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1.
JAMA Dermatol ; 160(1): 63-70, 2024 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-38055242

RESUMO

Importance: Hidradenitis suppurativa is a painful immune-mediated disorder with limited treatment options; hence, a need exists for new treatments. Objective: To evaluate the feasibility of heat shock protein 90 inhibition by RGRN-305 as a novel mechanism of action in treating moderate to severe hidradenitis suppurativa. Design, Setting, and Participants: This was a parallel-design, double-blind, proof-of-concept, placebo-controlled randomized clinical trial conducted between September 22, 2021, and August 29, 2022, at the Department of Dermatology, Aarhus University Hospital in Denmark. The study included a 1- to 30-day screening period, a 16-week treatment period, and a 4-week follow-up period. Eligibility criteria included age 18 years or older and moderate to severe hidradenitis suppurativa with 6 or more inflammatory nodules or abscesses in at least 2 distinct anatomic regions. Of 19 patients screened, 15 patients were enrolled in the study. Intention-to-treat analysis was performed. Interventions: Patients were randomly assigned (2:1) to receive oral RGRN-305, 250-mg tablet, or matching placebo once daily for 16 weeks. Main Outcomes and Measures: The primary efficacy end point was the percentage of patients achieving Hidradenitis Suppurativa Clinical Response 50 (HiSCR-50) at week 16. Secondary efficacy end points included HiSCR-75 or HiSCR-90, Hidradenitis Suppurativa Physician's Global Assessment, Dermatology Life Quality Index scores, and a pain numeric rating scale. Safety was assessed by adverse events, physical examinations, clinical laboratory measurements, and electrocardiograms. Results: A total of 15 patients were enrolled, completed the study, and were included in all analyses (10 [67%] female; median age, 29 [IQR, 23-41] years). The primary end point HiSCR-50 at week 16 was achieved by a higher percentage in the RGRN-305 group (60% [6 of 10]) than in the placebo group (20% [1 of 5]). Improvements were also observed across all secondary end points at week 16, including higher rates of the harder-to-reach HiSCR levels; 50% (5 of 10) achieved HiSCR-75 and 30% (3 of 10) achieved HiSCR-90, whereas none of the placebo-treated patients achieved HiSCR-75 or HiSCR-90. RGRN-305 was well tolerated, with no deaths or serious adverse events, and treatment-emergent adverse events were similarly frequent between the RGRN-305 and placebo groups. Conclusions and Relevance: The findings of this trial suggest that heat shock protein 90 inhibition by RGRN-305 offers a novel mechanism of action in treating hidradenitis suppurativa, warranting further evaluation in larger trials. Trial Registration: ClinicalTrials.gov Identifier: NCT05286567.


Assuntos
Proteínas de Choque Térmico , Hidradenite Supurativa , Adulto , Feminino , Humanos , Masculino , Método Duplo-Cego , Proteínas de Choque Térmico/efeitos adversos , Proteínas de Choque Térmico/agonistas , Hidradenite Supurativa/tratamento farmacológico , Índice de Gravidade de Doença , Resultado do Tratamento , Adulto Jovem
2.
Front Immunol ; 14: 1128897, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36825010

RESUMO

Introduction: Chronic inflammatory skin diseases may have a profound negative impact on the quality of life. Current treatment options may be inadequate, offering an unsatisfactory response or side effects. Therefore, ongoing efforts exist to identify novel effective and safe treatments. Heat shock protein (HSP) 90 is a chaperone that promotes the activity of a wide range of client proteins including key proinflammatory molecules involved in aberrant inflammation. Recently, a proof-of-concept clinical trial of 13 patients suggested that RGRN-305 (an HSP90 inhibitor) may be an oral treatment for psoriasis. However, HSP90 inhibition may be a novel therapeutic approach extending beyond psoriasis to include multiple immune-mediated inflammatory skin diseases. Methods: This study aimed to investigate (i) the anti-inflammatory effects and mechanisms of HSP90 inhibition and (ii) the feasibility of topical RGRN-305 administration (new route of administration) in models of inflammation elicited by 12-O-tetradecanoylphorbol-13-acetate (TPA) in primary human keratinocytes and mice (irritative dermatitis murine model). Results/Discussion: In primary human keratinocytes stimulated with TPA, a Nanostring® nCounter gene expression assay demonstrated that HSP90 inhibition with RGRN-305 suppressed many proinflammatory genes. Furthermore, when measured by quantitative real-time polymerase chain reaction (RT-qPCR), RGRN-305 significantly reduced the gene expression of TNF, IL1B, IL6 and CXCL8. We next demonstrated that topical RGRN-305 application significantly ameliorated TPA-induced skin inflammation in mice. The increase in ear thickness (a marker of inflammation) was significantly reduced (up to 89% inhibition). In accordance, RT-qPCR of the ear tissue demonstrated that RGRN-305 robustly reduced the gene expression of proinflammatory markers (Tnf, Il1b, Il6, Il17A and Defb4). Moreover, RNA sequencing revealed that RGRN-305 mitigated TPA-induced alterations in gene expression and suppressed genes implicated in inflammation. Lastly, we discovered that the anti-inflammatory effects were mediated, at least partly, by suppressing the activity of NF-κB, ERK1/2, p38 MAPK and c-Jun signaling pathways, which are consistent with previous findings in other experimental models beyond skin inflammation. In summary, HSP90 inhibition robustly suppressed TPA-induced inflammation by targeting key proinflammatory cytokines and signaling pathways. Our findings suggest that HSP90 inhibition may be a novel mechanism of action for treating immune-mediated skin disease beyond psoriasis, and it may be a topical treatment option.


