RESUMO
This work looks to improve the efficacy of Adriamycin (ADR) while mitigating its cardiotoxicity using combinations of micellar resveratrol (R): quercetin (Q) (mRQ) or R: curcumin (C) (mRC) in healthy mice and ovarian cancer xenograft models. Ovarian cancer cells, ES2-Luc, or A2780ADR are inoculated in mice (n =4/group) and sorted into eight cohorts. Mice are treated weekly for 4 weeks with ADR, ADR+mRQ, ADR+mRC, or controls (saline, empty micelles, ADR+EM, mRQ, or mRC). To evaluate the degree of cardioprotection, serum is collected to determine the cardiac Troponin I (cTnI). Cardiac tissue is collected for morphological evaluation and evaluation of creatine kinase levels. Our results indicate that mRQ+ADR is statistically significant in tumor reduction in xenograft models. In healthy mice, the left ventricular ejection fraction and fractional shortening in the ADR treated group is most compromised. Co-administration of mRQ with ADR can reduce ADR dosing through chemosensitization while being cardioprotective.
Assuntos
Cardiotoxicidade/tratamento farmacológico , Curcumina/uso terapêutico , Doxorrubicina/efeitos adversos , Micelas , Neoplasias Ovarianas/tratamento farmacológico , Polímeros/química , Quercetina/uso terapêutico , Resveratrol/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Cardiotoxicidade/diagnóstico por imagem , Cardiotoxicidade/fisiopatologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Curcumina/administração & dosagem , Curcumina/farmacologia , Sistemas de Liberação de Medicamentos , Feminino , Humanos , Concentração Inibidora 50 , Medições Luminescentes , Camundongos , Neoplasias Ovarianas/diagnóstico por imagem , Quercetina/administração & dosagem , Quercetina/farmacologia , Resveratrol/administração & dosagem , Resveratrol/farmacologia , Volume Sistólico/efeitos dos fármacos , Troponina I/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Successful liposomal formulations in the clinic are severely limited due to poor translational capability of the traditional bench techniques to clinical production settings. The gold standard for liposome bench manufacturing is a multi-step and parameter dependent extrusion method. Moreover, these parameters need re-optimization for clinical production. The microfluidics technique utilizes vigorous mixing of fluids at a nanoliter scale to produce liposomes in batches from milliliters to a couple liters. The fine control of process parameters results in improved reproducibility between batches. It is inherently scalable; however, the characteristics of liposomes produced by microfluidics both in vitro and in vivo have never been compared to those produced using extrusion. In this manuscript, we describe the comparison between the traditional extrusion method to microfluidics, the new paradigm in liposome production and scale-up.
Assuntos
Lipossomos/síntese química , Microfluídica/métodos , Animais , Sobrevivência Celular , Colesterol/química , Liberação Controlada de Fármacos , Feminino , Concentração Inibidora 50 , Cinética , Camundongos , Tamanho da Partícula , Soluções , Esfingomielinas/química , Testes de Toxicidade Aguda , VimblastinaRESUMO
In this work, a new sphingomyelin-cholesterol liposomal formulation (CPD100Li) for the delivery of a hypoxia activated prodrug of vinblastine, mon-N-oxide (CPD100), is developed. The optimized liposomal formulation uses an ionophore (A23187) mediated pH-gradient method. Optimized CPD100Li is characterized for size, drug loading, and stability. The in vitro toxicity of CPD100Li is assessed on different aspects of cell proliferation and apoptosis of ES2 ovarian cancer under normoxic and hypoxic conditions. The pharmacokinetics of CPD100Li in mice as well as the influence of A23187 on the retention of CPD100 are assessed. The dose limiting toxicity (DLT) and maximum tolerated dose (MTD) for CPD100Li are evaluated in nude mice. CPD100 is loaded in the liposome at 5.5â¯mg/mL. The sizes of CPD100Li using DLS, qNano and cryo-TEM techniques are 155.4⯱â¯4.2â¯nm, 132â¯nm, and 112.6⯱â¯19.8â¯nm, respectively. There is no difference between the in vitro characterization of CPD100Li with and without ionophore. Freshly prepared CPD100Li with ionophore are stable for 48â¯h at 4⯰C, while the freeze-dried formulation is stable for 3â¯months under argon at 4⯰C. The hypoxic cytotoxicity ratios (HCR) of CPD100 and CPD100Li are 0.16 and 0.11, respectively. CPD100Li under hypoxic conditions has a 9.2-fold lower IC50 value as compared to CPD100Li under normoxic conditions, confirming the hypoxia dependent activation of CPD100. CPD100Li treated ES2 cells show a time dependent enhanced cell death, along with caspase production and an increase in the number of cells in G0/G1 and higher cell arrest. The blood concentration profile of CPD100Li in mice without A23187 has a 12.6-fold lower area under the curve (AUC) and 1.6-fold lower circulation time compared to the CPD100Li with A23187. The DLT for both CPD100 and CPD100Li is 45â¯mg/kg and the MTD is 40â¯mg/kg in nude mice. Based on the preliminary data obtained, we clearly show that the presence of ionophore affects the in vivo stability of CPD100. CPD100Li presents a unique opportunity to develop a first-in-kind chemotherapy product based on achieving selective drug activation through the hypoxic physiologic microenvironment of solid tumors.
