RESUMO
P-glycoprotein (P-gp), a multidrug efflux pump encoded by the ABCB1 (formerly MDR1) gene, plays a crucial role in limiting drug absorption and eliminating toxic compounds in both humans and dogs. However, species-specific differences in P-gp substrates necessitate the development of canine-specific evaluation systems. Canine intestinal organoids derived monolayers offer a promising platform for studying drug transport, yet P-gp-mediated transport in these models remains unexplored.We generated canine colonoid-derived 2D monolayers to investigate ABCB1 gene expression and P-gp function. We employed widely recognised P-gp substrates, Rhodamine 123 and Doxorubicin, in conjunction with the P-gp inhibitor PSC833 at Days 5 and 10 of culture.A significant increase in gene expression of P-gp encoded by the ABCB1 was noted on Day 10 compared to Day 5 of culture. Despite this disparity in gene expression, the transport activity of P-gp, as assessed by the efflux of Rhodamine 123 and Doxorubicin with PSC833 inhibition, did not exhibit significant differences between these two time points. However, the inhibition of P-gp function by PSC833 confirms the presence of functional P-gp in our model.Canine intestinal organoid-derived monolayers provide a valuable tool for investigating P-gp-mediated drug transport. These findings highlight the potential for predicting drug bioavailability and adverse reactions in veterinary medicine, aligning with principles of ethical and sustainable research.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Doxorrubicina , Organoides , Rodamina 123 , Animais , Cães , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Rodamina 123/metabolismo , Organoides/metabolismo , Doxorrubicina/farmacologia , Mucosa Intestinal/metabolismo , Ciclosporinas/farmacologia , Transporte BiológicoRESUMO
Mechanical overloading (OVL) resulting from the ablation of muscle agonists, a supra-physiological model of resistance training, reduces skeletal muscle fragility, i.e. the immediate maximal force drop following lengthening contractions, and increases maximal force production, in mdx mice, a murine model of Duchene muscular dystrophy (DMD). Here, we further analyzed these beneficial effects of OVL by determining whether they were blocked by cyclosporin, an inhibitor of the calcineurin pathway, and whether there were also observed in the D2-mdx mice, a more severe murine DMD model. We found that cyclosporin did not block the beneficial effect of 1-month OVL on plantaris muscle fragility in mdx mice, nor did it limit the increases in maximal force and muscle weight (an index of hypertrophy). Fragility and maximal force were also ameliorated by OVL in the plantaris muscle of D2-mdx mice. In addition, OVL increased the expression of utrophin, cytoplamic γ-actin, MyoD, and p-Akt in the D2-mdx mice, proteins playing an important role in fragility, maximal force gain and muscle growth. In conclusion, OVL reduced fragility and increased maximal force in the more frequently used mild mdx model but also in D2-mdx mice, a severe model of DMD, closer to human physiopathology. Moreover, these beneficial effects of OVL did not seem to be related to the activation of the calcineurin pathway. Thus, this preclinical study suggests that resistance training could have a potential benefit in the improvement of the quality of life of DMD patients.
Assuntos
Ciclosporinas , Distrofia Muscular de Duchenne , Treinamento Resistido , Humanos , Animais , Camundongos , Distrofia Muscular de Duchenne/patologia , Camundongos Endogâmicos mdx , Calcineurina/metabolismo , Qualidade de Vida , Músculo Esquelético/metabolismo , Ciclosporinas/farmacologia , Modelos Animais de DoençasRESUMO
BACKGROUND: Letermovir (LTV), an antiviral with exclusive activity against Cytomegalovirus (CMV), is approved for prophylaxis of CMV infection and disease in adult hematopoietic cell transplant (HCT) patients. The use of LTV in the pediatric HCT population is off-label, and has limited literature to support its use. PROCEDURE: This was a single-center, retrospective, matched (1:1 LTV:non-LTV) cohort study of allogeneic HCT recipients transplanted at Children's Hospital Colorado from 2015 to 2022. The primary endpoint was clinically significant CMV DNAemia (defined as a CMV viral load >1000 copies/mL or any CMV DNAemia leading to preemptive treatment) through 6 months post transplant. Secondary outcomes included time to clinically significant CMV DNAemia, drug adverse effects, and dose adjustments of concomitant cyclosporine and voriconazole (known drug interactions). RESULTS: We compared 41 patients who received LTV prophylaxis to 41 patients who received no CMV prophylaxis. There was less clinically significant CMV DNAemia through D+180 in the LTV group (9.8% vs. 17.0%, p = .33). Overall, LTV was well tolerated, and 87.8% of patients experienced no adverse effects related to the drug. There was no observed pattern in LTV effect on cyclosporine serum concentrations, but LTV was associated with decreased voriconazole trough levels. CONCLUSIONS: In this retrospective study, the use of LTV prophylaxis in pediatric stem cell patients was associated with reduced clinically significant CMV DNAemia through D+180.
