RESUMO
KD033 is a clinical-stage immunocytokine composed of a high-affinity anti-human-PD-L1 antibody and the human IL-15/ IL-15 receptor sushi-domain complex. We have previously shown that KD033-surrogate, the anti-mouse-PD-L1/IL-15 immunocytokine, was efficacious in several syngeneic murine tumor models including those that were refractory to anti-PD-1/PD-L1 checkpoint blockers. KD033-surrogate showed better efficacy than the combination treatment of its component, anti-PD-L1 antibody with the non-targeting IL-15. KD033-surrogate was also efficacious in both low and high PD-L1-expressing tumors. In this study, we have utilized double knock-in mice expressing functional human PD-1/PD-L1 to show that the clinical molecule, KD033, reproduced the anti-tumor efficacy observed with KD033-surrogate in the syngeneic models. KD033 was equally efficacious in reducing the growth of human-PD-L1 positive (hPDL1+) and negative (hPDL1-) MC38 murine tumors. We observed similar peripheral pharmacodynamics changes in KD033-treated mice bearing either hPDL1+ or hPDL1- MC38 tumors. However, different transcriptomic profiles were observed between KD033-treated hPDL1+ and hPDL1- MC38 tumors with marked changes involving mostly downregulated genes in hPDL1- tumors in addition to the immune-related genes changes observed in both hPDL1+ and hPDL1- MC38 tumors. Cytotoxic and myeloid cell signatures were upregulated in both tumors with relatively greater increases observed in hPDL1- MC38 tumors. These effects of KD033 treatment in PD-L1 positive and negative tumors demonstrate the role of PD-L1 in targeting of IL-15 cytokine in vivo.
Assuntos
Antineoplásicos , Neoplasias , Animais , Humanos , Camundongos , Antineoplásicos/farmacologia , Antígeno B7-H1/genética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Interleucina-15/genética , Neoplasias/tratamento farmacológico , Neoplasias/genética , Proteínas Recombinantes de Fusão/farmacologia , Transdução de SinaisRESUMO
Detailed description of the mechanism of action of the therapeutic antibodies is essential for the functional characterization and future optimization of potential clinical agents. We recently developed KD035, a fully human antibody targeting vascular endothelial growth factor receptor 2 (VEGFR2). KD035 blocked VEGF-A, and VEGF-C-mediated VEGFR2 activation, as demonstrated by the in vitro binding and competition assays and functional cellular assays. Here, we report a computational model of the complex between the variable fragment of KD035 (KD035(Fv)) and the domains 2 and 3 of the extracellular portion of VEGFR2 (VEGFR2(D2-3)). Our modeling was guided by a priori experimental information including the X-ray structures of KD035 and related antibodies, binding assays, target domain mapping and comparison of KD035 affinity for VEGFR2 from different species. The accuracy of the model was assessed by molecular dynamics simulations, and subsequently validated by mutagenesis and binding analysis. Importantly, the steps followed during the generation of this model can set a precedent for future in silico efforts aimed at the accurate description of the antibody-antigen and more broadly protein-protein complexes.
Assuntos
Anticorpos , Fator A de Crescimento do Endotélio Vascular , Humanos , Simulação de Dinâmica Molecular , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Belumosudil is a selective rho-associated coiled-coil-containing protein kinase 2 inhibitor in clinical use for the treatment of chronic graft-versus-host disease. The current tablet formulation may be inappropriate for children or adults with dysphagia and/or upper gastrointestinal manifestations of chronic graft-versus-host disease. This study (NCT04735822) assessed the taste and palatability of oral suspensions of belumosudil, evaluated the relative bioavailability of an oral suspension versus the tablet formulation, and characterized the effect of food on the pharmacokinetics of an oral suspension. Addition of sweetener and/or flavor vehicle improved the taste. Relative bioavailability of 200-mg doses of the oral suspension and tablet in the fed state was similar for belumosudil and its metabolites (KD025m1 and KD025m2), but absorption was faster with the oral suspension (median time to maximum concentration: 2 vs 3 hours). Administration of the oral suspension with food increased exposure compared with fasted administration, with maximum observed concentration being increased by 16% and area under the concentration-time curve from time 0 to the last measurable concentration (AUC0-last) by 19%. Safety and tolerability were consistent with the known safety profile of belumosudil. These results may support administration of a 200-mg belumosudil oral suspension with or without food.
