RESUMEN
OBJECTIVES: Efavirenz (EFV), a non-nucleoside reverse transcriptase inhibitor, is a drug that is frequently included in highly active antiretroviral therapy for treatment of HIV infection. Decreased bioavailability and increased toxicity limit its use. We report a formulation of efavirenz-loaded lactoferrin nanoparticles (lacto-EFV-nano) for oral delivery which exhibited significantly improved pharmacological properties coupled with reduced toxicity compared with its free form. METHODS: Lacto-EFV-nano was prepared using the Sol-oil protocol and characterized using various sources of characterization. In vitro and in vivo studies were performed to test the stability, safety, efficacy, biodistribution and pharmacokinetics of lacto-EFV-nano. RESULTS: The nanoparticles prepared for the present study had an average size of 45-60 nm as revealed by field emission scanning electron microscope measurements. Further, dynamic light scattering data showed a hydrodynamic radius of 103 ± 5.3 nm, a zeta potential of -23 ± 1.2 mV and a polydispersity index of < 0.341. Lacto-EFV-nano was found to be stable as assessed using differential scanning calorimetry and Fourier-transform infrared spectroscopy. Cell viability studies showed that lacto-EFV-nano was at least 2-fold less toxic to peripheral blood mononuclear cells, Jurkat T cell and B16-F10 cell lines than free EFV. Furthermore, lacto-EFV-nano [50% inhibitory concentration (IC50 ) < 1.1 nM] showed > 2-fold enhanced anti-HIV-1 activity compared with free EFV (IC50 = 2.56 nM). Lacto-EFV-nano exhibited improved oral bioavailability and an improved in vivo pharmacokinetic profile, with a > 3-4-fold increase in the area under the plasma concentration-time curve (AUC), a 6-7-fold increase in the area under the first moment curve (AUMC), a > 30% increase in the peak plasma concentration of the drug after oral administration (Cmax ) and a 2-fold increase in the time to reach Cmax (Tmax ) and the time required for the concentration of the drug to reach half of its original value (t1/2 ). Furthermore, lacto-EFV-nano did not show any organ-related toxicity. A significant decrease in the concentrations of various parameters, elevated concentrations of which are markers of reduced safety, were also observed in rats treated with lacto-EFV-nano. CONCLUSIONS: Compared with free EFV, lacto-EFV-nano is a promising oral nanoformulation with enhanced bioavailability and efficacy of EFV and improved safety.
Asunto(s)
Antiinfecciosos/administración & dosificación , Antiinfecciosos/farmacocinética , Benzoxazinas/administración & dosificación , Benzoxazinas/farmacocinética , Portadores de Fármacos/administración & dosificación , Lactoferrina/administración & dosificación , Nanopartículas/administración & dosificación , Administración Oral , Alquinos , Animales , Antiinfecciosos/efectos adversos , Benzoxazinas/efectos adversos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Ciclopropanos , Portadores de Fármacos/efectos adversos , Femenino , Humanos , Lactoferrina/efectos adversos , Masculino , Nanopartículas/efectos adversos , Ratas WistarRESUMEN
In an attempt to establish the criteria for the length of simulation to achieve the desired convergence of free energy calculations, two studies were carried out on chosen complexes of FBPase-AMP mimics. Calculations were performed for varied length of simulations and for different starting configurations using both conventional- and QM/MM-FEP methods. The results demonstrate that for small perturbations, 1248 ps simulation time could be regarded a reasonable yardstick to achieve convergence of the results. As the simulation time is extended, the errors associated with free energy calculations also gradually tapers off. Moreover, when starting the simulation from different initial configurations of the systems, the results are not changed significantly, when performed for 1248 ps. This study carried on FBPase-AMP mimics corroborates well with our previous successful demonstration of requirement of simulation time for solvation studies, both by conventional and ab initio FEP. The establishment of aforementioned criteria of simulation length serves a useful benchmark in drug design efforts using FEP methodologies, to draw a meaningful and unequivocal conclusion.
Asunto(s)
Adenosina Monofosfato/análogos & derivados , Diabetes Mellitus/enzimología , Fructosa-Bifosfatasa/antagonistas & inhibidores , Adenosina Monofosfato/farmacología , Diseño de Fármacos , Simulación de Dinámica Molecular , TermodinámicaRESUMEN
Trans-imidazolium (bis imidazole) tetrachloro ruthenate (RuIm) and trans-indazolium (bis indazole) tetrachloro ruthenate (RuInd) are ruthenium coordination complexes, which were first synthesized and exploited for their anticancer activity. These molecules constitute two of the few most effective anticancer ruthenium compounds. The clinical use of these compounds however was hindered due to toxic side effects on the human body. Our present study on topoisomerase II poisoning by these compounds shows that they effectively poison the activity of topoisomerase II by forming a ternary cleavage complex of DNA, drug and topoisomerase II. The thymidine incorporation assays show that the inhibition of cancer cell proliferation correlates with topoisomerase II poisoning. The present study on topoisomerase II poisoning by these two compounds opens a new avenue for renewing further research on these compounds. This is because they could be effective lead candidates for the development of more potent and less toxic ruthenium containing topoisomerase II poisons. Specificity of action on this molecular target may reduce the toxic effects of these ruthenium-containing molecules and thus improve their therapeutic index.