Assuntos
Ciclosporinas , Infecções por Citomegalovirus , Transplante de Células-Tronco Hematopoéticas , Adulto , Humanos , Criança , Citomegalovirus , Estudos Retrospectivos , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Estudos de Coortes , Voriconazol , Infecções por Citomegalovirus/etiologia , Infecções por Citomegalovirus/prevenção & controle , Infecções por Citomegalovirus/tratamento farmacológico , Antivirais/uso terapêutico , Antivirais/efeitos adversos , Ciclosporinas/farmacologia , Ciclosporinas/uso terapêutico , TransplantadosRESUMO
Nephrotic syndrome (NS) is characterized by massive proteinuria; podocyte loss or altered function is a central event in its pathophysiology. Treatment with glucocorticoids is the mainstay of therapy, however, many patients experience one or multiple relapses and prolonged use may be associated with severe adverse effects. Recently the beneficial effects of glucocorticoids have been attributed to a direct effect on podocytes in addition to the well-known immunosuppressive effects. The molecular effects of glucocorticoid action have been studied using animal and cell models of NS. This review provides a comprehensive overview of different molecular mediators regulated by glucocorticoids, including an overview of the model systems that were used to study them. Glucocorticoids are described to stimulate podocyte recovery by restoring pro-survival signalling of slit diaphragm-related proteins and limiting inflammatory responses. Of special interest is the effect of glucocorticoids on stabilizing the cytoskeleton of podocytes, since these effects are also described for other therapeutic agents used in NS, such as cyclosporin. Current models provide much insight but do not fully recapitulate the human condition since the pathophysiology underlying NS is poorly understood. New and promising models include the glomerulus-on-a-chip and kidney organoids, which have the potential to be further developed into functional NS models in the future.
Assuntos
Ciclosporinas , Síndrome Nefrótica , Podócitos , Animais , Ciclosporinas/metabolismo , Ciclosporinas/farmacologia , Ciclosporinas/uso terapêutico , Glucocorticoides/farmacologia , Glucocorticoides/uso terapêutico , Humanos , Glomérulos Renais/metabolismo , Síndrome Nefrótica/tratamento farmacológico , Síndrome Nefrótica/metabolismo , Podócitos/metabolismoRESUMO
Drug-induced liver injury, including cholestasis, is an important clinical issue and economic burden for pharmaceutical industry and healthcare systems. However, human-relevant in vitro information on the ability of other types of chemicals to induce cholestatic hepatotoxicity is lacking. This work aimed at investigating the cholestatic potential of non-pharmaceutical chemicals using primary human hepatocytes cultured in 3D spheroids. Spheroid cultures were repeatedly (co-) exposed to drugs (cyclosporine-A, bosentan, macitentan) or non-pharmaceutical chemicals (paraquat, tartrazine, triclosan) and a concentrated mixture of bile acids for 4 weeks. Cell viability (adenosine triphosphate content) was checked every week and used to calculate the cholestatic index, an indicator of cholestatic liability. Microarray analysis was performed at specific time-points to verify the deregulation of genes related to cholestasis, steatosis and fibrosis. Despite the evident inter-donor variability, shorter exposures to cyclosporine-A consistently produced cholestatic index values below 0.80 with transcriptomic data partially supporting its cholestatic burden. Bosentan confirmed to be hepatotoxic, while macitentan was not toxic in the tested concentrations. Prolonged exposure to paraquat suggested fibrotic potential, while triclosan markedly deregulated genes involved in different types of hepatotoxicity. These results support the applicability of primary human hepatocyte spheroids to study hepatotoxicity of non-pharmaceutical chemicals in vitro.