Assuntos
Neoplasias Ovarianas/tratamento farmacológico , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Vimblastina/análogos & derivados , Vimblastina/farmacologia , Animais , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Colesterol/química , Liberação Controlada de Fármacos , Feminino , Humanos , Lipossomos/química , Camundongos , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Pró-Fármacos/farmacocinética , Pró-Fármacos/uso terapêutico , Esfingomielinas/química , Hipóxia Tumoral/efeitos dos fármacos , Vimblastina/farmacocinética , Vimblastina/uso terapêuticoRESUMO
Muscle atrophy occurs during chronic diseases, resulting in diminished quality of life and compromised treatment outcomes. There is a high demand for therapeutics that increase muscle mass while abrogating the need for special dietary and exercise requirements. Therefore, we developed an efficient nanomedicine approach capable of increasing muscle mass. Methods: The therapy is based on nanoparticle-mediated delivery of follistatin messenger RNA (mRNA) to the liver after subcutaneous administration. The delivered mRNA directs hepatic cellular machinery to produce follistatin, a glycoprotein that increases lean mass through inhibition of negative regulators of muscle mass (myostatin and activin A). These factors are elevated in numerous disease states, thereby providing a target for therapeutic intervention. Results: Animal studies validated that mRNA-loaded nanoparticles enter systemic circulation following subcutaneous injection, accumulate and internalize in the liver, where the mRNA is translated into follistatin. Follistatin serum levels were elevated for 72 h post injection and efficiently reduced activin A and myostatin serum concentrations. After eight weeks of repeated injections, the lean mass of mice in the treatment group was ~10% higher when compared to that of the controls. Conclusion: Based on the obtained results demonstrating an increased muscle mass as well as restricted fat accumulation, this nanoplatform might be a milestone in the development of mRNA technologies and the treatment of muscle wasting disorders.
Assuntos
Portadores de Fármacos/administração & dosagem , Folistatina/genética , Fígado/metabolismo , Desenvolvimento Muscular/efeitos dos fármacos , Nanopartículas/administração & dosagem , RNA Mensageiro/administração & dosagem , Animais , Injeções Subcutâneas , Camundongos , Resultado do TratamentoRESUMO
Solid tumors often contain hypoxic regions which are resistant to standard chemotherapy and radiotherapy. We have developed a liposomal delivery system for a prodrug of vinblastine (CPD100) which converts to the parent compound only in the presence of lower oxygen levels. As a part of this work we have developed and optimized two formulations of CPD100: one composed of sphingomyelin/cholesterol (55/45; mol/mol) (CPD100Li) and the other composed of sphingomyelin/cholesterol/PEG (55/40/5; mol/mol) (CPD100 PEGLi). We evaluated the antiproliferative effect of CPD100 and the two formulations against A549 non-small lung cancer cell. A549 cell line showed to be sensitive to CPD100 and the two formulations displayed a higher hypoxic: air cytotoxicity ratio compared to the pro-drug. CPD100 elimination from the circulation after injection in mouse was characterized by a very short circulation time (~0.44h), lower area under the curve (AUC) (33µgh/mL) and high clearance (916mL/h/kg) and lower volume of distribution (17.4mL/kg).Total drug elimination from the circulation after the administration of liposomal formulation was characterized by prolonged circulation time (5.5h) along with increase in the AUC (56µgh/mL) for CPD100 Li and (9.5h) with AUC (170µgh/mL) for CPD100PEGLi. This was observed along with increase in volume of distribution and decrease in clearance for the liposomes. The systemic exposure of the free drug was much lower than that achieved with the liposomes. When evaluated for the efficacy in A549 xenograft model in mice, both the liposomes demonstrated excellent tumor suppression and reduction for 3months. The blood chemistry panel and the comprehensive blood analysis showed no increase or decrease in the markers and blood count. In summary, the pharmacokinetic analysis along with the efficacy data emphasis on how the delivery vehicle modifies and enhances the accumulation of the drug and at the same time the increased systemic exposure is not related to toxicity.
Assuntos
Antineoplásicos Fitogênicos/administração & dosagem , Hipóxia/metabolismo , Óxidos/metabolismo , Pró-Fármacos/administração & dosagem , Vimblastina/administração & dosagem , Células A549 , Animais , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacocinética , Antineoplásicos Fitogênicos/uso terapêutico , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Lipossomos , Dose Máxima Tolerável , Camundongos , Camundongos Nus , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Polietilenoglicóis/química , Pró-Fármacos/química , Pró-Fármacos/farmacocinética , Pró-Fármacos/uso terapêutico , Carga Tumoral/efeitos dos fármacos , Vimblastina/química , Vimblastina/farmacocinética , Vimblastina/uso terapêuticoRESUMO
Amphiphilic block copolymers like polyethyleneglycol-block-polylactic acid (PEG-b-PLA) can self-assemble into micelles above their critical micellar concentration forming hydrophobic cores surrounded by hydrophilic shells in aqueous environments. The core of these micelles can be utilized to load hydrophobic, poorly water soluble drugs like docetaxel (DTX) and everolimus (EVR). Systematic characterization of the micelle structure and drug loading capabilities are important before in vitro and in vivo studies can be conducted. The goal of the protocol described herein is to provide the necessary characterization steps to achieve standardized micellar products. DTX and EVR have intrinsic solubilities of 1.9 and 9.6 µg/ml respectively Preparation of these micelles can be achieved through solvent casting which increases the aqueous solubility of DTX and EVR to 1.86 and 1.85 mg/ml, respectively. Drug stability in micelles evaluated at room temperature over 48 hr indicates that 97% or more of the drugs are retained in solution. Micelle size was assessed using dynamic light scattering and indicated that the size of these micelles was below 50 nm and depended on the molecular weight of the polymer. Drug release from the micelles was assessed using dialysis under sink conditions at pH 7.4 at 37 (o)C over 48 hr. Curve fitting results indicate that drug release is driven by a first order process indicating that it is diffusion driven.