Assuntos
Disponibilidade Biológica , Estudos Cross-Over , Interações Alimento-Droga , Voluntários Saudáveis , Suspensões , Comprimidos , Paladar , Humanos , Masculino , Administração Oral , Adulto , Adulto Jovem , Área Sob a Curva , Pessoa de Meia-Idade , Método Duplo-Cego , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/efeitos adversosRESUMO
Background: The goal of this research is to determine how well OrthAlign, a novel portable navigation system for total knee replacement, helps surgeons make accurate incisions. When comparing OrthoAlign, a portable accelerometer-based technique, to the gold standard of extramedullary jigs, the results are promising. This study aimed to evaluate the accuracy of distal femoral and proximal tibial cuts in total knee arthroplasty. Materials and Methods: This research analyzed data from a prospective cohort study. Participants in the study all had resections of the proximal tibia and distal femur using the OrthAlign portable navigations device. Total knee arthroplasty (TKA) patients throughout the same time period who used traditional medullary alignment jigs were included as a control group. Before and after surgery, full-length standing stitch radiographs of the patient's lower limbs were acquired so that the alignment of their knees could be assessed. Results and Conclusions: In the mechanical alignment exam done following the surgery, the OrthAlign group performed substantially better than the control group, although the difference was not statistically significant. Patients treated with OrthoAlign had considerably improved alignment of the tibial components in the coronal plane compared to those treated manually. When comparing the OrthAlign cohort to the conventional cohort, average sagittal plane alignment of tibial components was significantly different. Yet, when comparing femoral alignment after surgery, neither the mechanical alignment nor the OrthAlign groups fared better. Furthermore, there was no statistically significant difference between the two groups when it comes to the occurrence of outliers with postoperative mechanical axis alignment >3 degrees or tibial alignment in the coronal plane >2 degrees. Compared to OrthAlign, conventional alignment methods resulted in a higher percentage of postoperative tibial alignment in the sagittal plane (greater than 2 degrees). Patients whose femurs were misaligned by more than 2 degrees after surgery favored the OrthAlign method, albeit this was not statistically different from the control group. There was a significant reduction in tourniquet time for patients using OrthoAlign compared to those using mechanical alignment devices.
RESUMO
Background: Morbidity and mortality from tuberculosis, a significant infectious illness, are expected to rise worldwide. The projected number of new cases rose from 7.5 million in 1990 to 11.9 million in 2005, a 58.6% increase in 2011. The widespread belief that TB is no longer a public health concern is unfounded; on the contrary, the link between HIV/AIDS and antibiotic resistance has further exacerbated the crisis that already existed. Similar to the nations in sub-Saharan Africa, India is now considered a Group IV country, with an annual risk of infection between 1% and 2.5%. 2. Although 60% of TB cases occur in people who are HIV-positive, only 3-5% of cases in HIV-negative individuals are skeletal. The most frequent type of articuloskeletal tuberculosis is spinal tuberculosis. Aim: 1. The goal of this study is to evaluate the neurological outcome of anterior debridement, fusion, posterior instrumentation, and early rehabilitation in individuals with spinal cord injuries. 2. The goal of this study is to determine the prevalence of pressure ulcers, hypostatic pneumonia, and urinary tract infections urinary tract infections (UTIs) among these individuals. 3. The goal of this study is to determine the frequency of graft-related problems. 4. See how well these individuals are able to keep their corrected deformities from returning. Materials and Methods: Patients who had simultaneous anterior (anterior debridement and bone grafting) and posterior (posterior instrumentation and fusion) procedures were followed prospectively. Result: Thirty patients' films were examined. In addition, cord edema was suggested in 13 of the patients based on the presence of strong signal intensities there. Myelomalacia signs were seen in one patient, but he or she went on to make a complete neurological recovery. The average duration of operation was 355 minutes, and this included the time needed to position the patient for the two separate procedures. Conclusion: There was an 89.5% rate of neurological recovery with an average corrected loss of 6.98 degrees (0.20 degrees to 35.90 degrees), and the complication rate was acceptable in the group analyzed.