Assuntos
Ácidos e Sais Biliares/farmacologia , Paraquat/farmacologia , Esferoides Celulares/efeitos dos fármacos , Bosentana/farmacologia , Técnicas de Cultura de Células , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Ciclosporinas/farmacologia , Feminino , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo , Transcriptoma/efeitos dos fármacosRESUMO
Human cytomegalovirus (HCMV) is a ubiquitous pathogen that establishes a life-long infection affecting up to 80% of the US population. HCMV periodically reactivates leading to enhanced morbidity and mortality in immunosuppressed patients causing a range of complications including organ transplant failure and cognitive disorders in neonates. Therapeutic options for HCMV are limited to a handful of antivirals that target late stages of the virus life cycle and efficacy is often challenged by the emergence of mutations that confer resistance. In addition, these antiviral therapies may have adverse reactions including neutropenia in newborns and an increase in adverse cardiac events in HSCT patients. These findings highlight the need to develop novel therapeutics that target different steps of the viral life cycle. To this end, we screened a small molecule library against ion transporters to identify new antivirals against the early steps of virus infection. We identified valspodar, a 2nd-generation ABC transporter inhibitor, that limits HCMV infection as demonstrated by the decrease in IE2 expression of virus infected cells. Cells treated with increasing concentrations of valspodar over a 9-day period show minimal cytotoxicity. Importantly, valspodar limits HCMV plaque numbers in comparison to DMSO controls demonstrating its ability to inhibit viral dissemination. Collectively, valspodar represents a potential new anti-HCMV therapeutic that limits virus infection by likely targeting a host factor. Further, the data suggest that specific ABC transporters may participate in the HCMV life-cycle.
Assuntos
Transportadores de Cassetes de Ligação de ATP/farmacologia , Ciclosporinas/farmacologia , Infecções por Citomegalovirus/tratamento farmacológico , Citomegalovirus/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Antivirais/farmacologia , Linhagem Celular , Células Cultivadas , Infecções por Citomegalovirus/virologia , Humanos , Testes de Sensibilidade Microbiana , Replicação ViralRESUMO
Several anticancer drugs including cisplatin (CDDP) induce hypomagnesemia. However, it remains fully uncertain whether Mg2+ deficiency affects chemosensitivity of cancer cells. Here, we investigated the effect of low Mg2+ concentration (LM) on proliferation and chemosensitivity using human lung adenocarcinoma A549 cells. Cell proliferation was reduced by continuous culture with LM accompanied with the elevation of G1 phase proportion. The amounts of reactive oxygen species (ROS) and stress makers such as phosphorylated-ataxia telangiectasia mutated and phosphorylated-p53 were increased by LM. Cell injury was dose-dependently increased by anticancer drugs such as CDDP and doxorubicin (DXR), which were suppressed by LM. Similar results were obtained by roscovitine, a cell cycle inhibitor. These results suggest that LM induces chemoresistance mediated by ROS production and G1 arrest. The mRNA and protein levels of ATP binding cassette subfamily B member 1 (ABCB1) were increased by LM and roscovitine. The LM-induced elevation of ABCB1 and nuclear p38 expression was suppressed by SB203580, a p38 MAPK inhibitor. PSC833, an ABCB1 inhibitor, and SB203580 rescued the sensitivity to anticancer drugs. In addition, cancer stemness properties were suppressed by SB203580. We suggest that Mg2+ deficiency reduces the chemotherapy sensitivity of A549 cells, although it suppresses cell proliferation.
Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/metabolismo , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Magnésio/química , Células A549 , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Ciclo Celular , Proliferação de Células , Cisplatino/administração & dosagem , Ciclosporinas/farmacologia , Dano ao DNA , Doxorrubicina/administração & dosagem , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imidazóis/farmacologia , Linfonodos/patologia , Fosforilação , Piridinas/farmacologia , Espécies Reativas de Oxigênio , Roscovitina/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Two ATP-binding cassette transporters, ABCB1/MDR1 and ABCG2/BCRP, are considered the most critical determinants for chemoresistance in hepatocellular carcinoma. However, their roles in the chemoresistance in liver cancer stem cells remain elusive. Here we explored the role of inhibition of MDR1 or ABCG2 in sensitizing liver cancer stem cells to doxorubicin, the most frequently used chemotherapeutic agent in treating liver cancer. We show that the inhibition of MDR1 or ABCG2 in Huh7 and PLC/PRF/5 cells using either pharmacological inhibitors or RNAi resulted in the elevated level of intracellular concentration of doxorubicin and the accompanied increased apoptosis as determined by confocal microscopy, high-performance liquid chromatography, flow cytometry, and annexin V assay. Notably, the inhibition of MDR1 or ABCG2 led to the reversal of the chemoresistance, as evident from the enhanced death of the chemoresistant liver cancer stem cells in tumorsphere-forming assays. Thus, the elevation of effective intracellular concentration of doxorubicin via the inhibition of MDR1 or ABCG2 represents a promising future strategy that transforms doxorubicin from a traditional chemotherapy agent into a robust killer of liver cancer stem cells for patients undergoing transarterial chemoembolization.
Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Carcinoma Hepatocelular/metabolismo , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Neoplasias Hepáticas/metabolismo , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Carcinoma Hepatocelular/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ciclosporinas/farmacologia , Dicetopiperazinas/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Proteínas de Neoplasias/antagonistas & inibidores , Células-Tronco Neoplásicas/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND AND AIMS: The Hepatitis E virus hijacks the endosomal system for its release. These structures are highly dependent on cholesterol. Hence, this study investigates the impact of HEV on cholesterol-metabolism, the effect of intracellular cholesterol content on HEV-release and the potential of cholesterol-modulators to serve as antivirals. METHODS: Intracellular cholesterol-content of cells was modulated and impacts on HEV were monitored using qPCR, Western blot, microscopy, virus-titration and density-gradient centrifugation. Blood-lipids and HEV-RNA were routinely quantified in chronically infected patients during follow-up visits. RESULTS: In HEV-infected cells, decreased levels of cholesterol are found. In patients, HEV infection decreases serum-lipid concentrations. Importantly, statin treatment herein increases viral titers. Similarly, reduction of intracellular cholesterol via simvastatin treatment increases viral release in vitro. On the contrary, elevating intracellular cholesterol via LDL or 25-hydroxycholesterol strongly reduces viral release due to enhanced lysosomal degradation of HEV. Drug-induced elevation of intracellular cholesterol via fenofibrate or PSC833 impairs HEV release via the same mechanism. CONCLUSIONS: This study analyses the crosstalk between HEV and intracellular cholesterol. The results highlight the importance of an intact cholesterol homeostasis for HEV-release and thereby identify a potential target for antiviral strategies. Especially fenofibrate is considered a promising novel antiviral against HEV. Beyond this, the study may help clinicians evaluating co-treatments of HEV-infected patients with statins, as this may be counter indicated.
Assuntos
Antivirais/farmacologia , Colesterol/metabolismo , Ciclosporinas/farmacologia , Fenofibrato/farmacologia , Vírus da Hepatite E/efeitos dos fármacos , Antivirais/química , Sobrevivência Celular/efeitos dos fármacos , Ciclosporinas/química , Fenofibrato/química , Humanos , Testes de Sensibilidade Microbiana , Células Tumorais Cultivadas , Replicação Viral/efeitos dos fármacosRESUMO
We previously reported that the non-immunosuppressive cyclophilin inhibitors (CypIs)-cyclosporin A analog CRV431 and sanglifehrin analog NV556-efficiently inhibit HCV replication in vitro. In this study, we asked whether they can also reduce HCV replication in vivo. We found that a single oral administration of CRV431 and NV556 to HCV-infected humanized-liver mice drastically reduced HCV blood levels. The antiviral effect was observed when CRV431 or NV556 were each individually administered with HCV, 3, 6 weeks or even 3 months post-infection when viral replication is robust. These results were confirmed in chimeric mice implanted with human hepatocytes isolated from three distinct donors. Remarkably, no viral rebound was observed 5 months after a single dose administration of 50 mg/kg of CRV431 or NV556 four weeks post-HCV infection, indicating the possibility of suppression of an established viral infection. Since we recently demonstrated that both CRV431 and NV556 also inhibit the development of liver fibrosis and hepatocellular carcinoma in nonviral-induced non-alcoholic steatohepatitis mouse models, our present data suggest that the two entirely structurally different CypIs-CRV431 and NV556-derived from unrelated natural products, represent attractive partners to current direct-acting agent (DAA) regimens for the treatment of hepatitis C and liver diseases.
