Journal of Pharmacology and Experimental Therapeutics最新文献

MK-8189 is a novel phosphodiesterase 10A (PDE10A) inhibitor being evaluated in clinical studies for the treatment of schizophrenia. PDE10A is a cyclic nucleotide phosphodiesterase enzyme highly expressed in medium spiny neurons of the striatum. MK-8189 exhibits subnanomolar potency on the PDE10A enzyme and has excellent pharmaceutical properties. Oral administration of MK-8189 significantly increased cyclic guanosine monophosphate and phospho glutamate receptor 1 in rat striatal tissues. Activation of the dopamine D1 direct and D2 indirect pathways was demonstrated by detecting significant elevation of mRNA encoding substance P and enkephalin after MK-8189 administration. The PDE10A tracer [³H]MK-8193 was used to determine the PDE10A enzyme occupancy (EO) required for efficacy in behavioral models. In the rat-conditioned avoidance responding assay, MK-8189 significantly decreased avoidance behavior at PDE10A EO greater than ∼48%. MK-8189 significantly reversed an MK-801-induced deficit in prepulse inhibition at PDE10A EO of ∼47% and higher. Target engagement of MK-8189 in rhesus monkeys was examined with [¹¹C]MK-8193 in positron emission tomography studies, and plasma concentrations of 127 nM MK-8189 yielded ∼50% EO in the striatum. The impact of MK-8189 on cognitive symptoms was evaluated using the objective retrieval task in rhesus monkeys. MK-8189 significantly attenuated a ketamine-induced deficit in object retrieval performance at exposure that yielded ∼29% PDE10A EO. These findings demonstrate the robust impact of MK-8189 on striatal signaling and efficacy in preclinical models of symptoms associated with schizophrenia. Data from these studies were used to establish the relationship between preclinical efficacy, plasma exposures, and PDE10A EO to guide dose selection of MK-8189 in clinical studies. SIGNIFICANCE STATEMENT: We describe the primary pharmacology of MK-8189, a phosphodiesterase 10A (PDE10A) inhibitor under evaluation for the treatment of schizophrenia. We report efficacy in preclinical models that have been used to characterize other PDE10A inhibitors and atypical antipsychotics. The PDE10A occupancy achieved by MK-8189 in behavioral studies was used to support dose selection in clinical trials. This work provides evidence to support exploration of higher levels of PDE10A occupancy in clinical trials to determine if this translates to improved efficacy in patients.

Despite considerable efforts, there remains no Food and Drug Administration-approved medications for cocaine use disorder. One strategy to mitigate cocaine craving and relapse is to elevate dopamine. The dopamine transport inhibitor and releaser d-amphetamine has been shown to decrease cocaine self-administration (SA), although it has abuse liability. Recently, several modafinil analogs reduced cocaine SA in rats and monkeys, including JJC8-088, characterized as "cocaine-like" in rats, and JJC8-091, characterized as "atypical" and not SA by rats. The present study evaluated the reinforcing effects of both compounds in monkeys under several conditions. For experiment 1, 4 male cocaine-experienced rhesus monkeys self-administered cocaine (0.001-0.3 mg/kg per injection), JJC8-088 (0.001-0.3 mg/kg per injection), and JJC8-091 (0.1-3.0 mg/kg per injection) under a progressive-ratio schedule of reinforcement. Both JJC compounds functioned as reinforcers with equal reinforcing strength to cocaine. Although JJC8-091 was less potent than cocaine, JJC8-088 and cocaine had similar potencies. For experiment 2, 1 male and 2 female drug-naïve cynomolgus monkeys responded on a fixed-ratio schedule of food reinforcement. JJC8-091 was self-administered at rates higher than saline in all 3 monkeys. In experiment 3, monkeys from experiment 2 responded under a concurrent drug versus food choice paradigm and given access to cocaine or JJC8-091 under these conditions. At doses equal to or one-half log-units higher than doses used in experiment 2, cocaine, but not JJC8-091, was chosen over food. Together, these results demonstrate that although JJC8-091 may be reinforcing under some conditions, its reinforcing strength, in the presence of an alternative reinforcer, is substantially less than cocaine. SIGNIFICANCE STATEMENT: JJC8-088 and JJC8-091 have shown efficacy is reducing cocaine self-administration (SA) in rats and in nonhuman primates. This study found that both compounds maintained SA in monkeys responding under several conditions. However, when given access to an alternative reinforcer during the SA session, JJC8-091 was not reinforcing, suggesting that JJC8-091 may have lower abuse liability than cocaine and may be a viable candidate for cocaine use disorder because, in the human population, alternatives to drug use are often available.

