La evidencia en tiempos de coronavirus (COVID-19) / Evidence in times of coronavirus (COVID-19)

La pandemia de COVID-19 está generando información epidemiológica y clínica en una escala sin precedentes para una enfermedad de reciente aparición. Aunque ya se han iniciado numerosos ensayos clínicos de fármacos antiguos y nuevos como potenciales antivirales específicos, la mayor parte de la información publicada hasta ahora carece de los controles básicos para la evaluación de la eficacia de un medicamento. Los medios de comunicación amplifican estos resultados preliminares y suman presión a los médicos asistenciales y a los decisores de políticas públicas. Este artículo revisa las pruebas disponibles sobre los cuatro tratamientos antivirales específicos más prometedores: hidroxicloroquina, lopinavir/ritonavir, remdesvir e interferones alfa y beta. Se comprueba en todos ellos que no hay demostración suficiente de eficacia como para recomendar su uso fuera de una investigación experimental adecuadamente controlada. En el uso individual de un medicamento no hay forma de saber si está beneficiando o perjudicando al paciente. Es erróneo asumir que la eventual curación se debe al fármaco y un mal desenlace debe atribuirse a la enfermedad. Sólo la comparación entre grupos de pacientes asignados al tratamiento experimental o a un control adecuado permite conocer la eficacia y seguridad de las intervenciones. El desafío es conciliar la urgencia de actuar con la generación de nuevos conocimientos.Aunque no resulta sencillo organizar ensayos clínicos en este contexto, las instituciones pueden sumarse a los proyectos en marcha a nivel nacional e internacional. El uso de estos fármacos debe considerarse experimental, por lo que es necesario obtener el consentimiento informado del paciente. (AU)

The COVID-19 pandemic is generating epidemiological and clinical information on an unprecedented scale for a newly emerging disease. Although numerous clinical trials of old and new drugs as potential specific antivirals have already been started, most of the information published so far lacks basic controls for evaluating drug efficacy. The media amplify these preliminary results and add pressure to doctors and policymakers. This article reviews the available evidence for the four most promising specific antiviral treatments: hydroxychloroquine, lopinavir / ritonavir, remdesvir, and alpha and beta interferons. The findings show that none of them has sufficient demonstration of efficacy to recommend its use outside ofthe adequately controlled experimental study. In the individual use of a drug there is no way of knowing if it is benefiting orharming the patient. It is wrong to assume that the eventual cure is due to the drug and a poor outcome must be attributed to the disease. Only the comparison between groups of patients assigned to the experimental treatment or to an adequate control can establish the efficacy and safety of the interventions. The challenge is to reconcile the urgency to act with the generation of new knowledge. Although it is not easy to organize clinical trials in this context, the institutions can join theongoing projects at the national and international levels. The use of these drugs should be considered experimental, so it is necessary to obtain the informed consent of the patient. (AU)

Antiretroviral drugs saquinavir and ritonavir reduce inhibitory concentration values of itraconazole against Histoplasma capsulatum strains in vitro

Abstract Recent studies have shown that some drugs that are not routinely used to treat fungal infections have antifungal activity, such as protease inhibitor antiretroviral drugs. This study investigated the in vitro susceptibility of Histoplasma capsulatum var. capsulatum to saquinavir and ritonavir, and its combination with the antifungal itraconazole. The susceptibility assay was performed according to Clinical and Laboratory Standards Institute guidelines. All strains were inhibited by the protease inhibitor antiretroviral drugs. Saquinavir showed minimum inhibitory concentrations ranging from 0.125 to 1 μg mL−1 for both phases, and ritonavir presented minimum inhibitory concentrations ranging from 0.0312 to 4 μg mL−1and from 0.0625 to 1 μg mL−1 for filamentous and yeast phase, respectively. Concerning the antifungal itraconazole, the minimum inhibitory concentration values ranged from 0.0019 to 0.125 μg mL−1 and from 0.0039 to 0.0312 μg mL−1 for the filamentous and yeast phase, respectively. The combination of saquinavir or ritonavir with itraconazole was synergistic against H. capsulatum, with a significant reduction in the minimum inhibitory concentrations of both drugs against the strains (p < 0.05). These data show an important in vitro synergy between protease inhibitors and itraconazole against the fungus H. capsulatum.