Assuntos
Antineoplásicos , Dermatite , Proteínas de Choque Térmico HSP90 , Psoríase , Dermatopatias , Animais , Humanos , Camundongos , Anti-Inflamatórios/uso terapêutico , Antineoplásicos/uso terapêutico , Dermatite/tratamento farmacológico , Dermatite/metabolismo , Inflamação/metabolismo , Interleucina-6 , Psoríase/tratamento farmacológico , Qualidade de Vida , Dermatopatias/tratamento farmacológico , Proteínas de Choque Térmico HSP90/antagonistas & inibidores
3.
Front Immunol ; 14: 1289788, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38274815

RESUMO

Background: Heat shock protein 90 (HSP90) is an important chaperone supporting the function of many proinflammatory client proteins. Recent studies indicate HSP90 inhibition may be a novel mechanism of action for inflammatory skin diseases; however, this has not been explored in atopic dermatitis (AD). Objectives: Our study aimed to investigate HSP90 as a novel target to treat AD. Methods: Experimental models of AD were used including primary human keratinocytes stimulated with cytokines (TNF/IFNγ or TNF/IL-4) and a mouse model established by MC903 applications. Results: In primary human keratinocytes using RT-qPCR, the HSP90 inhibitor RGRN-305 strongly suppressed the gene expression of Th1- (TNF, IL1B, IL6) and Th2-associated (CCL17, CCL22, TSLP) cytokines and chemokines related to AD. We next demonstrated that topical and oral RGRN-305 robustly suppressed MC903-induced AD-like inflammation in mice by reducing clinical signs of dermatitis (oedema and erythema) and immune cell infiltration into the skin (T cells, neutrophils, mast cells). Interestingly, topical RGRN-305 exhibited similar or slightly inferior efficacy but less weight loss compared with topical dexamethasone. Furthermore, RNA sequencing of skin biopsies revealed that RGRN-305 attenuated MC903-induced transcriptome alterations, suppressing genes implicated in inflammation including AD-associated cytokines (Il1b, Il4, Il6, Il13), which was confirmed by RT-qPCR. Lastly, we discovered using Western blot that RGRN-305 disrupted JAK-STAT signaling by suppressing the activity of STAT3 and STAT6 in primary human keratinocytes, which was consistent with enrichment analyses from the mouse model. Conclusion: HSP90 inhibition by RGRN-305 robustly suppressed inflammation in experimental models mimicking AD, proving that HSP90 inhibition may be a novel mechanism of action in treating AD.


Assuntos
Dermatite Atópica , Humanos , Camundongos , Animais , Interleucina-6 , Inflamação/metabolismo , Citocinas/metabolismo , Proteínas de Choque Térmico
4.
Heliyon ; 8(11): e11359, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36387470

RESUMO

A critical target in age-related macular degeneration (AMD) is the retinal pigment epithelium (RPE), which forms the outer blood-retina barrier (BRB). RPE-barrier dysfunction might result from the disruption of intercellular tight junctions (TJs). A Connexin43 (Cx43)-based peptide, aCT1, has been shown to prevent VEGF-induced loss of transepithelial resistance, choroidal neovascularization (CNV) and RPE-cell damage via the stabilization of TJs. Here, we probe the relative efficacies of aCT1 alone, anti-VEGF alone, and aCT1 with anti-VEGF in treating AMD pathologies. aCT1 monotherapy administered as topical eye drops with and without a VEGF blocking antibody administered systemically was tested in a mouse model of laser-induced CNV. The CNV mouse is the standard neovascular AMD model, reproducing hallmarks of its pathology. CNV lesion size and fluid accumulation were assessed using optical coherence tomography. During the angiogenesis phase of CNV lesion development, single applications of anti-VEGF or aCT1 reduced lesion and fluid dome size equally. The combinatorial aCT1/anti-VEGF strategy demonstrated lack of additive effects in this model. These data suggest that TJ stabilization by aCT1 is effective in ameliorating RPE dysfunction in a model of AMD-like angiogenesis, and that this strategy is as effective as the current clinical standard of care, anti-VEGF therapy. Critically, aCT1 holds potential as a new neovascular AMD treatment that can be administered using eye drops, which is preferable to the intravitreal injections required for standard anti-VEGF therapy.

