Journal of Pharmacology and Experimental Therapeutics最新文献

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.

Progressive multiple sclerosis (MS) represents the worsening phase of the disease, characterized by increasing neurodegeneration and disability and mainly refractory to current treatments. Finding therapeutic options remains challenging partially not only because of the lack of understanding of pathogenic mechanisms but also because the early dogma was centered on neuroinflammation, overshadowing the critical role of the tissue repair process. The tissue repair target should start early in disease development, and therapeutic strategies for progressive MS should combine anti-inflammatory and neuroprotective aspects. Increasing preclinical evidence, together with the new era of omics applied on frozen human brain tissue, has shed light on some ligand receptor pairs, such as growth-arrest-specific 6 (GAS6)/protein tyrosine kinase receptor (TYRO3) and protein S (PROS1)/AXL receptor tyrosine kinase (AXL), required to dampen inflammation, promote tissue repair, and engage remyelination. Understanding the role of these proteins in the early stages of MS is a critical step toward preventing or stopping neurodegeneration. Herein, we will discuss the receptor/ligand pairs that might be targetable for therapeutic intervention in progressive MS. SIGNIFICANCE STATEMENT: The aim for progressive multiple sclerosis treatment should be to combine anti-inflammatory and neuroprotective therapeutic strategies based on early intervention. Targeting the TYRO3, AXL, and MER tyrosine kinase receptor (TAM) signaling axis, particularly as growth-arrest-specific 6/TYRO3 and protein S/AXL, which are involved in tempering inflammation, promoting tissue repair, and engaging remyelination, could significantly benefit patients in the early stages of progressive multiple sclerosis.

Amitriptyline, a pleiotropic tricyclic antidepressant, possesses antioxidant and anti-inflammatory properties. Despite its diverse benefits, the specific effects of amitriptyline on inflammatory bowel disease (IBD) are not yet well defined. To explore this, we used a dextran sulfate sodium (DSS)-induced colitis model to examine the anti-inflammatory effects of amitriptyline and the underlying mechanisms by which it operates. Our research revealed that amitriptyline is effective in alleviating several pathological manifestations associated with colitis. This includes improving body weight retention, reducing disease activity index, lessening of colon length shortening, and repairing of colonic mucosal damage. Treatment with amitriptyline significantly protected mucosal injury by preserving the population of goblet cells and increasing the expression of tight junction proteins. Furthermore, we observed that amitriptyline effectively countered immune cell infiltration, specifically neutrophils and macrophages, while simultaneously lowering the levels of inflammatory cytokines such as tumor necrosis factor α, interleukin (IL)-1β, and IL-6. Additionally, RNA sequencing analysis pointed to the potential involvement of the Toll-like receptor (TLR) pathway in the anticolitic effects induced by amitriptyline. Subsequent Western blot analysis indicated that amitriptyline significantly inhibited the TLR-4-mediated nuclear factor (NF)-κB signaling pathway. To bolster our findings, in vitro studies demonstrated that amitriptyline downregulated the TLR-4/NF-κB/mitogen-activated protein kinase signaling cascades in mouse macrophages stimulated with lipopolysaccharide. Further molecular investigations revealed that amitriptyline was able to suppress the elevated expression of myeloid differentiation factor 2 that lipopolysaccharide stimulation typically induces. In summary, our findings suggest that amitriptyline effectively mitigates DSS-induced colitis in mice through the inhibition of TLR-4/myeloid differentiation 2 pathway signaling, indicating its potential repurposing for IBD treatment. SIGNIFICANCE STATEMENT: The potential of using amitriptyline in treating inflammatory bowel disease appears promising, leveraging its established safety and dosing profile as an antidepressant. The study results show that amitriptyline can alleviate pathological symptoms, inflammation, and intestinal mucosal damage in mice with colitis induced by DSS. The protective effect observed appears to be linked to the inhibition of TLR-4/myeloid differentiation 2 signaling pathway. By exploring novel applications for existing medications, we can optimize amitriptyline's efficacy and broaden its impact in both medical and commercial contexts.

Tyrosine kinase inhibitors (TKIs) targeting the breakpoint cluster region-ABL fusion protein, such as imatinib (Gleevec), have revolutionized targeted cancer therapies. However, drug resistance and side effects, particularly those affecting hemostasis, continue to pose significant challenges for TKI therapies. As tyrosine kinases serve pivotal roles in platelet hemostatic function, we investigated the potential impact of both established and emerging ABL TKIs on human platelet activities ex vivo. Our study included standard-of-care agents (eg, imatinib and nilotinib) and second-generation ABL inhibitors, including ponatinib and bosutinib, designed to mitigate drug resistance. Additionally, we explored the effects of allosteric inhibitors targeting the myristoyl pocket of ABL (eg, asciminib and GNF-2) and novel agents in preclinical development, including ELVN-919, which uniquely exhibits high specificity for the ABL kinase active site. Our findings reveal that while ABL inhibitors such as ponatinib and bosutinib impede platelet activity, highly specific new-generation ABL inhibitors, including first-in-class therapeutics, do not impact platelet function ex vivo. Overall, these new insights around the effects of ABL TKIs on platelet function could inform the development of targeted therapies with reduced hematologic toxicities. SIGNIFICANCE STATEMENT: This study examines the effects of clinically relevant small molecule breakpoint cluster region (BCR)-ABL tyrosine kinase inhibitors (TKIs) on platelet activity. This analysis includes first-time assessments of agents such as asciminib and ELVN-919 on human platelet function ex vivo, alongside established therapies (eg, imatinib, ponatinib) with well characterized effects on platelet function, to discern potential antiplatelet and other effects of BCR-ABL TKIs and inform clinical safety.