Bidirectional signaling through platelet integrin α_IIbβ₃ is essential in hemostasis and thrombosis. In quiescent platelets, α_IIbβ₃ is in a low-affinity ligand binding state. However, on platelet activation by agonists through inside-out signaling, a rapid switch in the conformation of the integrin results in a high affinity ligand binding state capable of binding soluble fibrinogen. Ligand binding to the α_IIbβ₃ induces a signaling termed outside-in signaling that regulate platelet spreading and clot retraction. These events are often interchangeably used to represent outside-in signaling pathway. Using pharmacological inhibitors of known signaling molecules that have been implicated to regulate outside-in signaling, we assessed human platelet spreading and clot retraction. We found that inhibition of phosphoinositide-3-kinase, phospholipase C, protein kinase C, and focal adhesion kinase strongly attenuated both platelet spreading and clot retraction suggesting that they are essential for both clot retraction and platelet spreading, whereas inhibition of Rac1, rho-associated, coiled-coil containing protein kinase, p38, and MEK did not affect platelet spreading but significantly delayed clot retraction suggesting that these signaling molecules do not participate in platelet spreading. Interestingly, Src family kinases are required for platelet spreading and FAK activation but suppress clot retraction because their inhibition causes faster clot retraction. Thus, it becomes evident that platelet spreading, and clot retraction are differently regulated through α_IIbβ₃ outside-in signaling and should not be used interchangeably as readout for α_IIbβ₃ outside-in signaling assessment. SIGNIFICANCE STATEMENT: Current antiplatelet drugs have increased risk of bleeding and low efficacy. There is an increased effort to identify novel antiplatelet agents that have improved efficacy with reduced risk of bleeding. It is increasingly felt that inhibition of α_IIbβ_3-induced outside-in signaling may inhibit thrombosis without compromising hemostasis. However, the signaling entities regulating outside-in signaling are poorly understood. The work included in this article delineates the distinct signaling pathways involved in outside-in signaling and identify potential novel targets for intervention of thrombosis.

Estrogen receptor (ER)-negative breast cancers are known to be aggressive and unresponsive to antiestrogen therapy, and triple-negative breast cancers are associated with poor prognosis and metastasis. Thus, new targeted therapies are needed. Forkhead box M1 (FOXM1) is abundantly expressed in human cancers and implicated in protecting tumor cells from oxidative stress by reducing the levels of intracellular reactive oxygen species (ROS). Aspirin, a prototypical anticancer agent with deleterious side effects that has been modified to release nitric oxide and hydrogen sulfide is called nitric oxide-hydrogen sulfide-releasing aspirin (NOSH-aspirin, NOSH-ASA), generating a "safer" class of new anti-inflammatory agents. We evaluated NOSH-ASA against ER-negative breast cancer using cell lines and a xenograft mouse model. NOSH-ASA strongly inhibited growth of MDA-MB-231 and SKBR3 breast cancer cells with low IC₅₀s of 90 ± 5 and 82 ± 5 nM, respectively, with marginal effects on a normal breast epithelial cell line. NOSH-ASA inhibited cell proliferation, caused G₀/G₁ phase arrest, increased apoptosis, and was associated with increases in ROS. In MDA-MB-231 cell xenografts, NOSH-ASA reduced tumor size markedly, which was associated with reduced proliferation (decreased proliferating cell nuclear antigen expression), induction of apoptosis (increased terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells), and increased ROS, whereas nuclear factor κ-light-chain-enhancer of activated B cells and FoxM1 that were high in untreated xenografts were significantly reduced. mRNA data for FoxM1, p21, and cyclin D1 corroborated with the respective protein expressions and arrest of cells. Taken together, these molecular events contribute to NOSH-ASA-mediated growth inhibition and apoptotic death of ER-negative breast cells in vitro and in vivo. Additionally, as a ROS inducer and FOXM1 inhibitor, NOSH-ASA has potential as a targeted therapy. SIGNIFICANCE STATEMENT: We examined the cellular effects and xenograft tumor inhibitory potential of NOSH-aspirin, a nitric oxide- and hydrogen sulfide-donating hybrid, against estrogen receptor-negative breast cancer, which currently lacks effective therapeutic options. Inducing reactive oxygen species and downregulating forkhead box M1 are plausible mechanisms contributing to decreased cell proliferation and increased apoptosis. NOSH-aspirin reduced tumor size by 90% without inducing any observable gross toxicity, underscoring its promising translational potential.