5.
Oncogenesis ; 11(1): 2, 2022 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-35022385

RESUMO

Circumventing chemoresistance is crucial for effectively treating cancer including glioblastoma, a lethal brain cancer. The gap junction protein connexin 43 (Cx43) renders glioblastoma resistant to chemotherapy; however, targeting Cx43 is difficult because mechanisms underlying Cx43-mediated chemoresistance remain elusive. Here we report that Cx43, but not other connexins, is highly expressed in a subpopulation of glioblastoma and Cx43 mRNA levels strongly correlate with poor prognosis and chemoresistance in this population, making Cx43 the prime therapeutic target among all connexins. Depleting Cx43 or treating cells with αCT1-a Cx43 peptide inhibitor that sensitizes glioblastoma to the chemotherapy temozolomide-inactivates phosphatidylinositol-3 kinase (PI3K), whereas overexpression of Cx43 activates this signaling. Moreover, αCT1-induced chemo-sensitization is counteracted by a PI3K active mutant. Further research reveals that αCT1 inactivates PI3K without blocking the release of PI3K-activating molecules from membrane channels and that Cx43 selectively binds to the PI3K catalytic subunit ß (PIK3CB, also called PI3Kß or p110ß), suggesting that Cx43 activates PIK3CB/p110ß independent of its channel functions. To explore the therapeutic potential of simultaneously targeting Cx43 and PIK3CB/p110ß, αCT1 is combined with TGX-221 or GSK2636771, two PIK3CB/p110ß-selective inhibitors. These two different treatments synergistically inactivate PI3K and sensitize glioblastoma cells to temozolomide in vitro and in vivo. Our study has revealed novel mechanistic insights into Cx43/PI3K-mediated temozolomide resistance in glioblastoma and demonstrated that targeting Cx43 and PIK3CB/p110ß together is an effective therapeutic approach for overcoming chemoresistance.

6.
FASEB J ; 35(8): e21762, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34246197

RESUMO

Phase II clinical trials have reported that acute treatment of surgical skin wounds with the therapeutic peptide alpha Connexin Carboxy-Terminus 1 (αCT1) improves cutaneous scar appearance by 47% 9-month postsurgery. While Cx43 and ZO-1 have been identified as molecular targets of αCT1, the mode-of-action of the peptide in scar mitigation at cellular and tissue levels remains to be further characterized. Scar histoarchitecture in αCT1 and vehicle-control treated skin wounds within the same patient were compared using biopsies from a Phase I clinical trial at 29-day postwounding. The sole effect on scar structure of a range of epidermal and dermal variables examined was that αCT1-treated scars had less alignment of collagen fibers relative to control wounds-a characteristic that resembles unwounded skin. The with-in subject effect of αCT1 on scar collagen order observed in Phase I testing in humans was recapitulated in Sprague-Dawley rats and the IAF hairless guinea pig. Transient increase in histologic collagen density in response to αCT1 was also observed in both animal models. Mouse NIH 3T3 fibroblasts and primary human dermal fibroblasts treated with αCT1 in vitro showed more rapid closure in scratch wound assays, with individual cells showing decreased directionality in movement. An agent-based computational model parameterized with fibroblast motility data predicted collagen alignments in simulated scars consistent with that observed experimentally in human and the animal models. In conclusion, αCT1 prompts decreased directionality of fibroblast movement and the generation of a 3D collagen matrix postwounding that is similar to unwounded skin-changes that correlate with long-term improvement in scar appearance.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Cicatriz/metabolismo , Conexina 43/metabolismo , Derme/metabolismo , Fibroblastos/metabolismo , Peptídeos/farmacologia , Animais , Cicatriz/patologia , Matriz Extracelular/metabolismo , Feminino , Cobaias , Humanos , Masculino , Ratos , Ratos Sprague-Dawley
7.
Exp Dermatol ; 30(6): 773-781, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33583094

