPase, FR202126, prevents alveolar bone destruction in experimental periodontitis in rats.

An acidic microenvironment formed by vacuolar ATPase (V-ATPase) expressed in plasma membranes of osteoclasts is thought to be indispensable for bone resorption. This study examined the efficacy of a novel V-ATPase inhibitor, FR202126, in reducing alveolar bone loss caused by experimental periodontitis in rats. FR202126 inhibited H+ transport in plasma membrane vesicles of murine osteoclasts, whereas FR202126 exerted no effect on H+ transport of mitochondrial ATPase or gastric H+,K+-ATPase, indicating that FR202126 is a specific inhibitor of V-ATPase. As expected from the mechanism, FR202126 remarkably inhibited in vitro bone resorption whatever bone resorptive factors were added. Moreover, FR202126 was also able to exert an inhibitory effect on in vivo bone resorption. Experimental periodontitis was induced by ligature wire tied around the contact between the first and second maxillary molars. Insertion of ligature wire for 7 days induced alveolar bone destruction by activating osteoclasts. Oral administration of FR202126 (u.i.d.) significantly prevented alveolar bone loss in experimental periodontitis which may offer a new approach to treatment of periodontal disease.

Effect of novel PKCθ selective inhibitor AS2521780 on acute rejection in rat and non-human primate models of transplantation.

Selective inhibition of protein kinase Cθ (PKCθ) may be useful in inducing T cell-specific immunosuppression with a reduced rate of side effects. To our knowledge, however, no reports have been published regarding the selective inhibition of PKCθ by small-molecule compounds in animal models of allograft rejection. Here, we investigated the effect of the newly synthesized PKCθ selective inhibitor AS2521780 in mono- and combination therapies on acute rejection in ACI-to-Lewis rat cardiac and non-human primate (NHP) renal transplantation models. In the rat cardiac transplantation model, AS2521780 significantly prolonged graft survival to 14days at 10mg/kg twice daily (b.i.d.) and to 20days at 30mg/kg b.i.d. In contrast, acute rejection occurred in all recipients in the non-treated group by Days 5 or 6 post-transplantation. Significant improvements (P<0.001) in graft survival were observed following treatment with a combination of AS2521780 at 3mg/kg b.i.d. and a suboptimal dose of tacrolimus (0.02mg/kg) or mycophenolate mofetil (15mg/kg). In the NHP renal transplantation model, AS2521780 at 3mg/kg b.i.d. and tacrolimus at 1mg/kg (suboptimal dose) significantly improved graft survival compared to tacrolimus alone (P<0.05). The present study of AS2521780 in rat cardiac and NHP renal transplantation models demonstrates the potential of PKCθ as a novel drug target for organ transplantation. As AS2521780 was well tolerated and the dose of tacrolimus or mycophenolate mofetil can be reduced when used in combination with this drug, immunosuppressive regimens containing selective inhibitors of PKCθ might have good safety profiles.

Effect of AS2521780, a novel PKCθ selective inhibitor, on T cell-mediated immunity.

T cell-mediated immunity is central to the pathogenesis of autoimmune diseases, and is a target in the development of alternative therapeutic strategies with reduced adverse effects on other cell types and organs. Protein kinase C (PKC) is a family of serine/threonine kinases, with knockout of the PKCθ isoform in mice resulting in defective T cell activation. However, the effects of selective inhibition of PKCθ by small-molecule compounds on T cell signaling are still unknown. Here, we evaluated the effect of the novel PKCθ inhibitor AS2521780 on T cell activation and joint inflammation in a rat model of arthritis. AS2521780 exerted potent inhibition of recombinant human PKCθ enzyme activity (IC50=0.48 nM), which was more than 30-fold higher than that of other PKC isoforms. Further, AS2521780 exerted little or no inhibition on other protein kinases. AS2521780 suppressed CD3/CD28-induced Interleukin-2 (IL-2) gene transcription in Jurkat T cells and proliferation of human primary T cells. AS2521780 also suppressed concanavalin A-induced cytokine production by rat splenocytes and monkey peripheral blood mononuclear cells with similar potency. Moreover, AS2521780 significantly reduced paw swelling in a dose-dependent manner in a rat model of adjuvant-induced arthritis. These results indicate that PKCθ is an attractive drug target and AS2521780 is a potential immunosuppressant for T cell-mediated autoimmune diseases.