Assuntos
Antivirais/uso terapêutico , Ciclosporinas/uso terapêutico , Hepacivirus/efeitos dos fármacos , Hepatite C/tratamento farmacológico , Animais , Antivirais/farmacologia , Ciclosporinas/farmacologia , Hepacivirus/fisiologia , Hepatite C/virologia , Humanos , Fígado/efeitos dos fármacos , Fígado/virologia , Camundongos , Camundongos SCID , Camundongos Transgênicos , Replicação Viral/efeitos dos fármacosRESUMO
Biologicals like anti-tumor necrosis factor (TNF) therapy for Crohn's disease (CD) are safe and effective but there is a significant rate of primary and secondary nonresponse in the patients. In this study, we applied a computational approach to discover novel drug therapies for anti-TNF refractory CD in silico. We use a transcriptome dataset (GSE100833) for the anti-TNF refractory CD patients from NCBI GEO. After co-expression analysis, we specifically investigated the extent of protein-protein interactions among genes in clusters based on a protein-protein interaction database, STRING. Pathway analysis was performed using the clEnrich function based on KEGG gene sets. Co-expressed genes in cluster 1, 2, 3, 4, up or down-regulated genes and all differentially expressed genes are highly connected. Among them, cluster 1, which is highly enriched for chemokine signaling, also showed enrichment for cytokine-cytokine receptor interaction and identifies several drugs including cyclosporin with known efficacy in CD. Vorinostat, histone deacetylase inhibitors, and piperlongumine, which is known to have inhibitory effect on activity of NF-κB, were also identified. Some alkaloids were also selected as potential therapeutic drugs. These finding suggest that they might serve as a novel therapeutic option for anti-TNF refractory CD and support the use of public molecular data and computational approaches to discover novel therapeutic options for CD.
Assuntos
Biologia Computacional/métodos , Doença de Crohn/tratamento farmacológico , Descoberta de Drogas/métodos , Reposicionamento de Medicamentos/métodos , Anticorpos Monoclonais/farmacologia , Ciclosporinas/farmacologia , Dioxolanos/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Humanos , Família Multigênica/genética , Mapas de Interação de Proteínas , Transcriptoma/genética , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Vorinostat/farmacologiaAssuntos
Doenças Autoimunes/tratamento farmacológico , Betacoronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Ciclosporinas/farmacologia , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Replicação Viral/efeitos dos fármacos , Betacoronavirus/efeitos dos fármacos , COVID-19 , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Ciclosporinas/uso terapêutico , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Pneumonia Viral/epidemiologia , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/efeitos dos fármacos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , SARS-CoV-2 , Síndrome Respiratória Aguda Grave/epidemiologia , Síndrome Respiratória Aguda Grave/imunologia , Síndrome Respiratória Aguda Grave/prevenção & controle , Síndrome Respiratória Aguda Grave/virologiaRESUMO
Aortic valve stenosis (AS) is a pathological condition that affects about 3% of the population, representing the most common valve disease. The main clinical feature of AS is represented by the impaired leaflet motility, due to calcification, which leads to the left ventricular outflow tract obstruction during systole. The formation and accumulation of calcium nodules are driven by valve interstitial cells (VICs). Unfortunately, to date, the in vitro and in vivo studies were not sufficient to fully recapitulate all the pathological pathways involved in AS development, as well as to define a specific and effective pharmacological treatment for AS patients. Cyclophilin A (CyPA), the most important immunophilin and endogenous ligand of cyclosporine A (CsA), is strongly involved in several detrimental cardiovascular processes, such as calcification. To date, there are no data on the CyPA role in VIC-mediated calcification process of AS. Here, we aimed to identify the role of CyPA in AS by studying VIC calcification, in vitro. In this study, we found that (i) CyPA is up-regulated in stenotic valves of AS patients, (ii) pro-calcifying medium promotes CyPA secretion by VICs, (iii) in vitro treatment of VICs with exogenous CyPA strongly stimulates calcium deposition, and (iv) exogenous CyPA inhibition mediated by CsA analogue MM284 abolished in vitro calcium potential. Thus, CyPA represents a biological target that may act as a novel candidate in the detrimental AS development and its inhibition may provide a novel pharmacological approach for AS treatment.