RESUMO

Keratinocytes are the key cellular target for IL-17A-mediated effects in psoriasis and HSP90 is important for IL-17A-mediated signalling. RGRN-305 is a novel HSP90 inhibitor reported to reduce psoriatic phenotypes in preclinical animal models. The aim of this study was to investigate the effect of RGRN-305 on a psoriasis-like inflammatory response in human keratinocytes in vitro. Using RT-qPCR, we demonstrated a significantly increased expression of the HSP90 isoforms HSP90AB1, HSP90B1 and TRAP1 in lesional compared with non-lesional psoriatic skin. In a psoriasis-like setting where keratinocytes were stimulated with TNFα and/or IL-17A, we analysed the mRNA expression using the NanoString nCounter technology and demonstrated that the HSP90 inhibitor RGRN-305 significantly reduced the IL-17A- and TNFα-induced gene expression of a number of proinflammatory genes, including the psoriasis-associated genes CCL20, NFKBIZ, IL36G and IL23A. In agreement with the mRNA data, the protein level of CCL20, IκBζ and IL-36γ were inhibited by RGRN-305 as demonstrated by western blotting and ELISA. Interestingly, when keratinocytes were stimulated with a TLR3 agonist, RGRN-305 also demonstrated potent immunomodulatory effects, significantly inhibiting poly(I:C)-induced expression of the proinflammatory genes TNFα, IL1B, IL6 and IL23A. Taken together, our data support a role for HSP90 not only in the pathogenesis of psoriasis, but also in broader immune responses. Therefore, HSP90 provides an attractive target for the treatment of psoriasis and other diseases where the innate immune system plays an important role.


Assuntos
Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Queratinócitos/efeitos dos fármacos , Queratinócitos/imunologia , Psoríase/tratamento farmacológico , Psoríase/imunologia , Linhagem Celular , Humanos
8.
Mater Sci Eng C Mater Biol Appl ; 108: 110191, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31923988

RESUMO

Effective therapeutic delivery of peptide and protein drugs is challenged by short in vivo half-lives due to rapid degradation. Sustained release formulations of αCT1, a 25 amino acid peptide drug, would afford lower dosing frequency in indications that require long term treatment, such as chronic wounds and cancers. In this study, rhodamine B (RhB) was used as a model drug to develop and optimize a double emulsion-solvent evaporation method of poly(lactic-co-glycolic acid) (PLGA) nanoparticle synthesis. Encapsulation of αCT1 in these nanoparticles (NPs) resulted in a sustained in vitro release profile over three weeks, characterized by an initial burst release of approximately 50% of total encapsulated drug over the first three days followed by sustained release over the remaining two and a half weeks. NP uptake by glioblastoma stem cells was through endocytosis and RhB and αCT1 were observed in cells after at least 4 days.


Assuntos
Materiais Biomiméticos , Conexina 43 , Glioblastoma , Nanopartículas , Peptídeos , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Linhagem Celular Tumoral , Conexina 43/química , Conexina 43/farmacologia , Preparações de Ação Retardada/química , Preparações de Ação Retardada/farmacologia , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Humanos , Nanopartículas/química , Nanopartículas/uso terapêutico , Peptídeos/química , Peptídeos/farmacologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/farmacologia
9.
Med Sci (Basel) ; 7(9)2019 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-31480425

RESUMO

Kappa-opioid agonists (KOAs) enhance cardiac performance, as well as reduce infarct size and prevent deleterious cardiac remodeling following myocardial infarction. Additionally, KOAs promote diuresis; however, there has been limited development of KOAs as a class due to the promotion of untoward central nervous system (CNS)-mediated side effects. Our laboratory has developed a peripherally-restricted, orally-active, KOA (JT09) for the treatment of pain and cardiovascular disease. Peripherally-restricted KOAs possess a limited side-effect profile and demonstrate potential in preventing heart failure. The aim of this study was to assess the diuretic activity of lead compound JT09 relative to vehicle control and Tolvaptan through single oral administration to adult male Sprague-Dawley rats. JT09-administered rats demonstrated significantly increased urine output relative to vehicle control. However, the effect persisted for 8 h, whereas Tolvaptan-administered rats demonstrated diuretic activity for 24 h. Relative to Tolvaptan, urine output was significantly reduced in JT09 administered animals at all-time points, suggesting that the overall diuretic effect of JT09 is less profound than Tolvaptan. Additionally, JT09-administered rats demonstrated alterations in clinical chemistry; reduced urine specific gravity; and increased urine pH relative to vehicle control. The following study establishes a preliminary diuretic profile for JT09.