RESUMO
Belumosudil is a selective Rho-associated, coiled-coil-containing protein kinase-2 inhibitor. In this crossover design thorough QT/QTc study, single therapeutic (200 mg) and supratherapeutic (1000 mg) oral doses of belumosudil, moxifloxacin (positive control), and placebo were administered to 34 subjects. Twelve-lead electrocardiograms and serial pharmacokinetic sampling were acquired. The effect of belumosudil on the placebo-corrected, change-from-baseline QTcF was small, and an effect exceeding 10 ms could be excluded across all time points with both doses. Using concentration-QTc analysis, an effect on ΔΔQTcF >10 ms can be excluded up to belumosudil concentrations of ≈12 080 ng/mL, more than 2-fold above mean Cmax after the supratherapeutic dose. There was no clinically relevant effect on heart rate or cardiac conduction (ie, the PR and QRS intervals) for belumosudil. No differences in safety were noted between belumosudil and placebo treatment. Assay sensitivity was demonstrated by moxifloxacin's effect on the QTc interval. In conclusion, belumosudil at therapeutic and supratherapeutic doses did not have a clinically meaningful effect on electrocardiogram parameters.
Assuntos
Proteínas Quinases , Acetamidas , Relação Dose-Resposta a Droga , Voluntários Saudáveis , Humanos , MoxifloxacinaRESUMO
Belumosudil is a selective Rho-associated protein kinase 2 inhibitor. Inhibition of Rho-associated protein kinase 2 has emerged as a promising treatment for chronic graft-versus-host disease by restoring immune homeostasis and reducing fibrosis. In vitro assessments have suggested that metabolism of belumosudil is primarily dependent on cytochrome P450 (CYP) 3A4 activity and that the solubility of belumosudil is pH dependent. As such, this 2-part clinical drug-drug interaction study was conducted to assess the effect of itraconazole (a strong CYP3A4 inhibitor), rifampicin (a strong CYP3A4 inducer), rabeprazole, and omeprazole (both proton pump inhibitors) on the pharmacokinetics of belumosudil. No clinically relevant change in belumosudil exposure was observed following a 200-mg single oral dose of belumosudil with itraconazole; however, exposure of main metabolite, KD025m2, was decreased. Consistent with the proposed metabolic pathway of belumosudil, the strong CYP3A4 inducer rifampicin significantly decreased exposure of belumosudil and KD025m2 and increased KD025m1 exposure. When a 200-mg single oral dose of belumosudil was coadministered with both rabeprazole and omeprazole, parent and metabolite exposures were largely reduced, suggesting that belumosudil dosage should be increased when given with PPIs. Administration of belumosudil with and without perpetrator drugs was safe, and no notable adverse events were reported.
Assuntos
Inibidores do Citocromo P-450 CYP3A , Inibidores da Bomba de Prótons , Acetamidas , Citocromo P-450 CYP3A/metabolismo , Indutores do Citocromo P-450 CYP3A/farmacologia , Inibidores do Citocromo P-450 CYP3A/farmacologia , Indutores das Enzimas do Citocromo P-450 , Interações Medicamentosas , Humanos , Itraconazol , Omeprazol/farmacologia , Proteínas Quinases , Inibidores da Bomba de Prótons/farmacologia , Rabeprazol/farmacologia , RifampinaRESUMO
Belumosudil is a selective Rho-associated coiled-coil containing protein kinase 2 inhibitor. A capsule formulation was used during early clinical development of belumosudil; it was later replaced by a tablet formulation, which mimicked the capsule's release properties and facilitated manufacturing scalability. To assess belumosudil's pharmacokinetics, including potential food effects, and evaluate the relative bioavailability of the 2 formulations, 2 phase 1 clinical trials were conducted. Administration of both belumosudil tablets and capsules with food increased exposure ≈2× as compared to the fasted state and delayed time to maximum concentration by 0.5 hour, indicating a decrease in the rate but increase in the extent of absorption with fed administration. Relative bioavailability was slightly higher when belumosudil was administered as tablets vs capsules, although the difference was not clinically meaningful. Safety and tolerability were generally consistent with the known safety profile of belumosudil. The results of these studies support administration of belumosudil with food.
Assuntos
Disponibilidade Biológica , Acetamidas , Adulto , Cápsulas , Estudos Cross-Over , Humanos , ComprimidosRESUMO
Belumosudil is a selective Rho-associated coiled-coil containing protein kinase 2 (ROCK2) inhibitor. ROCK2 has been shown to drive proinflammatory response and fibrosis that occurs with chronic graft-versus-host disease; therefore, inhibition of ROCK2 has emerged as a therapeutic target for chronic graft-versus-host disease. In this phase 1 two-part study, the pharmacokinetics, mass balance, and metabolic profile of belumosudil were evaluated after single doses of unlabeled belumosudil oral tablets (200 mg), radiolabeled belumosudil intravenous (IV) microtracer infusions (100 µg), and radiolabeled oral capsules (200 mg). Absolute bioavailability based on area under the plasma concentration-time curve from time 0 to infinity for the oral dose/area under the plasma concentration-time curve from time 0 to infinity for the IV dose was calculated as 63.7%. Radiolabeled IV microtracer dosing demonstrated a low extraction ratio and distribution of belumosudil into tissues. The majority of total radioactivity was recovered in feces, with minimal amounts recovered in urine, suggesting minimal renal elimination of belumosudil. In addition to parent and main metabolite KD025m2, metabolites identified in plasma included the phase 2 metabolites O-dealkylated belumosudil sulfate and belumosudil glucuronide. These metabolites (with the exception of the glucuronide) in addition to monohydroxy-belumosudil, and belumosudil diol were identified in feces. No metabolites in urine accounted for >10% of the radioactive dose.