Assuntos
Estenose da Valva Aórtica/tratamento farmacológico , Estenose da Valva Aórtica/cirurgia , Valva Aórtica/patologia , Calcinose/tratamento farmacológico , Calcinose/cirurgia , Ciclofilina A/antagonistas & inibidores , Ciclosporinas/farmacologia , Ciclosporinas/uso terapêutico , Idoso , Idoso de 80 Anos ou mais , Valva Aórtica/metabolismo , Valva Aórtica/cirurgia , Estenose da Valva Aórtica/metabolismo , Calcinose/metabolismo , Células Cultivadas , Ciclofilina A/metabolismo , Feminino , Humanos , Masculino , Resultado do TratamentoRESUMO
P-glycoprotein (Pgp), a member of the ATP-binding cassette family, is one of the major causes of multidrug resistance in tumors. Current clinical treatments to overcome MDR involve the co-delivery of a Pgp inhibitor and a chemotherapeutic. A concern for this treatment that has led to varied clinical trial success is the associated systemic toxicities involving endogenous Pgp. Local drug delivery systems, such as in situ forming implants (ISFIs), alleviate this problem by delivering a high concentration of the drug directly to the target site without the associated systemic toxicities. ISFIs are polymeric drug solutions that undergo a phase transition upon injection into an aqueous environment to form a solid drug eluting depot allowing for a high initial intratumoral drug concentration. In this study, we have developed an ISFI capable of overcoming the Pgp resistance by co-delivering a chemotherapeutic, Doxorubicin (Dox), with a Pgp inhibitor, either Pluronic P85 or Valspodar (Val). Studies investigated in vitro cytotoxicity of Dox when combined with either Pgp inhibitor, effect of the inhibitors on release of Dox from implants in PBS, in vivo Dox distribution and retention in a subcutaneous flank colorectal murine tumor, and therapeutic response characterized by tumor growth curves and histopathology. Dox + Val showed a 4-fold reduction in the 50% lethal dose (LD50) after 48 hours. Concurrent delivery of Dox and Val showed the greatest difference at 16 days post injection for both Dox penetration and retention. This treatment group had a 5-fold maximum Dox penetration compared to Dox alone ISFIs (0.53 ± 0.22 cm vs 0.11 ± 0.11 cm, respectively, from the center of the ISFI). Additionally, there was a 3-fold increase in normalized total intratumoral Dox intensity with the Dox + Val ISFIs compared to Dox alone ISFIs (0.54 ± 0.11 vs 0.18 ± 0.09, respectively). Dox + Val ISFIs showed a 2-fold reduction in tumor growth and a 27.69% increase in necrosis 20 days post-injection compared to Dox alone ISFIs. These findings demonstrate that co-delivery of Dox and Val via ISFI can avoid systemic toxicity issues seen with clinical Pgp inhibitors.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Neoplasias Colorretais/tratamento farmacológico , Ciclosporinas/farmacologia , Poloxaleno/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Doxorrubicina/farmacologia , Sistemas de Liberação de Medicamentos , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistência a Múltiplos Medicamentos/genética , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Camundongos , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: New therapeutic drugs are urgently needed against visceral leishmaniasis because current drugs, such as pentavalent antimonials and miltefosine, produce severe side effects and development of resistance. Whether cyclosporine A (CsA) and its derivatives can be used as therapeutic drugs for visceral leishmaniasis has been controversial for many years. METHODS: In this study, we evaluated the efficacy of CsA and its derivative, dihydrocyclosporin A (DHCsA-d), against promastigotes and intracellular amastigotes of Leishmania donovani. Sodium stibogluconate (SSG) was used as a positive control. RESULTS: Our results showed that DHCsA-d was able to inhibit the proliferation of L. donovani promastigotes (IC50: 21.24 µM and 12.14 µM at 24 h and 48 h, respectively) and intracellular amastigotes (IC50: 5.23 µM and 4.84 µM at 24 and 48 h, respectively) in vitro, but CsA treatment increased the number of amastigotes in host cells. Both DHCsA-d and CsA caused several alterations in the morphology and ultrastructure of L. donovani, especially in the mitochondria. However, DHCsA-d showed high cytotoxicity towards cells of the mouse macrophage cell line RAW264.7, with CC50 values of 7.98 µM (24 h) and 6.65 µM (48 h). Moreover, DHCsA-d could increase IL-12, TNF-α and IFN-γ production and decrease the levels of IL-10, IL-4, NO and H2O2 in infected macrophages. On the contrary, CsA decreased IL-12, TNF-α, and IFN-γ production and increased the levels of IL-10, IL-4, NO and H2O2 in infected macrophages. The expression of L. donovani cyclophilin A (LdCyPA) in promastigotes and intracellular amastigotes and the expression of cyclophilin A (CyPA) in RAW 264.7 cells were found to be significantly downregulated in the CsA-treated group compared to those in the untreated group. However, no significant changes in LdCyPA and CyPA levels were found after DHCsA-d or SSG treatment. CONCLUSIONS: Our findings initially resolved the dispute regarding the efficacy of CsA and DHCsA-d for visceral leishmaniasis treatment. CsA showed no significant inhibitory effect on intracellular amastigotes. DHCsA-d significantly inhibited promastigotes and intracellular amastigotes, but it was highly cytotoxic. Therefore, CsA and DHCsA-d are not recommended as antileishmanial drugs.