10.
Int J Mol Sci ; 19(6)2018 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-29914066

RESUMO

The most ubiquitous gap junction protein within the body, connexin 43 (Cx43), is a target of interest for modulating the dermal wound healing response. Observational studies found associations between Cx43 at the wound edge and poor healing response, and subsequent studies utilizing local knockdown of Cx43 found improvements in wound closure rate and final scar appearance. Further preclinical work conducted using Cx43-based peptide therapeutics, including alpha connexin carboxyl terminus 1 (αCT1), a peptide mimetic of the Cx43 carboxyl terminus, reported similar improvements in wound healing and scar formation. Clinical trials and further study into the mode of action have since been conducted on αCT1, and Phase III testing for treatment of diabetic foot ulcers is currently underway. Therapeutics targeting connexin activity show promise in beneficially modulating the human body's natural healing response for improved patient outcomes across a variety of injuries.


Assuntos
Cicatriz/metabolismo , Conexina 43/metabolismo , Pé Diabético/tratamento farmacológico , Pele/metabolismo , Animais , Cicatriz/tratamento farmacológico , Conexina 43/química , Conexina 43/genética , Pé Diabético/metabolismo , Humanos , Fragmentos de Peptídeos/farmacologia , Fragmentos de Peptídeos/uso terapêutico , Pele/efeitos dos fármacos
11.
Curr Opin Pharmacol ; 41: 79-88, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29803991

RESUMO

Resistance of malignant glioma, including glioblastoma (GBM), to the chemotherapeutic temozolomide (TMZ) remains a key obstacle in treatment strategies. The gap junction protein connexin43 (Cx43) has complex roles in the establishment, progression, and persistence of malignant glioma. Recent findings demonstrate that connexins play an important role in the microenvironment of malignant glioma and that Cx43 is capable of conferring chemotherapeutic resistance to GBM cells. Carboxyl-terminal Cx43 peptidomimetics show therapeutic promise in overcoming TMZ resistance via mechanisms that may include modulating junctional activity between tumor cells and peritumoral cells and/or downstream molecular signaling events mediated by Cx43 protein binding. High levels of intra-tumor and inter-tumor heterogeneity make it difficult to clearly define specific populations for Cx43-targeted therapy; hence, development of in vitro models that better mimic the microenvironment of malignant glioma, and the incorporation of patient-derived stem cells, could provide opportunities for patient-specific drug screening. This review summarizes recent advances in understanding the roles of Cx43 in malignant glioma, with a special focus on tumor microenvironment, TMZ resistance, and therapeutic opportunity offered by Cx43 peptidomimetics.


Assuntos
Neoplasias do Sistema Nervoso Central/tratamento farmacológico , Conexina 43 , Resistencia a Medicamentos Antineoplásicos , Glioblastoma/tratamento farmacológico , Glioma/tratamento farmacológico , Peptidomiméticos , Temozolomida/uso terapêutico , Antineoplásicos Alquilantes/uso terapêutico , Conexina 43/metabolismo , Conexina 43/fisiologia , Humanos , Terapia de Alvo Molecular , Peptidomiméticos/farmacologia , Peptidomiméticos/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Microambiente Tumoral
12.
Artigo em Inglês | MEDLINE | ID: mdl-28512447

RESUMO

Diabetic peripheral neuropathy (DPN) remains one of the most common and serious complications of diabetes. Currently, pharmacological agents are limited to treating the pain associated with DPN, and do not address the underlying pathological mechanisms driving nerve damage, thus leaving a significant unmet medical need. Interestingly, research conducted using exercise as a treatment for DPN has revealed interleukin-6 (IL-6) signaling to be associated with many positive benefits such as enhanced blood flow and lipid metabolism, decreased chronic inflammation, and peripheral nerve fiber regeneration. IL-6, once known solely as a pro-inflammatory cytokine, is now understood to signal as a multifunctional cytokine, capable of eliciting both pro- and anti-inflammatory responses in a context-dependent fashion. IL-6 released from muscle in response to exercise signals as a myokine and as such has a unique kinetic profile, whereby levels are transiently elevated up to 100-fold and return to baseline levels within 4 h. Importantly, this kinetic profile is in stark contrast to long-term IL-6 elevation that is associated with pro-inflammatory states. Given exercise induces IL-6 myokine signaling, and exercise has been shown to elicit numerous beneficial effects for the treatment of DPN, a causal link has been suggested. Here, we discuss both the clinical and preclinical literature related to the application of IL-6 as a treatment strategy for DPN. In addition, we discuss how IL-6 may directly modulate Schwann and nerve cells to explore a mechanistic understanding of how this treatment elicits a neuroprotective and/or regenerative response. Collectively, studies suggest that IL-6, when administered in a low-dose pulsatile strategy to mimic the body's natural response to exercise, may prove to be an effective treatment for the protection and/or restoration of peripheral nerve function in DPN. This review highlights the studies supporting this assertion and provides rationale for continued investigation of IL-6 for the treatment of DPN.