The objective of this study was to investigate whether phosphoinositide 3-kinase (PI3K) and p38 mitogen-activated kinase contribute to the protection of irisin during hemorrhage/resuscitation. Experimental groups were divided based on the different treatments during resuscitation as follows: (1) hemorrhage: adult male CD-1 mice were subjected to hemorrhage at a mean arterial blood pressure of 35-45 mm Hg for 60 minutes, followed by resuscitation with shed blood and lactated Ringer's solution (n = 13); (2) hemorrhage + irisin: receiving irisin (5 μg/kg; n = 13); (3) hemorrhage + irisin + PI3K inhibitor: receiving both Ly294002 (1 mg/kg, i.v.) and irisin (n = 6); and (4) hemorrhage + irisin + p38 inhibitor: receiving SB202190 (1 mg/kg, i.v.) and irisin (n = 6). Compared with hemorrhage/resuscitation control, irisin improved cardiac function and the recovery of hemodynamics in association with the decreased systemic interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-α, which were completely abrogated by PI3K or p38 inhibitions. Furthermore, the inhibition of PI3K or p38 abolished irisin-induced reduction of the inflammatory cell infiltration and terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine nick-end labeling-positive apoptosis in the cardiac and skeletal muscles. Irisin reduced TNF-α and IL-6 expression in cardiac and skeletal muscles, which was abrogated by the inhibition of PI3K or p38. Irisin-treated hemorrhage increases the phosphorylation of PI3K and p38 in both cardiac and skeletal muscles, which was mitigated by the inhibition of PI3K or p38. PI3K and p38 play an important role in modulating the protective effect of irisin during the hemorrhage/resuscitation. SIGNIFICANCE STATEMENT: This study has identified a critical pathway in the regulation of trauma/hemorrhage by using a preclinical trauma model, in which irisin, as a hormone factor, stimulates PI3K and p38 pathways to induce protection against traumatic conditions. The study holds promise for developing a new therapeutic strategy to target irisin and its pathways related to PI3K and p38 to treat trauma and its comorbidities to reduce mortality for clinical implications.

Advanced-stage endometrial cancer patients typically receive a combination of platinum and paclitaxel chemotherapy. However, limited treatment options are available for those with recurrent disease, and there is a need to identify alternative treatment options for the advanced setting. Our goal was to evaluate the preclinical efficacy and mechanism of action of the anticancer drug Oklahoma Nitrone-007 (OKN-007) alone and in combination with carboplatin and paclitaxel in endometrial cancer. The effect of OKN-007 on the metabolic viability of endometrial cancer cells in both two- and three-dimensional (2D and 3D) cultures, as well as on clonogenic growth, in vitro was assessed. We also evaluated OKN-007 in vivo using an intraperitoneal xenograft model and targeted gene expression profiling to determine the molecular mechanism and gene expression programs altered by OKN-007. Our results showed that endometrial cancer cells were generally sensitive to OKN-007 in both 2D and 3D cultures. OKN-007 displayed a reduction in 3D spheroid and clonogenic growth. Subsequent targeted gene expression profiling revealed that OKN-007 significantly downregulated the immunosuppressive immunometabolic regulatory enzyme indolamine 2,3-dioxygenase 1 (IDO1) (-11.27-fold change) and modulated upstream inflammatory pathways that regulate IDO1 expression (interferon-gamma [IFN-γ], Janus kinase/signal transducer and activator of transcription [JAK-STAT], transforming growth factor beta [TGF-β], and nuclear factor-kappa B [NF-κB]), downstream IDO1 effector pathways (mammalian target of rapamycin [mTOR] and aryl hydrocarbon receptor [AhR]), and altered T cell signaling pathways. OKN-007 treatment reduced IDO1, sulfatase 2 (SULF2), and TGF-β protein expression in vivo and inhibited TGF-β, NF-κB, and AhR-mediated nuclear signaling in vitro. These findings indicate that OKN-007 surmounts proinflammatory, immunosuppressive, and protumorigenic pathways and is a promising approach for the effective treatment of endometrial cancer. SIGNIFICANCE STATEMENT: Women with advanced and recurrent endometrial cancer have limited therapeutic options. Oklahoma Nitrone-007 (OKN-007), which has minimal toxicity and is currently being evaluated in early-phase clinical trials for the treatment of cancer, is a potential new strategy for the treatment of endometrial cancer.