Metformin's potential in treating ischemic stroke and neurodegenerative conditions is of growing interest. Yet, the absence of established systemic and brain pharmacokinetic (PK) parameters at relevant preclinical doses presents a significant knowledge gap. This study highlights these PK parameters and the importance of using pharmacologically relevant preclinical doses to study pharmacodynamics in stroke and related neurodegenerative diseases. A liquid chromatography with tandem mass spectrometry method to measure metformin levels in plasma, brain, and cerebrospinal fluid was developed and validated. In vitro assays examined brain tissue binding and metabolic stability. Intravenous bolus administration of metformin to C57BL6 mice covered a low- to high-dose range maintaining pharmacological relevance. Quantification of metformin in the brain was used to assess brain PK parameters, such as unidirectional blood-to-brain constant (K_in) and unbound brain-to-plasma ratio (K_{p, uu, brain}). Metformin exhibited no binding in the mouse plasma and brain and remained metabolically stable. It rapidly entered the brain, reaching detectable levels in as little as 5 minutes. A K_in value of 1.87 ± 0.27 μL/g/min was obtained. As the dose increased, K_{p, uu, brain} showed decreased value, implying saturation, but this did not affect an increase in absolute brain concentrations. Metformin was quantifiable in the cerebrospinal fluid at 30 minutes but decreased over time, with concentrations lower than those in the brain across all doses. Our findings emphasize the importance of metformin dose selection based on PK parameters for preclinical pharmacological studies. We anticipate further investigations focusing on PKs and pharmacodynamics in disease conditions, such as stroke. SIGNIFICANCE STATEMENT: The study establishes crucial pharmacokinetic parameters of metformin for treating ischemic stroke and neurodegenerative diseases, addressing a significant knowledge gap. It further emphasizes the importance of selecting pharmacologically relevant preclinical doses. The findings highlight metformin's rapid brain entry, minimal binding, and metabolic stability. The necessity of considering pharmacokinetic parameters in preclinical studies provides a foundation for future investigations into metformin's efficacy for neurodegenerative disease(s).

Ovarian cancer is the most lethal gynecological malignancy, with a 5-year survival rate of approximately 50%. The dismal prognosis is due in part to metastatic disease and acquired drug resistance to conventional chemotherapies such as taxanes. Colchicine binding site inhibitors (CBSIs) are attractive alternatives to taxanes because they could potentially achieve oral bioavailability and overcome drug resistance associated with the prolonged use of taxanes. VERU-111 is one of the most advanced CBSIs that is orally available, potent, and well tolerated and has shown good efficacy in several preclinical solid tumor models. Here, we demonstrate for the first time the in vitro potency of VERU-111 as well as its efficacy at inhibiting tumor growth and metastasis in an orthotopic ovarian cancer mouse model. VERU-111 has nanomolar potency against ovarian cancer cell lines and strongly inhibits colony formation, proliferation, invasion, and migration. VERU-111 disrupts microtubule formation to induce mitotic catastrophe and ultimately apoptosis in a concentration-dependent manner. The efficacy of VERU-111 was comparable with standard chemotherapy paclitaxel, the current first-line treatment of ovarian cancer, with no observed synergy with combination paclitaxel + VERU-111 treatment. In vivo, VERU-111 markedly suppressed ovarian tumor growth and completely suppressed distant organ metastasis. Together, these results support VERU-111 for its potential as a novel therapy for ovarian cancer, particularly for late-stage metastatic disease. SIGNIFICANCE STATEMENT: VERU-111 is an investigational new drug and has comparable efficacy as paclitaxel in suppressing tumor cell proliferation, colony formation, and migration in ovarian cancer models in vitro and has potent in vivo antitumor and antimetastatic activity in an orthotopic ovarian cancer mouse model. VERU-111 has low systemic toxicity and, unlike paclitaxel, is orally bioavailable and is not a substrate for the major drug efflux transporters, making it a promising and attractive alternative to taxane-based therapy.