13.
J Mol Med (Berl) ; 95(5): 535-552, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28132078

RESUMO

A critical target tissue in age-related macular degeneration (AMD) is the retinal pigment epithelium (RPE), which forms the outer blood-retina barrier (BRB). RPE-barrier dysfunction might result from attenuation/disruption of intercellular tight junctions. Zonula occludens-1 (ZO-1) is a major structural protein of intercellular junctions. A connexin43-based peptide mimetic, αCT1, was developed to competitively block interactions at the PDZ2 domain of ZO-1, thereby inhibiting ligands that selectively bind to this domain. We hypothesized that targeting ZO-1 signaling using αCT1 would maintain BRB integrity and reduce RPE pathophysiology by stabilizing gap- and/or tight-junctions. RPE-cell barrier dysfunction was generated in mice using laser photocoagulation triggering choroidal neovascularization (CNV) or bright light exposure leading to morphological damage. αCT1 was delivered via eye drops. αCT1 treatment reduced CNV development and fluid leakage as determined by optical coherence tomography, and damage was correlated with disruption in cellular integrity of surrounding RPE cells. Light damage significantly disrupted RPE cell morphology as determined by ZO-1 and occludin staining and tiling pattern analysis, which was prevented by αCT1 pre-treatment. In vitro experiments using RPE and MDCK monolayers indicated that αCT1 stabilizes tight junctions, independent of its effects on Cx43. Taken together, stabilization of intercellular junctions by αCT1 was effective in ameliorating RPE dysfunction in models of AMD-like pathology. KEY MESSAGE: The connexin43 mimetic αCT1 accumulates in the mouse retinal pigment epithelium following topical delivery via eye drops. αCT1 eye drops prevented RPE-cell barrier dysfunction in two mouse models. αCT1 stabilizes intercellular tight junctions. Stabilization of cellular junctions via αCT1 may serve as a novel therapeutic approach for both wet and dry age-related macular degeneration.


Assuntos
Conexina 43/química , Peptídeos/química , Peptídeos/farmacologia , Epitélio Pigmentado da Retina/metabolismo , Junções Íntimas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo , Animais , Linhagem Celular , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Epitélio Pigmentado da Retina/efeitos dos fármacos , Junções Íntimas/efeitos dos fármacos
14.
J Invest Dermatol ; 137(3): 620-630, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27856288

RESUMO

The transmembrane protein Cx43 has key roles in fibrogenic processes including inflammatory signaling and extracellular matrix composition. aCT1 is a Cx43 mimetic peptide that in preclinical studies accelerated wound closure, decreased inflammation and granulation tissue area, and normalized mechanical properties after cutaneous injury. We evaluated the efficacy and safety of aCT1 in the reduction of scar formation in human incisional wounds. In a prospective, multicenter, within-participant controlled trial, patients with bilateral incisional wounds (≥10 mm) after laparoscopic surgery were randomized to receive acute treatment (immediately after wounding and 24 hours later) with an aCT1 gel formulation plus conventional standard of care protocols, involving moisture-retentive occlusive dressing, or standard of care alone. The primary efficacy endpoint was average scarring score using visual analog scales evaluating incision appearance and healing progress over 9 months. There was no significant difference in scar appearance between aCT1- or control-treated incisions after 1 month. At month 9, aCT1-treated incisions showed a 47% improvement in scar scores over controls (Vancouver Scar Scale; P = 0.0045), a significantly higher Global Assessment Scale score (P = 0.0009), and improvements in scar pigmentation, thickness, surface roughness, and mechanical suppleness. Adverse events were similar in both groups. aCT1 has potential to improve scarring outcome after surgery.


Assuntos
Cicatriz/tratamento farmacológico , Cicatriz/metabolismo , Conexina 43/química , Fragmentos de Peptídeos/uso terapêutico , Peptídeos/química , Pele/efeitos dos fármacos , Adulto , Idoso , Conexina 43/uso terapêutico , Feminino , Humanos , Inflamação , Laparoscopia , Masculino , Pessoa de Meia-Idade , Segurança do Paciente , Estudos Prospectivos , Índice de Gravidade de Doença , Estresse Mecânico , Cicatrização , Adulto Jovem
15.
Cancer Lett ; 374(1): 117-126, 2016 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-26884256

