Dissertação (mestrado)—Universidade de Brasília, Faculdade de Medicina, 2008. / Submitted by Jaqueline Oliveira (jaqueoliveiram@gmail.com) on 2008-11-19T19:26:40Z No. of bitstreams: 1 DISSERTACAO_2008_EricaAlessandraRAlves.pdf: 1225470 bytes, checksum: 1bdf066ff4cc5dac11f9085df9118f7d (MD5) / Approved for entry into archive by Georgia Fernandes(georgia@bce.unb.br) on 2009-03-05T14:09:36Z (GMT) No. of bitstreams: 1 DISSERTACAO_2008_EricaAlessandraRAlves.pdf: 1225470 bytes, checksum: 1bdf066ff4cc5dac11f9085df9118f7d (MD5) / Made available in DSpace on 2009-03-05T14:09:36Z (GMT). No. of bitstreams: 1 DISSERTACAO_2008_EricaAlessandraRAlves.pdf: 1225470 bytes, checksum: 1bdf066ff4cc5dac11f9085df9118f7d (MD5) / A introdução da terapia antiretroviral de alta eficácia em 1996 determinou uma queda substancial da morbidade e mortalidade associadas ao Vírus da Imunodeficiência Humana (VIH). Embora a infecção seja controlada pelos antiretrovirais, observam-se efeitos colaterais indesejados, fato que demonstra a capacidade de interação destes fármacos com as células dos indivíduos que os recebem. Recentemente, estes medicamentos têm sido relacionados à modulação do sistema imunitário inato e adaptativo, entretanto pouco se sabe acerca da influência dos protocolos rotineiramente utilizados pelos indivíduos infectados por este vírus, já que a maior parte das pesquisas investiga a influência de drogas isoladas sobre o sistema imunitário. Portanto, o objetivo deste trabalho foi investigar a influência, in vitro, da zidovudina (AZT), da lamivudina (3TC), do efavirenz (EFV) e do lopinavir/ritonavir (LPV/RTV) isoladamente ou das associações AZT/3TC/EFV e AZT/3TC/LPV/RTV sobre a capacidade fagocitária e produção de ânions superóxido por neutrófilos e monócitos de indivíduos saudáveis. Foram retirados 20 ml do sangue periférico de 20 voluntários, após consentimento informado, para realização dos ensaios e avaliadas tanto a capacidade fagocitária dos neutrófilos e monócitos pelos receptores para padrões moleculares de patógenos (rPMP) quanto pelos receptores para opsoninas. Os fármacos foram utilizados nas concentrações plasmáticas máximas observadas após ingestão, sendo para o AZT 1,64 µg/ml, para o 3TC 1,5µg/ml, para o EFV 3,7 µg/ml, para o LPV 11,45 µg/ml e para o RTV 2,86 µg/ml. O índice fagocitário foi determinado pela multiplicação da média de Saccharomyces cerevisiae aderidas/ingeridas por fagócito pelo percentual de fagócitos envolvidos na fagocitose. A avaliação da capacidade oxidativa dos fagócitos foi feita pelo teste do nitroblue tetrazolium. O EFV isoladamente aumentou o percentual de neutrófilos envolvidos na fagocitose pelos rPMP (p=0,004) e para opsoninas (p=0,020) enquanto que a préincubação com LPV/RTV (p=0,024) e com a associação AZT/3TC/LPV/RTV (p=0,008) por 60 minutos diminuiu o índice fagocitário dos neutrófilos quando avaliada a capacidade fagocitária destas células pelos rPMP. A pré-incubação dos fagócitos por 30 minutos com LPV/RTV aumentou a capacidade fagocitária dos monócitos pelos rPMP (p=0,003) tanto pelo aumento da média de leveduras aderidas/ingeridas por monócito quanto pelo percentual de monócitos envolvidos na fagocitose, resultado semelhante ao da pré-incubação dos monócitos com a associação AZT/3TC/LPV/RTV por 30 minutos e tendeu a aumentar o percentual de neutrófilos envolvidos na fagocitose (p=0,077) quando avaliada a fagocitose por meio dos receptores para opsoninas. Entretanto, quando avaliada a influência do pré-tratamento dos monócitos com LPV/RTV por 60 minutos, a capacidade fagocitária destas células pelos rPMP diminuiu (p=0,013) devido ao decréscimo do percentual de monócitos envolvidos na fagocitose. Estes fármacos isoladamente também diminuíram a capacidade fagocitária dos monócitos (p=0,020) mediada por receptores para opsoninas pela diminuição da média de leveduras aderidas/ingeridas por monócito, enquanto que o AZT tendeu a aumentar a capacidade fagocitária destas células pelos receptores para padrões moleculares de patógenos (p=0,097), pois tendeu a aumentar o percentual de monócitos envolvidos na fagocitose. A produção de radicais de oxigênio foi mais abrangentemente influenciada pelos antiretrovirais estudados. O AZT (p=0,027) e o 3TC (p=0,004) isoladamente e as associações LPV/RTV (p=0,0001) e AZT/3TC/LPV/RTV (p=0,0001) diminuíram a produção estimulada do ânion superóxido pelos fagócitos. O percentual de redução do NBT basal também foi deprimido pela associação LPV/RTV (p=0,0007) e AZT/3TC/LPV/RTV (p<0,0001) e tendeu a ser reduzido pelo 3TC isoladamente (p=0,071). Os demais tratamentos não influenciaram a capacidade fagocitária ou a produção de ânions superóxido pelos fagócitos. Os resultados sugerem que o protocolo AZT/3TC/EFV, por não interferir com a capacidade fagocitária, seja mais adequado para o tratamento inicial dos indivíduos infectados pelo VIH enquanto