A novel validated UPLC method for quantitation of lopinavir and ritonavir in bulk drug and pharmaceutical formulation with its impurities

A simple gradient Ultra Performance liquid chromatographic method (UPLC) was developed for determination of lopinavir and ritonavir from its related impurities and assay for the first time. This method involves the use of a C18 (Acquity UPLC BEH C18, 50 × 2.1 mm, 1.7 µm) column thermostated at 30 oC using triethylamine (pH 2.2): 0.1% H3PO4 in acetonitrile and methanol (85:15) as mobile phase in gradient elution mode. A Photo Diode Array (PDA) detector set at 215 nm was used for detection with flow rate 0.4 mL/min. This method was validated over the range of limit of quantitation (LOQ) to 50 to 150% of impurity specification limit and of working concentration for assay. The developed method was validated for linearity, range, precision, accuracy and specificity. This method was successfully applied for content determination of lopinavir and ritonavir in pharmaceutical formulations. This method can be conveniently used in quality control laboratory for routine analysis for assay and related substances as well as for evaluation of stability samples of bulk drugs and pharmaceutical formulations.

Desenvolveu-se, pela primeira vez, método de cromatografia líquida de ultra-eficiência (UPLC), com gradiente simples, para a determinação de lopinavir e ritonavir e suas impurezas relativas. Esse método envolve o uso de C18 (Acquity UPLC BEH C18, 50 × 2,1 mm, 1,7 µm), termostatizada a 30 oC, utilizando-se trietilamina (pH 2,2) e fase móvel de H3PO4 0,1% em acetonitrila e metanol (85:15), em eluição por gradiente. Detector de arranjo de fotodiiodo (PDA), fixado a 215 nm, foi utilizado para a detecção, com fluxo de 0,4 mL/min. Esse método foi validado em faixa de limite de quantificação (LOQ) de 50 a 150% de limite de impureza e da concentração de trabalho para o ensaio. O método desenvolvido foi validado quanto à linearidade, faixa, precisão, exatidão e especificidade. Esse método foi aplicado com sucesso para a determinação de lopinavir e de ritonavir em formulações farmacêuticas. Tal método pode ser convenientemente utilizado em laboratório de controle de qualidade, não só para análise de rotina e ensaio de substâncias relacionadas, como também para a avaliação da estabilidade de amostras a granel ou em formulações farmacêuticas.

Análise morfológica e fisiológica dos fígados e rins de ratas prenhes e seus fetos tratados pela associação zidovudina, lamivudina e ritonavir durante toda a prenhez / Morphological and physiological analysis of livers and kidneys of pregnant rats and their fetuses treated by the association of zidovudine, lamivudine and ritonavir for the whole period of pregnancy