RESUMO

Connexins are a family of transmembrane proteins that are characterized by their capacity to form intercellular channels called gap junctions that directly link the cytoplasm of adjacent cells. The formation of gap junctions by connexin proteins facilitates intercellular communication between neighboring cells by allowing for the transfer of ions and small signaling molecules. Communication through gap junctions is key to cellular equilibrium, where connexins, and the gap junction intercellular communication that connexins propagate, have roles in cellular processes such as cell growth, differentiation, and tissue homeostasis. Due to their importance in maintaining cellular functions, the disruption of connexin expression and function underlies the etiology and progression of numerous pathologies, including cancer. Over the past half a century, the role of connexins and gap junction intercellular communication have been highlighted as critical areas of research in cellular malignancies, and much research effort has been geared toward understanding their dysfunction in human cancers. Although ample evidence supports the role of connexins in a variety of human cancers, detailed examination in specific cancers, such as breast cancer, is still lacking. This review highlights the most abundant gap junction connexin isoform in higher vertebrate organisms, Connexin 43, and its role in breast cancer.


Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Conexina 43/metabolismo , Animais , Feminino , Humanos , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia
16.
J Vis Exp ; (107): e53329, 2016 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-26862835

RESUMO

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death. Common sites of metastatic spread include lung, lymph node, brain, and bone. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.


Assuntos
Neoplasias Pulmonares/secundário , Neoplasias Mamárias Experimentais/patologia , Neoplasias Experimentais , RNA Neoplásico/genética , Animais , Feminino , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Neoplasias Mamárias Experimentais/genética , Camundongos , Reação em Cadeia da Polimerase em Tempo Real
17.
BMC Cancer ; 15: 296, 2015 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-25881004

RESUMO

BACKGROUND: Treatment failure is a critical issue in breast cancer and identifying useful interventions that optimize current cancer therapies remains a critical unmet need. Expression and functional studies have identified connexins (Cxs), a family of gap junction proteins, as potential tumor suppressors. Studies suggest that Cx43 has a role in breast cancer cell proliferation, differentiation, and migration. Although pan-gap junction drugs are available, the lack of specificity of these agents increases the opportunity for off target effects. Consequently, a therapeutic agent that specifically modulates Cx43 would be beneficial and has not been tested in breast cancer. In this study, we now test an agent that specifically targets Cx43, called ACT1, in breast cancer. METHODS: We evaluated whether direct modulation of Cx43 using a Cx43-directed therapeutic peptide, called ACT1, enhances Cx43 gap junctional activity in breast cancer cells, impairs breast cancer cell proliferation or survival, and enhances the activity of the targeted inhibitors tamoxifen and lapatinib. RESULTS: Our results show that therapeutic modulation of Cx43 by ACT1 maintains Cx43 at gap junction sites between cell-cell membrane borders of breast cancer cells and augments gap junction activity in functional assays. The increase in Cx43 gap junctional activity achieved by ACT1 treatment impairs proliferation or survival of breast cancer cells but ACT1 has no effect on non-transformed MCF10A cells. Furthermore, treating ER+ breast cancer cells with a combination of ACT1 and tamoxifen or HER2+ breast cancer cells with ACT1 and lapatinib augments the activity of these targeted inhibitors. CONCLUSIONS: Based on our findings, we conclude that modulation of Cx43 activity in breast cancer can be effectively achieved with the agent ACT1 to sustain Cx43-mediated gap junctional activity resulting in impaired malignant progression and enhanced activity of lapatinib and tamoxifen, implicating ACT1 as part of a combination regimen in breast cancer.


Assuntos
Neoplasias da Mama/metabolismo , Conexina 43/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Quinazolinas/administração & dosagem , Tamoxifeno/administração & dosagem , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/administração & dosagem , Proteínas Adaptadoras de Transdução de Sinal , Antineoplásicos/administração & dosagem , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Lapatinib , Quinazolinas/metabolismo , Tamoxifeno/metabolismo , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/metabolismo
18.
Wound Repair Regen ; 23(2): 203-12, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25703647

RESUMO

Nonhealing neuropathic foot ulcers remain a significant problem in individuals with diabetes. The gap-junctional protein connexin43 (Cx43) has roles in dermal wound healing and targeting Cx43 signalling accelerates wound reepithelialization. In a prospective, randomized, multicenter clinical trial we evaluated the efficacy and safety of a peptide mimetic of the C-terminus of Cx43, alpha connexin carboxy-terminal (ACT1), in accelerating the healing of chronic diabetic foot ulcers (DFUs) when incorporated into standard of care (SOC) protocols. Adults with DFUs of at least four weeks duration were randomized to receive SOC with or without topical application of ACT1. Primary outcome was mean percent ulcer reepithelialization and safety variables included incidence of treatment related adverse events (AEs) and detection of ACT1 immunogenicity. ACT1 treatment was associated with a significantly greater reduction in mean percent ulcer area from baseline to 12 weeks (72.1% vs. 57.1%; p = 0.03). Analysis of incidence and median time-to-complete-ulcer closure revealed that ACT1 treatment was associated with a greater percentage of participants that reached 100% ulcer reepitheliazation and a reduced median time-to-complete-ulcer closure. No AEs reported were treatment related, and ACT1 was not immunogenic. Treatment protocols that incorporate ACT1 may present a therapeutic strategy that safely augments the reepithelialization of chronic DFUs.