Assuntos
Antiprotozoários/farmacologia , Ciclosporina/farmacologia , Ciclosporinas/farmacologia , Leishmania donovani/efeitos dos fármacos , Leishmaniose Visceral/parasitologia , Animais , Avaliação Pré-Clínica de Medicamentos , Humanos , Interferon gama/imunologia , Interleucina-10/imunologia , Interleucina-2/imunologia , Leishmania donovani/crescimento & desenvolvimento , Leishmania donovani/fisiologia , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/imunologia , Macrófagos/imunologia , Macrófagos/parasitologia , Camundongos , Células RAW 264.7RESUMO
Introduction: Cyclophilins are a family of diverse regulatory enzymes that have been studied for over 30 years; they participate in many pathophysiological processes. Genetic deletion or pharmacologic inhibition of cyclophilins has shown therapeutic effects in a wide spectrum of disease models, including liver disorders, and hence may be beneficial in treating nonalcoholic steatohepatitis (NASH).Areas Covered: This articles briefly describes cyclophilin isomerases and the main classes of cyclophilin antagonists; it then summarizes data showing cyclophilin participation in the major pathophysiological activities that occur in NASH.Expert Opinion: Optimization of therapeutic outcomes in the treatment of NASH may be best realized by targeting multiple pathologic pathways, especially when treating advanced stages of the disease. A preferred approach for achieving this goal is to use compounds such as cyclophilin inhibitors that simultaneously target multiple disease processes. The pleiotropic benefits of this drug class derive from the extraordinary functionality of prolyl isomerization as a regulatory mechanism and its evolutionary diversification into many biochemical pathways. Nonimmunosuppressive analogs of cyclosporine A are the most thoroughly characterized cyclophilin inhibitors and show significant potential to attenuate several of the major pathophysiological events in NASH - mitochondrial dysfunction, cellular injury and death, inflammation, and in particular, fibrosis.
Assuntos
Ciclofilinas/antagonistas & inibidores , Inibidores Enzimáticos/uso terapêutico , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Animais , Ciclofilinas/metabolismo , Ciclosporinas/farmacologia , Ciclosporinas/uso terapêutico , Desenvolvimento de Medicamentos , Inibidores Enzimáticos/farmacologia , Humanos , Hepatopatia Gordurosa não Alcoólica/fisiopatologiaRESUMO
Previous studies show that cyclophilins contribute to many pathologic processes, and cyclophilin inhibitors demonstrate therapeutic activities in many experimental models. However, no drug with cyclophilin inhibition as the primary mode of action has advanced completely through clinical development to market. In this study, we present findings on the cyclophilin inhibitor, CRV431, that highlight its potential as a drug candidate for chronic liver diseases. CRV431 was found to potently inhibit all cyclophilin isoforms tested-A, B, D, and G. Inhibitory constant or IC50 values ranged from 1 to 7 nM, which was up to 13 times more potent than the parent compound, cyclosporine A (CsA), from which CRV431 was derived. Other CRV431 advantages over CsA as a nontransplant drug candidate were significantly diminished immunosuppressive activity, less drug transporter inhibition, and reduced cytotoxicity potential. Oral dosing to mice and rats led to good blood exposures and a 5- to 15-fold accumulation of CRV431 in liver compared with blood concentrations across a wide range of CRV431 dosing levels. Most importantly, CRV431 decreased liver fibrosis in a 6-week carbon tetrachloride model and in a mouse model of nonalcoholic steatohepatitis (NASH). Additionally, CRV431 administration during a late, oncogenic stage of the NASH disease model resulted in a 50% reduction in the number and size of liver tumors. These findings are consistent with CRV431 targeting fibrosis and cancer through multiple, cyclophilin-mediated mechanisms and support the development of CRV431 as a safe and effective drug candidate for liver diseases. SIGNIFICANCE STATEMENT: Cyclophilin inhibitors have demonstrated therapeutic activities in many disease models, but no drug candidates have yet advanced completely through development to market. In this study, CRV431 is shown to potently inhibit multiple cyclophilin isoforms, possess several optimized pharmacological properties, and decrease liver fibrosis and tumors in mouse models of chronic liver disease, which highlights its potential to be the first approved drug primarily targeting cyclophilin isomerases.