OBJETIVO: avaliar os efeitos da administração da associação zidovudina-lamivudina-ritonavir nos fígados e rins de ratas prenhes e seus conceptos do ponto de vista morfológico e fisiológico. MÉTODOS: 40 ratas albinas prenhes foram aleatoriamente divididas em 4 grupos: 1 controle (Ctrl: controle de veículo) e 3 experimentais (Exp1x, Exp3x e Exp9x). Estes últimos foram tratados por solução oral de zidovudina/lamivudina/ritonavir (Exp1x: 10/5/20 mg/kg; Exp3x: 30/15/60 mg/kg; Exp9x: 90/45/180 mg/kg). As drogas e o veículo foram administrados por gavagem, desde o 1º até o 20º dia de prenhez. No último dia do experimento, todos os animais foram anestesiados e sangue foi retirado da cavidade cardíaca para avaliação sérica das enzimas aspartato aminotransferase (AST) e alanina aminotransferase (ALT), por método calorimétrico, bem como da ureia, determinada por método cinético-enzimático, e creatinina, por método cinético-colorimétrico. Em seguida, fragmentos dos fígados e rins maternos e fetais foram coletados, fixados em formol a 10 por cento e processados segundo os métodos histológicos para inclusão em parafina. Cortes com 5 µm de espessura foram corados pela hematoxilina-eosina (HE) e analisados por microscopia de luz. Na leitura das lâminas, considerou-se o padrão de normalidade para fígado e rins, tais como: hepatócitos, espaço porta íntegros e veias hepáticas bem definidas. Nos rins, a presença de corpúsculos renais, túbulos contorcidos e alças de Henle típicos. Nos fígados fetais considerou-se, ainda, a morfologia das células da linhagem eritrocitária nas diferentes fases do desenvolvimento, bem como os megacariócitos. Quando houve alteração da coloração padrão estabelecida para as estruturas hepáticas e renais, alteração na morfologia de núcleos, rompimento de limites de alguma organela citoplasmática, presença de congestão vascular, tudo isso foi entendido como provavelmente provocado pelas drogas em sua(s) dose(s) de aplicação. A avaliação estatística foi realizada por análise de variância (ANOVA), completada pelo teste de Tukey-Kramer (p<0,05). RESULTADOS: os fígados maternos dos grupos Ctrl, Exp1x e Exp3x mostraram hepatócitos típicos, espaço porta íntegros e veias hepáticas com aspecto normal. No fígado materno do grupo Exp9x, foram encontrados hepatócitos com sinais de atrofia e apoptose (eosinofilia citoplasmática e núcleos picnóticos). Além disso, identificou-se vasodilatação dos capilares sinusoides (congestão). Os rins maternos dos grupos Ctrl e Exp1x apresentaram-se normais, com corpúsculos renais, túbulos contorcidos e alças de Henle típicos. Já nos grupos Exp3x e Exp9x, foram encontrados congestão vascular, glomérulos pequenos ricos em células contendo núcleos hipercromáticos, sendo mais intensos no Exp9x. Com relação aos fígados e rins fetais, não foram observadas alterações morfológicas ou fisiológicas nos grupos estudados. Encontrou-se aumento significante nos níveis da AST (305,70±55,80; p<0,05) e da creatinina (0,50±0,09; p<0,05) no grupo Exp9x. CONCLUSÕES: nossos resultados evidenciam que a administração da associação zidovudina/lamivudina/ritonavir a ratas prenhes em altas doses causa alterações morfológicas e funcionais nos fígados e rins maternos. Não houve alterações nem morfológicas nem fisiológicas nos fígados e rins fetais.

PURPOSE: to evaluate the effect of administration of three different doses of the zidovudine/lamivudine/ritonavir combination on the liver and kidneys of pregnant rats and their concepts from a morphological and physiological standpoint. METHODS: 40 pregnant EPM-1 Wistar rats were randomly divided into 4 groups: 1 control (Ctrl: drug vehicle control, n=10) and 3 experimental groups: Exp1x, Exp3x and Exp9x. An oral solution of the zidovudine/lamivudine/ritonavir combination was administered to the experimental groups from the day 0 to day 20 of pregnancy: Exp1x=10/5/20 mg/kg; Exp3x=30/15/60 mg/kg; Exp9x=90/45/180 mg/kg. On the 20th pregnancy day the rats were anesthetized and blood was taken directly from the ventricular chambers for further biochemical determinations: aspartate-(AST) and alanine-(ALT) aminotransferases (Calorimetric method), urea nitrogen (BUN) by an enzymatic-kinetic method, and creatinine by a kinetic-calorimetric method. Maternal and fetal liver and kidney samples were taken, fixed in 10 percent formaldehyde and processed histologically for paraffin embedding. Five µm-thick fragments of maternal and fetal livers and kidneys were stained with hematoxilyn-eosin, being analyzed by light microscopy. To interpret the results, the well-known pattern of normality for livers and kidneys was considered on the basis of the following structures: hepatocytes, portal structure, hepatic veins, renal corpuscles, renal tubules and loop of Henle. Regarding the fetal livers, we also considered the erythrocytes in their different stages of development as well as the megacariocytes. If there was a change in the established staining pattern for liver and kidney structures, changes in nuclear morphology, rupture of some cytoplasmic organelles, and presence of vascular congestion, this was considered to be due to the drug doses. Results were submitted to analysis of variance (ANOVA) and to the Tukey-Kramer multiple comparisons test (p<0.05). RESULTS: no morphological changes were observed in the maternal livers of the Ctrl, Exp1x and Exp3x groups. In the maternal liver of the Exp9x group, hepatocytes showed signs of atrophy and apoptosis (eosinophilic cytoplasm and pycnotic nuclei) and marked sinusoid capillary vasodilation (congestion) was observed. The maternal kidneys of the Ctrl and Exp1x groups were normal, with renal corpuscles, convoluted tubules and typical loops of Henle. In contrast, the Exp3x and Exp9x groups showed vascular congestion and small glomeruli rich in cells containing hyperchromatic nuclei which were more intense in Exp9x. Regarding the fetal organs, no morphological or physiological changes were observed. A significant increase of AST (305.70±55.80, p<0.05) and creatinine (0.50±0.09, p<0.05) was observed in group Exp9x. CONCLUSIONS: our results show that the administration of the zidovudine, lamivudine and ritonavir combination to pregnant rats at high doses caused morphological and physiological changes in the maternal liver and kidneys. On the other hand, there were no changes in fetal organs.