Post-traumatic stress disorder (PTSD) is caused by exposure to a traumatic or stressful event. Symptoms related to this disorder include persistent re-experiencing of memories and fear of generalization. Current pharmacological treatments for PTSD are insufficient, with fewer than 30% of patients reporting symptom remission. This study aims to determine the efficacy of acute (R,S)-ketamine and chronic fluoxetine (FLX) in reducing fear memory and fear generalization. In rodents, fear conditioning (FC) is commonly used in the literature to induce behaviors related to symptoms of PTSD, and the open field test (OFT) can assess anxiety and fear generalization behaviors during the exploration of a novel environment. In this study, FC consisted of a white noise cue stimulus and 4 inescapable foot shocks. Treatments began 4 hours after FC. Fear and anxiety behaviors were recorded during re-exposure to the FC stimuli at 24 hours and 2 weeks. The OFT was conducted 1 day before the last FC re-exposure. Results support the combined use of acute ketamine and chronic FLX as a treatment for reducing behaviors indicative of fear memory during re-exposure at 2 weeks, but not behaviors indicative of anxiety and fear generalization in the OFT. FLX alone was most effective in reducing behaviors related to fear generalization. This study contributes to the existing literature on pharmacological treatment for fear and anxiety behaviors relating to fear memory and fear generalization. Continued research is necessary to replicate results, optimize treatment protocols, and investigate the molecular adaptations to trauma and treatment. SIGNIFICANCE STATEMENT: Up to 6% of people in the United States will develop PTSD within their lifetime, and less than half of those individuals will find relief from their symptoms given the current therapeutic options. This study offers preliminary support for the efficacy of ketamine and FLX in reducing PTSD-like behaviors induced by fear-conditioning in mice. Compared with current standard treatments, the results of the current study indicate the potential for a more effective therapeutic option for those with stress-related disorders, such as PTSD.

Estrogen receptors (ERs) are essential pharmacological targets for treating hormonal disorders and estrogen-dependent malignancies. Selective activation of ERβ is hypothesized to provide therapeutic benefit with reduced risk of unwanted estrogenic side-effects associated with ERα activity. However, activating ERβ without activating ERα is challenging due to the high sequence and structural homology between the receptor subtypes. We assessed the impact of structural modifications to the parent compound OSU-ERβ-12 on receptor subtype binding selectivity using cell-free binding assays. Functional selectivity was evaluated by transactivation in HEK-293 cells overexpressing human or murine ERs. In vivo selectivity was examined through the uterotrophic effects of the analogs after oral administration in estrogen-naïve female mice. Furthermore, we evaluated the in vivo pharmacokinetics of the analogs following single-dose intravenous and oral administration. Regarding selectivity, a single compound exhibited greater functional selectivity than OSU-ERβ-12 for human ERβ. However, like others in the meta-carborane series, its poor in vivo pharmacokinetics limit its suitability for further development. Surprisingly, and at odds with their pharmacokinetic and in vitro human activity data, most analogs potently induced uterotrophic effects in estrogen-naïve female mice. Further investigation of activity in HEK-293 cells expressing murine ERs revealed species-specific differences in the ER subtype selectivity of these analogs. Our findings highlight species-specific receptor pharmacology and the challenges it poses to characterizing developmental therapeutics in preclinical species. SIGNIFICANCE STATEMENT: This study investigates para- and meta-substituted carborane analogs targeting estrogen receptors (ERs), revealing the greater selectivity of carborane analogs for human ERβ compared to the mouse ortholog. These findings shed light on the intricacies of using preclinical species in drug development to predict human pharmacology. The report also provides insights for the refinement and optimization of carborane analogs as potential therapeutic agents for estrogen-related disease states.