Assuntos
Anti-Infecciosos/administração & dosagem , Conexina 43/administração & dosagem , Conexina 43/farmacologia , Pé Diabético/tratamento farmacológico , Infecção da Ferida Cirúrgica/tratamento farmacológico , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/administração & dosagem , Cicatrização/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal , Administração Tópica , Anti-Infecciosos/farmacologia , Pé Diabético/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Peptídeos , Estudos Prospectivos , Infecção da Ferida Cirúrgica/patologia , Resultado do Tratamento , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/farmacologia
19.
J Invest Dermatol ; 135(1): 289-298, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25072595

RESUMO

The gap junction protein, connexin43 (Cx43), has critical roles in the inflammatory, edematous, and fibrotic processes following dermal injury and during wound healing, and is abnormally upregulated at the epidermal wound margins of venous leg ulcers (VLUs). Targeting Cx43 with ACT1, a peptide mimetic of the carboxyl-terminus of Cx43, accelerates fibroblast migration and proliferation, and wound reepithelialization. In a prospective, multicenter clinical trial conducted in India, adults with chronic VLUs were randomized to treatment with an ACT1 gel formulation plus conventional standard-of-care (SOC) protocols, involving maintaining wound moisture and four-layer compression bandage therapy, or SOC protocols alone. The primary end point was mean percent ulcer reepithelialization from baseline to 12 weeks. A significantly greater reduction in mean percent ulcer area from baseline to 12 weeks was associated with the incorporation of ACT1 therapy (79% (SD 50.4)) as compared with compression bandage therapy alone (36% (SD 179.8); P=0.02). Evaluation of secondary efficacy end points indicated a reduced median time to 50 and 100% ulcer reepithelialization for ACT1-treated ulcers. Incorporation of ACT1 in SOC protocols may represent a well-tolerated, highly effective therapeutic strategy that expedites chronic venous ulcer healing by treating the underlying ulcer pathophysiology through Cx43-mediated pathways.


Assuntos
Conexina 43/metabolismo , Úlcera da Perna/tratamento farmacológico , Peptídeos/administração & dosagem , Cicatrização/efeitos dos fármacos , Adulto , Doença Crônica , Conexina 43/química , Feminino , Seguimentos , Humanos , Estimativa de Kaplan-Meier , Úlcera da Perna/patologia , Masculino , Pessoa de Meia-Idade , Peptídeos/efeitos adversos , Estudos Prospectivos , Estrutura Terciária de Proteína , Resultado do Tratamento
20.
FEBS Lett ; 588(8): 1349-64, 2014 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-24607540

RESUMO

Gap junctions and their connexin components are indispensable in mediating the cellular coordination required for tissue and organ homeostasis. The critical nature of their existence mandates a connection to disease while at the same time offering therapeutic potential. Therapeutic intervention may be offered through the pharmacological and molecular disruption of the pathways involved in connexin biosynthesis, gap junction assembly, stabilization, or degradation. Chemical inhibitors aimed at closing connexin channels, peptide mimetics corresponding to short connexin sequences, and gene therapy approaches have been incredibly useful molecular tools in deciphering the complexities associated with connexin biology. Recently, therapeutic potential in targeting connexins has evolved from basic research in cell-based models to clinical opportunity in the form of human trials. Clinical promise is particularly evident with regards to targeting connexin43 in the context of wound healing. The following review is aimed at highlighting novel advances where the pharmacological manipulation of connexin biology has proven beneficial in animals or humans.


Assuntos
Doenças Cardiovasculares/metabolismo , Conexina 43/metabolismo , Terapia de Alvo Molecular , Neoplasias/metabolismo , Peptídeos/farmacologia , Animais , Doenças Cardiovasculares/tratamento farmacológico , Doenças do Sistema Nervoso Central/tratamento farmacológico , Doenças do Sistema Nervoso Central/metabolismo , Conexina 43/antagonistas & inibidores , Conexina 43/genética , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/metabolismo , Junções Comunicantes/patologia , Humanos , Neoplasias/tratamento farmacológico , Peptídeos/uso terapêutico , Dermatopatias/tratamento farmacológico , Dermatopatias/metabolismo , Cicatrização
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