Assuntos
Glucuronídeos , Doença Enxerto-Hospedeiro , Acetamidas , Administração Oral , Disponibilidade Biológica , Humanos , MasculinoRESUMO
Efforts to target glucose metabolism in cancer have been limited by the poor potency and specificity of existing anti-glycolytic agents and a poor understanding of the glucose dependence of cancer subtypes in vivo. Here, we present an extensively characterized series of potent, orally bioavailable inhibitors of the class I glucose transporters (GLUTs). The representative compound KL-11743 specifically blocks glucose metabolism, triggering an acute collapse in NADH pools and a striking accumulation of aspartate, indicating a dramatic shift toward oxidative phosphorylation in the mitochondria. Disrupting mitochondrial metabolism via chemical inhibition of electron transport, deletion of the malate-aspartate shuttle component GOT1, or endogenous mutations in tricarboxylic acid cycle enzymes, causes synthetic lethality with KL-11743. Patient-derived xenograft models of succinate dehydrogenase A (SDHA)-deficient cancers are specifically sensitive to KL-11743, providing direct evidence that TCA cycle-mutant tumors are vulnerable to GLUT inhibitors in vivo.
Assuntos
Ciclo do Ácido Cítrico , Neoplasias , Ácido Aspártico/metabolismo , Glucose/metabolismo , Humanos , Mitocôndrias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismoRESUMO
Immunocytokines hold great potential as anticancer agents, as they use a specific antitumor antibody to deliver an immune-activating cytokine directly to the immunosuppressive tumor microenvironment (TME). We have developed a novel immunocytokine (KD033) composed of a fully human, high-affinity antiprogrammed death-ligand 1 (PD-L1) linked to the sushi-domain of the human IL-15/IL-15 receptor alpha (IL-15/IL-15Rα) complex. A murine PD-L1 cross-reactive KD033 surrogate (srKD033) and a nontargeting antibody (ntKD033) were also developed to investigate mechanism of action in murine tumor models. Efficacy analyses showed a robust antitumor effect of single-dose srKD033 in several diverse syngeneic murine tumor models. In a CT26 murine colon tumor model, single-dose srKD033 produced durable antitumor immunity as evidenced by resistance to subsequent tumor rechallenges. Mice responding to srKD033 treatment showed increased retention of PD-L1/IL-15 in the TME which likely facilitated prolonged IL-15-induced expansion of cytotoxic cells. Importantly, target-based PD-L1/IL-15 delivery via srKD033 was well-tolerated and induced significant antitumor activity in murine carcinoma models that are non- or minimally responsive to IL-15 or anti-PD-L1/PD-1 monotherapy.
Assuntos
Antígeno B7-H1/antagonistas & inibidores , Sinergismo Farmacológico , Imunoterapia/métodos , Interleucina-15/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , CamundongosRESUMO
PURPOSE: The rho-associated coiled-coil-containing protein kinase-2 (ROCK2) signaling pathway regulates the Th17/regulatory T cells balance and controls profibrotic pathways. Selective ROCK2 inhibition with belumosudil (KD025) may offer a novel approach to the management of chronic graft-versus-host disease (cGVHD). PATIENTS AND METHODS: A phase IIa, open-label, dose-finding study of belumosudil enrolled 54 patients with cGVHD who had received one to three prior lines of therapy (LOTs). The primary end point was overall response rate (ORR). RESULTS: The median time from cGVHD diagnosis to enrollment was 20 months. Seventy-eight percent of patients had severe cGVHD, 50% had ≥ 4 organs involved, 73% had cGVHD refractory to their last LOT, and 50% had received ≥ 3 prior LOTs. With an overall median follow-up of 29 months, the ORR (95% CI) with belumosudil 200 mg once daily, 200 mg twice daily, and 400 mg once daily was 65% (38% to 86%), 69% (41% to 89%), and 62% (38% to 82%), respectively. Responses were clinically meaningful, with a median duration of response of 35 weeks, and were associated with quality-of-life improvements and corticosteroid (CS) dose reductions. CS treatment was discontinued in 19% of patients. The failure-free survival rate was 76% (62% to 85%) and 47% (33% to 60%) at 6 and 12 months, respectively. The 2-year overall survival rate was 82% (69% to 90%). Belumosudil was well-tolerated, with low rates of cytopenia. There were no unexpected adverse events and no apparent increased risk of infection, including cytomegalovirus infection and reactivation. CONCLUSION: Belumosudil treatment resulted in a high ORR and overall survival rate and demonstrated quality-of-life improvements, CS dose reductions, and limited toxicity. Data from the study indicated that belumosudil may prove to be an effective therapy for patients with treatment-refractory cGVHD.
