In vitro effectivity of three approved drugs and their synergistic interaction against Leishmania infantum. / Efectividad in vitro de tres fármacos aprobados y su interacción sinérgica contra Leishmania infantum

INTRODUCTION: Leishmaniasis remains one of the neglected tropical diseases. Repurposing existing drugs has proven to be successful for treating neglected tropical diseases while combination therapy is a strategic alternative for the treatment of infectious diseases. Auranofin, lopinavir/ritonavir, and sorafenib are FDA approved drugs used in the treatment of diverse diseases by acting on different essential biological enzymes. OBJECTIVE: To evaluate the effects of monotherapy and combined therapies with the three drugs against Leishmania infantum. MATERIALS AND METHODS: We compared the leishmanicidal effects of the three drugs on promastigotes in vitro as regards the parasite count, the drug concentration providing a half-maximal response, and the ultrastructural changes of the parasite. We determined the fractional inhibitory concentration index of combined drugs in two ways, as well as the activity of the three drugs together to establish their synergetic effect. RESULTS: The monotherapy with the three drugs was effective with auranofin showing the best leishmanicidal effect (EC50=1.5 µM), whereas sorafinib reduced parasite growth at EC50=2.5 µM. The scanning electron microscopy of promastigotes from all treated media showed distortion in the shape with loss of flagella and bleb formation. Acidocalcinosis was evident by transmission electron microscopy with all treatments suggesting apoptosis. Treatment with lopinavir/ritonavir showed signs of autophagy. The two-way combination of the drugs led to additive interactions while the combination of the three drugs showed synergistic action. CONCLUSION: Each drug when used as monotherapy against Leishmania spp. was effective, but the combination therapy was more effective than the individual drugs due to the additive or synergistic effects.

Introducción. La leishmaniasis sigue siendo una de las enfermedades tropicales desatendidas. La reutilización de medicamentos existentes ha demostrado ser exitosa para tratar enfermedades tropicales desatendidas y la terapia combinada es una alternativa estratégica para el tratamiento de enfermedades infecciosas. Auranofin, lopinavir/ritonavir y sorafenib son medicamentos aprobados por la Food and Drug Administration (FDA) de Estados Unidos utilizados en el tratamiento de diversas enfermedades, pues actúan sobre diferentes enzimas biológicas esenciales. Objetivo. Evaluar los efectos terapéuticos de la monoterapia y de los tres fármacos combinados contra Leishmania infantum. Materiales y métodos. Los efectos leishmanicidas de los tres fármacos sobre los promastigotes se compararon in vitro en cuanto al recuento de parásitos, la concentración del fármaco que proporcionara una respuesta semimáxima y los cambios ultraestructurales del parásito. Se calculó el índice de concentración inhibitoria de fracciones de fármacos combinados de dos maneras y la actividad de los tres fármacos juntos para determinar el efecto sinérgico. Resultados. La monoterapia con los tres medicamentos fue efectiva, pero la auranofina tuvo el mejor efecto antileishmanicida con un CE50 de 1,5 µM, en tanto que el sorafinib redujo el crecimiento del parásito con un CE50 de 2,5 µM. La microscopía electrónica de barrido de promastigotes de todos los medios tratados mostró una distorsión en la forma, con pérdida de flagelos y formación de ampollas. La acidocalcinosis fue evidente por microscopía electrónica de transmisión con todos los tratamientos, lo que sugiere apoptosis. El tratamiento con lopinavir/ritonavir mostró signos de autofagia. La combinación de dos medicamentos condujo a interacciones aditivas, mientras que la combinación de las tres drogas produjo una acción sinérgica. Conclusión. Los tres medicamentos usados como monoterapia contra Leishmania spp. fueron efectivos, pero el tratamiento combinado lo fue en mayor medida debido a los efectos aditivos o sinérgicos.

Antiarthritic synergism of combined oral and parenteral chrysotherapy. II. Increased inhibition of activated leukocyte oxygen burst by combined gold action.

We have observed an antiarthritic effect of combined chrysotherapy in adjuvant arthritis. Since superoxide radicals (O2-) are potent mediators of rheumatoid inflammation, we studied the combined effect of auranofin (AF) and injectable golds on luminol-dependent chemiluminescence (LDCL) and O2- generation by cytochrome-c reduction of activated leukocytes by different receptor-mediated stimuli: phorbol myristic acetate, 10(-6) M; f-Met-Leu-Phe, 10(-6) M; and poly-L-histidine, 10(-5) M. AF, 0.6 and 0.9 micrograms Au/ml, inhibited 34 and 58% of O2- generation, respectively; the addition to AF of 0.3 micrograms Au/ml of gold thiosulfate (GTS) increased this inhibition to 84 and 97% of the oxygen burst. Similar synergistic potentiation inhibition was obtained by LDCL. When the inhibition of O2- generation by the combined action of AF and GTS was compared with AF + gold sodium thiomalate (GTM), only GTS showed an activation on AF's inhibition of the oxygen burst of human leukocytes. The ligand thiosulfate in equimolar concentrations to GTS had a statistically significant (P less than 0.01) inhibitory effect on AF's blockade of O2- generation during the first 5 min of the interaction with the PMNs; thiomalate had no effect. Sequential pretreatment of PMNs with AF and GTS on O2- generation revealed that for synergism of combined gold action to take place, the cell membrane had to be subjected first to the action of oral gold or to the simultaneous combined action of oral and parenteral gold.(ABSTRACT TRUNCATED AT 250 WORDS)

Antiarthritic synergism of combined oral and parenteral chrysotherapy. I. Studies in adjuvant-induced arthritis in rats.

In comparative clinical studies of auranofin (AF, oral gold) and parenteral gold in the treatment of rheumatoid arthritis, no difference in efficacy was detected. Since the pharmacologic profiles of these compounds are different, we studied their combined effect on adjuvant arthritis (AA). The effect of AF alone and combined with gold sodium thiomalate (GTM) or gold sodium thiosulfate (GTS) on the excretion of urinary hydroxyproline (UHP) and urinary calcium (UCa), and the articular index of arthritic rats was followed during five weeks of treatment. The excretion of UHP and UCa was significantly inhibited (P less than 0.005) in rats treated with AF combined with GTM or GTS as compared with animals treated with the individual gold compounds. However, the articular index only decreased significantly (P less than 0.02) in the group of rats treated with AF + GTS. The present studies open the possibility that combined treatment with oral and injectable gold provide a new approach for chrysotherapy with an increased antiarthritic potency.

Acción de la auroterapia oral sobre la respuesta inmune de pacientes con artritis reumatoidea / Action of oral aurotherapy in immune response of rheumatoid arthritis patients

Se han estudiado las respuestas inmunes celular (RIC) y humoral (RIH) en 13 pacientes afectados de artritis reumatoides (AR) "definida" o "clásica", antes y después de 6 a 12 meses de tratamiento con 6mg/día de auranofin. Se ha observado un aumento en la frecuencia de positividad de algunas pruebas cutáneas (PPD, PHA, candidina) y de estimulación cutánea con DNCB, una reducción o negativización de los títulos de factor reumatoideo y del número de linfocitos B (rosetas EAC) y una normalización de los niveles séricos de IgG y complemento. Se concluye que el auranofin es capaz de influir positivamente en la RIC y normalizar algunos parámetros de la RIH, actuando como un inmunomodulador en el tratamiento de la AR