Aneurysmal subarachnoid hemorrhage (SAH) may be associated with cerebral vasospasm, which can lead to delayed cerebral ischemia, infarction, and worsened functional outcomes. The delayed nature of cerebral ischemia secondary to SAH-related vasculopathy presents a window of opportunity for the evaluation of well tolerated neuroprotective agents administered soon after ictus. Secondary ischemic injury in SAH is associated with increased extracellular glutamate, which can overactivate N-methyl-d-aspartate receptors (NMDARs), thereby triggering NMDAR-mediated cellular damage. In this study, we evaluated the effect of the pH-sensitive GluN2B-selective NMDAR inhibitor NP10679 on neurologic impairment after SAH. This compound demonstrates a selective increase in potency at the acidic extracellular pH levels that occur in the setting of ischemia. We found that NP10679 produced durable improvement of behavioral deficits in a well characterized murine model of SAH, and these effects were greater than those produced by nimodipine alone, the current standard of care. In addition, we observed an unexpected reduction in SAH-induced luminal narrowing of the middle cerebral artery. Neither nimodipine nor NP10679 alters each other's pharmacokinetic profile, suggesting no obvious drug-drug interactions. Based on allometric scaling of both toxicological and efficacy data, the therapeutic margin in humans should be at least 2. These results further demonstrate the utility of pH-dependent neuroprotective agents and GluN2B-selective NMDAR inhibitors as potential therapeutic strategies for the treatment of aneurysmal SAH. SIGNIFICANCE STATEMENT: This report describes the properties and utility of the GluN2B-selective pH-sensitive N-methyl-d-aspartate receptor inhibitor, NP10679, in a well characterized rodent model of subarachnoid hemorrhage. We show that the administration of NP10679 improves long-term neurological function following subarachnoid hemorrhage and that in rats, there are no drug-drug interactions between NP10679 and nimodipine, the standard of care for this indication.

The advent of HER2-targeted monoclonal antibodies such as trastuzumab has significantly improved the clinical outcomes for patients with breast cancer overexpressing HER2 and, more recently, also for gastric cancers. However, the development of resistance, as is frequently the case for other antineoplastic modalities, constrains their clinical efficacy. Multiple molecular mechanisms and signaling pathways have been investigated for their potential involvement in the development of resistance to HER2-targeted therapies, among which is autophagy. Autophagy is an inherent cellular mechanism whereby cytoplasmic components are selectively degraded to maintain cellular homeostasis via the generation of energy and metabolic intermediates. Although the cytoprotective form of autophagy is thought to predominate, other forms of autophagy have also been identified in response to chemotherapeutic agents in various tumor models; these include cytotoxic, cytostatic, and nonprotective functional forms of autophagy. In this review, we provide an overview of the autophagic machinery induced in response to HER2-targeted monoclonal antibodies, with a focus on trastuzumab and trastuzumab-emtansine, in an effort to determine whether autophagy targeting or modulation could be translated clinically to increase their effectiveness and/or overcome the development of resistance. SIGNIFICANCE STATEMENT: This manuscript is one in a series of papers that interrogate the role(s) of the autophagy induced in response to antineoplastic agents in various cancer models. This series of papers was developed in an effort to establish whether autophagy targeting or modulation is likely to be an effective adjuvant strategy to increase the efficacy of cancer chemotherapeutic agents. This review explores the relationship between the autophagic machinery and HER2-targeted therapies.

Interleukin (IL)-33 has been shown to centrally regulate, among other processes, inflammation and fibrosis. Both intracellular full-length (FLIL33) precursor and extracellular mature cytokine (MIL33) forms exert such regulation, albeit differentially. Drug development efforts to target the IL-33 pathway have focused mostly on MIL33 and its specific cell-surface receptor, ST2, with limited attempts to negotiate the pathophysiological contributions from FLIL33. Furthermore, even a successful strategy for targeting MIL33 effects would arguably benefit from a simultaneous attenuation of the levels of FLIL33, which remains the continuous source of MIL33 supply. We therefore sought to develop an approach to depleting FLIL33 protein levels. We previously reported that the steady-state levels of FLIL33 are controlled in part through its proteasomal degradation and that such regulation can be mapped to a segment in the N-terminal portion of FLIL33. We hypothesized that disruption of this regulation would lead to a decrease in FLIL33 levels, thus inducing a beneficial therapeutic effect in an IL-33-dependent pathology. To test this hypothesis, we designed and tested cell-permeable decoy peptides, which mimic the target N-terminal FLIL33 region. We argued that such mimic peptides would compete with FLIL33 for the components of the native FLIL33 production and maintenance molecular machinery. Administered in the therapeutic regimen to bleomycin-challenged mice, the tested cell-permeable decoy peptides alleviated the overall severity of the disease by restoring body weight loss and attenuating accumulation of collagen in the lungs. This proof-of-principle study lays the foundation for future work toward the development of this prospective therapeutic approach. SIGNIFICANCE STATEMENT: An antifibrotic therapeutic approach is proposed and preclinically tested in mice in vivo based on targeting the full-length IL-33 precursor protein. Peptide fusion constructs consisted of a cell-permeable sequence fused with a sequence mimicking an N-terminal segment of IL-33 precursor that is responsible for this protein's stability. Systemic administration of such peptides to mice in either the acute intratracheal or chronic systemic bleomycin challenge models leads to a decrease in the bleomycin-induced elevations of pulmonary IL-33 and collagen.