que o protocolo contendo AZT/3TC/LPV/RTV, por sua propriedade imunomoduladora, deveria ser reservado para aqueles pacientes que apresentem intolerância ou resistência ao EFV. É possível também que pacientes com resistência a múltiplos esquemas antiretrovirais se beneficiem de protocolos que contenham LPV/RTV, pois como evidenciado diminuem a produção de radicais de oxigênio, podendo contribuir para taxas mais baixas de replicação do viral, de apoptose de linfócitos e da neurodegeneração relacionada ao VIH. __________________________________________________________________________________ ABSTRACT / The introduction of highly active antiretroviral therapy (HAART) in 1996 has decreased the morbidity and the mortality rates associated with the HIV infection. Though the infection can be controlled by antiretrovirals, these medicines may cause undesirable side effects due to their capacity to interact with cells of patients. It has been reported that these drugs may modulate the innate and adaptative immnune system. However, few is known about the influences on immune functions by protocols routinely used in the treatment of HIV infected patients, constituted by reverse transcriptase and protease inhibitors, because most of researches have just investigated the influence restricted to isolated drugs. So, this work aimed to evaluate the influence of the isolate treatment with zidovudine (AZT), lamivudine (3TC), efavirenz (EFV), lopinavir/ritonavir (LPV/RTV) and of the associations AZT/3TC/EFV or AZT/3TC/LPV/RTV on phagocytic capacity and superoxide production by neutrophils and monocytes of healthy individuals, in vitro. It was collected 20 ml of peripheral blood from 20 healthy volunteers and the phagocytic capacity was assessed through pathogen-associated molecular patterns receptors (PAMPr) and through opsonin receptors. The drugs were used on maximum plasma levels considering pharmacokinetic studies after ingestion and the concentration applied for each one was 1,64 µg/ml for AZT, 1,5µg/ml for 3TC, 3,7 µg/ml for EFV and 11,45 µg/ml and 2,86 µg/ml for LPV and RTV, respectively. The phagocytic index was calculated as the average number of ingested Saccharomyces cerevisiae per phagocyte multiplied by the percentage of these cells engaged in phagocytosis. The oxidative capacity was assessed by the nitroblue tetrazolium (NBT) test. The isolated treatment with EFV increased the percentual of neutrophils engaged in phagocytosis through PAMPr (p=0,004) and through opsonin receptors (p=0,020), while the preincubation with LPV/RTV (p=0,024) and AZT/3TC/LPV/RTV (p=0,008) for 60 minutes decreased neutrophil phagocytic index when phagocytosis through PAMPr was investigated. The pretreatment of phagocytes for 30 minutes with LPV/RTV increased the monocyte phagocytic capacity through PAMPr (p=0,003) due to both the increase of the ingested yeasts average and the percentual of monocytes involved in phagocytosis, similar to the result observed to the pretreatment of these cells with AZT/3TC/LPV/RTV for 30 minutes and tended to increase (p=0,077) the percentual of neutrophils engaged in phagocytosis through opsonin receptors. However, when LPV/RTV was incubated with monocytes for 60 minutes, the monocyte phagocytic index through PAMPr decreased (p=0,013) due to the diminished percentual of monocytes engaged in phagocytosis. The LPV/RTV isolately decreased the monocyte phagocytic capacity (p=0,020) due to the reduction of the number of ingested yeasts by these cells. The AZT tended to increase the monocyte phagocytic capacity when phagocytosis through PAMPr was analyzed (p=0,097), because it tended to increase the percentual of monocytes engaged in phagocytosis. The production of reactive oxygen species (ROS) were more intensely influenced by the studied antiretrovirals. The AZT (p=0,027) and the 3TC (p=0,004) isolately and the associations LPV/RTV (p=0,0001) and AZT/3TC/LPV/RTV (p=0,0001) decreased the stimulated production of ROS by phagocytes. The percentual of baseline NBT reduction was also depressed by the associations LPV/RTV (p=0,0007) e AZT/3TC/LPV/RTV (p<0,0001) and tended to be reduced by the treatment with 3TC alone (p=0,071). These results suggest that the protocol AZT/3TC/EFV can be more adequate to the initial treatment of those individuals infected by HIV because it has not changed the phagocytic capacity or ROS production by neutrophils and monocytes, while the association AZT/3TC/LPV/RTV could be more interesting to patients with intolerance or viral resistance to EFV. It is also possible that patients with multiple resistances to antiretrovirals can be benefited by protocols containing LPV/RTV, because the reduction of ROS determined by these drugs, as demonstrated in this research, can contribute for the lowest rates of viral replication, lymphocyte apoptosis and HIV-related neurodegeneration.