Assuntos
Acetamidas/uso terapêutico , Doença Enxerto-Hospedeiro/tratamento farmacológico , Quinases Associadas a rho/antagonistas & inibidores , Acetamidas/efeitos adversos , Adulto , Idoso , Estudos de Coortes , Feminino , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Inibidores de Proteínas Quinases/efeitos adversos , Inibidores de Proteínas Quinases/uso terapêutico , Adulto JovemRESUMO
Aerobic glycolysis, originally identified by Warburg as a hallmark of cancer, has recently been implicated in immune cell activation and growth. Glucose, the starting material for glycolysis, is transported through the cellular membrane by a family of glucose transporters (GLUTs). Therefore, targeting glucose transporters to regulate aerobic glycolysis is an attractive approach to identify potential therapeutic agents for cancers and autoimmune diseases. Herein, we describe the discovery and optimization of a class of potent, orally bioavailable inhibitors of glucose transporters, targeting both GLUT1 and GLUT3.
Assuntos
Descoberta de Drogas/métodos , Proteínas Facilitadoras de Transporte de Glucose/antagonistas & inibidores , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Glucose/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Células CACO-2 , Relação Dose-Resposta a Droga , Descoberta de Drogas/tendências , Glicólise/efeitos dos fármacos , Glicólise/fisiologia , Humanos , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , RatosRESUMO
CXCR4, the specific receptor for the chemokine SDF-1 alpha that also binds CXCR4-using HIV gp120s, affects survival of different cell types, including neurons. However, current data show that the outcome of CXCR4 activation on neuronal survival may vary depending on the ligand and/or the cellular conditions. In this study, we have systematically compared the effects of SDF-1 alpha and gp120(IIIB) (with or without CD4) on several intracellular pathways involved in cell survival, including MAP kinases and Akt-dependent pathways. Our data show that gp120(IIIB) and SDF-1 alpha are both potent activators of MAP kinases in neuronal and non-neuronal cells, though the kinetic of these responses is slightly different. Furthermore, unlike SDF-1 alpha, and independently of CD4, gp120(IIIB) is unable to stimulate Akt and some of its antiapoptotic targets (NF-kappa B and MDM2)--despite its ability to activate other signaling pathways in the same conditions. Finally, the viral protein is more efficient in recruiting some effectors (e.g., JNK) than others in comparison with SDF-1 alpha (EC(50) = 0.1 vs. 0.6 nM). We conclude that the intrinsic efficacy of the two ligands is significantly different and is pathway dependent. These findings have important implications for our understanding of CXCR4-mediated responses in the CNS, as well as the role of this coreceptor in HIV neuropathogenesis.
Assuntos
Apoptose , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/patogenicidade , Neurônios/fisiologia , Receptores CXCR4/metabolismo , Animais , Astrócitos/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular , Células Cultivadas , Quimiocina CXCL12 , Quimiocinas CXC/metabolismo , Ativação Enzimática , Regulação da Expressão Gênica , Proteína gp120 do Envelope de HIV/genética , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Ratos , Receptores CXCR4/genética , Transdução de SinaisRESUMO
The chemokine receptor CXCR4 functions as human immunodeficiency virus (HIV)-1 coreceptor and is involved in acquired immunodeficiency virus (AIDS) neuropathogenesis. CXCR4 is expressed by most cell types in the brain, including microglia, astrocytes, and neurons. Studies have shown that the HIV envelope protein gp120 binds to neuronal CXCR4 and activates signal transduction pathways leading to apoptosis. However, the natural CXCR4 ligand (CXCL12) has been referred to induce both neuronal survival and death. Here the authors used flow cytometry to determine whether gp120 and CXCL12 differ in their ability to induce CXCR4 internalization in the human neuroblastoma cells SH-SY5Y, which constitutively express CXCR4. As expected, increasing concentration of CXCL12 reduced surface expression of CXCR4 in a time-and concentration-dependent manner. Conversely, gp120IIIB (monomeric or oligomeric, in presence or absence of soluble CD4) did not change CXCR4 membrane levels. Similar results were obtained in a murine lymphocyte cell line (300-19) stably expressing human CXCR4. Nevertheless, gp120IIIB was still able to activate intracellular signaling and proapoptotic pathways, via CXCR4. These results show that gp120IIIB toxicity and signaling do not require CXCR4 internalization in SH-SY5Y cells, and suggest that the viral protein may alter normal CXCR4 trafficking thus, interfering with activation of prosurvival pathways.