Asunto(s)
ADN/metabolismo , Imidazoles/farmacología , Indazoles/farmacología , Compuestos Organometálicos/farmacología , Compuestos de Rutenio/farmacología , Inhibidores de Topoisomerasa II , Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfatasas/metabolismo , Animales , Antineoplásicos/farmacología , Antineoplásicos/toxicidad , ADN/química , ADN-Topoisomerasas de Tipo II/metabolismo , Humanos , Imidazoles/toxicidad , Indazoles/toxicidad , Estructura Molecular , Conformación de Ácido Nucleico , Compuestos Organometálicos/toxicidad , Ratas , Compuestos de Rutenio/toxicidad , Timidina/metabolismo , Células Tumorales CultivadasRESUMEN
Topoisomerase II is a major molecular target for a number of DNA-binding anticancer drugs. In the present study, we report topoisomerase II inhibition and anticancer activity by four substituted ferrocene derivatives which do not bind to DNA. The first derivative, acetyl-substituted ferrocene (monoacetylferrocene), showed a minor inhibition of topoisomerase II activity along with a consequent inhibition of cancer cell proliferation. The second derivative (diacetylferrocene) showed a higher potency of action compared to the monosubstituted derivative. The third and fourth derivatives, with mono- and disubstituted carboxaldoxime groups (ferrocenecarboxaldoxime and ferrocenedicarboxaldoxime), showed a higher anticancer action and stronger topoisomerase II inhibition. To understand their molecular mechanism of action, cleavage assays were carried out to monitor the drug-induced, topoisomerase II mediated DNA cleavage. The results show that diacetylferrocene and ferrocenedicarboxaldoxime could form an enzyme-drug-DNA ternary complex, called a "cleavage complex," resulting in DNA cleavage. These results along with those of an immunoprecipitation assay indicate that the two compounds interact with topoisomerase II alone and poison its activity by trapping the enzyme and enzyme-cleaved DNA in the covalently closed cleavage complex. The formation of such a complex has numerous genetic implications, which ultimately results in neoplastic cell death.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Compuestos Ferrosos/farmacología , Inhibidores de Topoisomerasa II , Animales , Sitios de Unión , Bovinos , División Celular/efectos de los fármacos , ADN/química , ADN/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Compuestos Ferrosos/química , Humanos , Metalocenos , Termodinámica , Células Tumorales CultivadasRESUMEN
Topoisomerase II is a cellular target for a number of clinically relevant antitumor drugs. To elucidate the possible cellular target for the antiproliferation activity of cobalt salicylaldoxime (CoSAL), which inhibits 50% of leukemic cell proliferation at a concentration of 60 microM, DNA binding studies and studies of the action of this complex on topoisomerase II catalytic activities were carried out. The results from DNA binding studies show that CoSAL binds DNA strongly with a stoichiometric ratio of two drug molecules for five nucleotide bases and shows a mode of interaction similar to that of DNA groove binding agents. The results from topoisomerase II inhibition studies show that the complex inhibits the relaxation activity of topoisomerase II in a dose-dependent manner and poisons its activity through cleavage complex formation. To see if the hydroxyl group present on imine nitrogen is involved in topoisomerase II poisoning, we synthesized an analogue of CoSAL in which the hydroxyl group was replaced with semicarbazone. This complex too binds DNA with an affinity similar to that of CoSAL, but with a small difference in the mode of interaction; however, it marginally inhibits leukemic cell proliferation and does not inhibit topoisomerase II activity, which suggests the involvement of a hydroxyl group. An immunoprecipitation assay was conducted which showed that the cleavage complex formed in the presence of CoSAL contained 75% of the complex, while the other complex shows only 7. 65%. Cyclic voltametric spectra of the complexes in the presence of DNA show that they do not oxidize DNA. These results suggest that CoSAL shows a bidirectional mode of interaction with enzyme and DNA and inhibits topoisomerase II activity by forming a drug-mediated cleavage complex. Our data strongly suggest that topoisomerase II may be one of the cellular targets for antiproliferation activity of CoSAL.
Asunto(s)
ADN-Topoisomerasas de Tipo II/metabolismo , ADN/metabolismo , Hígado/citología , Compuestos Organometálicos/farmacología , Oximas/farmacología , Adenosina Trifosfatasas/metabolismo , Animales , Sitios de Unión , División Celular/efectos de los fármacos , ADN/química , ADN/efectos de los fármacos , ADN-Topoisomerasas de Tipo II/efectos de los fármacos , Cinética , Hígado/efectos de los fármacos , Hígado/enzimología , Estructura Molecular , Compuestos Organometálicos/síntesis química , Compuestos Organometálicos/química , Oximas/síntesis química , Oximas/química , Ratas , Termodinámica , Timidina/metabolismoRESUMEN
The ability of two structurally different ruthenium complexes to interfere with the catalytic activity of topoisomerase II was studied to elucidate their molecular mechanism of action and relative antineoplastic activity. The first complex, [RuCl2(C6H6)(dmso)], could completely inhibit DNA relaxation activity of topoisomerase II and form a drug-induced cleavage complex. This strongly suggests that the drug interferes with topoisomerase II activity by cleavage complex formation. The bi-directional binding of [RuCl2(C6H6)(dmso)] to DNA and topoisomerase II was verified by immunoprecipitation experiments which confirmed the presence of DNA and ruthenium in the cleavage complex. The second complex, Ruthenium Salicylaldoxime, could not inhibit topoisomerase II relaxation activity appreciably and also could not induce cleavage complex formation, though its DNA-binding characteristics and antiproliferation activity were almost comparable to those of [RuCl2(C6H6)(dmso)]. The results suggest that the difference in ligands and their orientation around a metal atom may be responsible for topoisomerase II poisoning by the first complex and not by the second. A probable mechanism is proposed for [RuCl2(C6H6)(dmso)], where the ruthenium atom interacts with DNA and ligands of the metal atom form cross-links with topoisomerase II. This may facilitate the formation of a drug-induced cleavage complex.