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Ciclofilinas/antagonistas & inibidores , Ciclosporinas/uso terapêutico , Modelos Animais de Doenças , Doença Hepática Terminal/tratamento farmacológico , Cirrose Hepática/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Ciclosporinas/farmacologia , Relação Dose-Resposta a Droga , Doença Hepática Terminal/patologia , Feminino , Humanos , Células Jurkat , Cirrose Hepática/patologia , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Ratos , Ratos Sprague-Dawley , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/fisiologiaRESUMO
Hepatitis B virus (HBV) infection is a major health burden worldwide with 240 million chronically infected individuals. Nucleos(t)ide analogs and interferons are the current standards of care due to their suppression of HBV replication, but the treatments rarely eradicate HBV from individuals. Similar to current treatments for human immunodeficiency virus type-1 (HIV-1) and hepatitis C virus (HCV) patients, improved HBV therapies will require the combination of multiple drugs which target distinct steps of the HBV life cycle. In this study, we tested the potential of a cyclophilin inhibitor, CRV431, to affect HBV replication in transgenic mice. We found that oral treatment with CRV431 (50 mg/kg/day) for a period of 16 days significantly reduced liver HBV DNA levels and moderately decreased serum HBsAg levels. We observed an additive inhibitory effect on liver HBV DNA levels in mice treated with a combination of low doses of CRV431 (10 mg/kg/day) and the nucleotide prodrug, tenofovir exalidex (TXL), (5 mg/kg/day). No toxicity was observed in CRV431-treated mice. Although it is well known that CRV431 neutralizes the peptidyl-prolyl isomerase activity of cyclophilins, its anti-HBV mechanism(s) of action remains unknown. Nevertheless, this study provides the first demonstration of a beneficial effect of a cyclophilin inhibitor in vivo in an HBV transgenic mouse model. Altogether our data reveal the potential of CRV431 to be part of improved new therapies for HBV patients.
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Antivirais/farmacologia , Ciclofilinas/antagonistas & inibidores , Ciclosporinas/farmacologia , DNA Viral/efeitos dos fármacos , Antígenos de Superfície da Hepatite B/metabolismo , Vírus da Hepatite B/efeitos dos fármacos , Animais , Feminino , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Hepacivirus/efeitos dos fármacos , Hepatite B/tratamento farmacológico , Hepatite B/virologia , Hepatite B Crônica/tratamento farmacológico , Hepatite B Crônica/virologia , Hepatite C/tratamento farmacológico , Hepatite C/virologia , Humanos , Fígado/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Nucleotídeos/farmacologia , Pró-Fármacos/farmacologia , Replicação Viral/efeitos dos fármacosRESUMO
Overcoming drug resistance in ovarian cancer is the overarching goal in gynecologic oncology. One way to increase drug cytotoxicity without increasing the drug dose is to simultaneously apply multidrug resistance modulator. Valspodar is the second generation P-glycoprotein 1 modulator capable of reversing multidrug resistance in different cancers. In this study, we evaluated the effect of valspodar and cisplatin co-treatment on cell viability, cell death and oxidative status in ovarian cancer cells. Two human ovarian cancer cell lines SK-OV-3 and MDAH-2774 were treated with cisplatin, valspodar, or cisplatin + valspodar for 24 or 48 hours. Untreated cells were used as control group. Cell viability was evaluated by MTT assay. Cell death was assessed by TUNEL and comet assay. Lipid peroxidation (malondialdehyde) and protein thiol groups were analyzed as oxidative stress markers. The expression of mitochondrial superoxide dismutase (MnSOD) was assessed by immunocytochemistry. Valspodar effectively reduced the resistance of SK-OV-3 cells to cisplatin, as demonstrated by increased oxidative stress, decreased cell viability and increased apoptosis in SK-OV-3 cells co-treated with valspodar and cisplatin compared to other groups. However, valspodar did not significantly affect the resistance of MDAH-2774 cells to cisplatin. Stronger staining for MnSOD in MDAH-2774 vs. SK-OV-3 cells after co-treatment with cisplatin and valspodar may determine the resistance of MDAH-2774 cell line to cisplatin.
Assuntos
Antineoplásicos/farmacologia , Ciclosporinas/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cisplatino/uso terapêutico , Ensaio Cometa , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Compostos de Sulfidrila/metabolismo , Superóxido Dismutase/metabolismoRESUMO
Four novel benzophenone derivatives, cytosporins A-D (1-4), hemiterpene-conjugated phenolics with an unprecedented benzo[b][1,5]dioxocane skeleton, were isolated from Cytospora rhizophorae A761. The structures of the new compounds were fully characterized on the basis of extensive spectroscopic analysis. The deduced structure represents the first example of natural meroterpenoids which bear a benzo[b][1,5]dioxocane framework embodying hemiterpene and benzophenone moieties. Moreover, compounds 1-4 were evaluated for in vitro antimicrobial activity.