Assuntos
Apoptose/fisiologia , Proteína gp120 do Envelope de HIV/metabolismo , Neurônios/patologia , Neurônios/virologia , Receptores CXCR4/metabolismo , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Quimiocina CXCL12 , Quimiocinas CXC/farmacologia , Endocitose/fisiologia , Citometria de Fluxo , Proteína gp120 do Envelope de HIV/farmacologia , Humanos , Neuroblastoma , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
The chemokine receptor CXCR4 regulates neuronal survival and differentiation and is involved in a number of pathologies, including cancer and human immunodeficiency virus (HIV). Recent data suggest that chemokines act in concert with neurotransmitters and neuropeptides, such as opioids. This study aimed to determine whether mu-opioid agonists alter the effect of CXCL12 (the specific CXCR4 ligand) on central neurons. Neuronal expression of CXCR4 and micro-opioid receptors (MORs) was analyzed by Western blot, immunostaining, and flow cytometry. Single-cell studies showed that all CXCR4-positive neurons coexpress MORs. Treatment of neuronal cultures with the selective MOR agonist DAMGO or the endogenous peptide endomorphin-1 inhibited intracellular signaling pathways (ERK1/2 and Akt) activated by CXCL12. Furthermore, DAMGO abolished the neuroprotective effect of CXCL12 in N-methyl-d-aspartate (NMDA) neurotoxicity studies. The effects of DAMGO and endomorphin-1 were inhibited by a general or a micro-specific opioid receptor antagonist, and not caused by changes in neuronal CXCR4 levels. DAMGO did not affect CXCL12-induced internalization of CXCR4. The authors propose that interactions between MOR and CXCR4 signaling can modulate the action of CXCL12 on neuronal survival-which may have important implications to neuroAIDS as well as other neuroinflammatory disorders.
Assuntos
Analgésicos Opioides/farmacologia , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , Neurônios/efeitos dos fármacos , Receptores CXCR4/metabolismo , Receptores Opioides mu/metabolismo , Animais , Western Blotting , Sobrevivência Celular , Células Cultivadas , Quimiocina CXCL12 , Quimiocinas CXC/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Microscopia Confocal , Antagonistas de Entorpecentes/farmacologia , Neurônios/metabolismo , Oligopeptídeos/metabolismo , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Receptor Cross-Talk/efeitos dos fármacos , Receptor Cross-Talk/fisiologia , Receptores CXCR4/efeitos dos fármacos , Receptores Opioides mu/efeitos dos fármacosRESUMO
Abnormal activation of CXCR 4 during inflammatory/infectious states may lead to neuronal dysfunction or damage. The major goal of this study was to determine the coupling of CXCR 4 to p53-dependent survival pathways in primary neurons. Neurons were stimulated with the HIV envelope protein gp120(IIIB) or the endogenous CXCR 4 agonist, SDF-1 alpha. We found that gp120 stimulates p53 activity and induces expression of the p53 pro-apoptotic target Apaf-1 in cultured neurons. Inhibition of CXCR 4 by AMD 3100 abrogates the effect of gp120 on both p53 and Apaf-1. Moreover, gp120 neurotoxicity is markedly reduced by the p53-inhibitor, pifithrin-alpha. The viral protein also regulates p53 phosphorylation and expression of other p53-responsive genes, such as MDM 2 and p21. Conversely, SDF-1 alpha, which can promote neuronal survival, increases p53 acetylation and p21 expression in neurons. Thus, the stimulation of different p53 targets could be instrumental in determining the outcome of CXCR 4 activation on neuronal survival in neuro-inflammatory disorders.