Clinical evaluation of the efficacy and tolerability of ritonavir in combination with two reverse transcriptase inhibitors

A retrospective study of 76 patients was carried out using ritonavir in an antiretroviral regimen combined with two reverse transcriptase inhibitors to treat outpatients from July, 1996, to April, 1998, with the objective of evaluating clinical efficacy and tolerability,. Seventy-six percent of the patients had been diagnosed with AIDS, an average number of CD4 cells =233.7 cells/mmü and viral load = 144,084 RNA copies/mmü. The majority of patients (76.3 percent) were antiretroviral treatment-experienced, 21.4 percent having taken protease inhibitors. A positive clinical response was found in 86.7 percent (including an average weight gain of 4.41kg in 58.5 percent), an average CD4 count increase of 169.5 cells/mmü in 83.3 percent and an average viral load decrease of approximately 2.28 log in 75 percent of patients. A high percentage of adverse effects (76.3 percent) was detected, with most slight or moderate, but they significantly impacted adherence to treatment as 31.6 percent stopped taking the drug as a result. We conclude that this antiretroviral regimen has good clinical efficacy, but relatively poor tolerability.

Safety and efficacy of reduced doses of ritonavir (RTV) plus saquinavir (SQV) in the treatment of AIDS patients in Brazil

The use of reduced doses of Ritonavir (RIT) and Saquinavir (SQV) is considered a potent alternative in treating patients infected by HIV-1. We tested a combination of 300mg of RIT plus 600mg of SQV, twice daily, in association with two reverse transcriptase inhibitors to treat AIDS patients for a period of 6 monts. Evaluation of HIV-1 RNA plasma levels, CD4+/CD8+ cell count and biochemical/hematological parameters (liver enzymes, serum electrolytes, creatinin, blood glucose, uric acid, white blood cell count, platelet count, and hemoglobin level) were performed after 30, 90 and 180 days of therapy. Clinical failure and adverse reactions were also recorded in order to assess safety and efficacy of the treatment. A total of 30 AIDS patients (25 male; 5 female) were enrolled in the study. Eight patients discontinuede the therapy due to intolerance, 2 patients presented clinical failure (onset of AIDS defining events during the study period), 2 patients were excluded due to protocol violation. Five patients tolerated only a lower dose of RIT (400mg/day). Patients who completed 6 months of therapy had a drop in viral load from 4.8ñ.7log10median4.9log) to 3.4ñ1.0log10(median 2.6log), and an increase in CD4+ count from 109ñ86 cells/ml(median 84 cells/ml) to 249ñ114 cells/ml(median 265cells/ml), compared to baseline values. However, patients who used a lower dose of RIT (400mg/day) had a less impressive drop in viral load values(mean0.6log10RNA copies/ml) when compared with those using the 600mg/day of the drug(mean 2.4log10). The percentage of patients presenting undetectable levels of HIV-1 RNA in plasma was quite different for the 2 groups: 92 percent of patients with a viral load <400 RNA copies/ml were using 600mg of RIT. The combination of reduced doses of RIT and SQV reduced viral load >1.0log10 after 6 months in 83 percent of study patients. The dose of 600mg/day of RIT was more effective in reducing viral load than 400mg/day, but was less well-tolerated. CD4+ cell counts increased in all patients regardless of the RIT dose used.