Metabolomics approaches have recently been used to understand the complex molecular interactions of biological systems. One popular area in which these methods are being developed is to understand the biochemical changes during abiotic and biotic stresses; for example, how a cell may respond to a drug. Since metabolites are the end products of gene expression, these can be used to indicate the result of the activities and interaction of the cell or organism with its environment. The investigation of the level and compositional changes of metabolites against metabolic stresses such as chemotherapeutic treatment (drug exposure) are required to understand more fully abiotic perturbation to biological systems. The aim of this project was to understand the metabolic effect that the anti-viral drugs indinavir and lopinavir (currently used by HIV patients) have on HPV-related cervical cancer cell lines by measuring changes in metabolism using a wide range of analytical techniques; including Fourier transform infrared (FT-IR) and Raman spectroscopies, and gas and liquid chromatography-mass spectrometry (GC and LC-MS). The analyses and interpretation of the large volumes of complex multidimensional data generated by metabolomics approaches were performed with a combination of multivariate data analysis techniques such as principal components analysis (PCA) and canonical variates analysis (CVA), as well as univariate approaches such as N-Way analysis of variance (ANOVA). By combining biochemical imaging, metabolite fingerprinting and footprinting, and metabolite profiling, with multi- and uni-variate analyses, the actions and effects of the anti-viral drugs were investigated. FT-IR spectroscopy was initially used to generate global biochemical finger- and foot-prints, and Raman spectroscopy was employed to investigate intracellular distribution of metabolites, and other cellular species, as well as the localisation of drug molecules within cells. FT-IR spectroscopy ascertained that the intra- and extra-cellular metabolomes were being directly influenced in a fashion that correlated with increasing anti-viral dosing; these effects were phenotypic rather than measurements of the drug level. Raman imaging spectroscopy indicated that the indinavir but not lopinavir was being compartmentalised within the cell nucleus, but only in HPV early protein 6 (E6) expressing cells. This observation was further confirmed by fractionation of cell samples into nuclear and cytoplasmic fractions and assessing the indinavir concentrations via LC-MS. Finally, LC-MS and GC-MS metabolite profiling were employed to investigate changes in the intracellular metabolome in response to the anti-viral compounds across a range of physiologically relevant concentrations and in the presence and absence of the E6 oncoprotein. General effects of both anti-viral compounds included the regulation of metabolites such as glutathione, octenedionoic and octadecenoic acids, which may be involved in stress related responses, reduced levels of sugars and sugar-phosphates indicating a potential arrest of glycolysis, and reduced levels of malic acid indicating potential decreased flux into the TCA cycle; all indicating that central metabolism was being reduced. Finally, LC-MS based quantification indicated that in the presence of E6, lopinavir was actively removed from the cell, whereas the indinavir intracellular concentration increased concomitantly with the level of dosing. These investigations have revealed that metabolomics approaches are an apt tool for the study of anti-viral effects within cell cultures, but improvements need to be made with respect to the major limitation of metabolite identification.

The novel coronavirus disease 2019 (COVID-19), appeared in the United States over 1 year ago. This virus has a wide range of presentations, from being asymptomatic to causing severe acute respiratory syndrome, which can lead to death. It has led to a worldwide effort to find effective treatments, from repurposed medications to new discoveries, as well as the push to develop effective vaccines. As the race to fight this pandemic unfolds, this column provides what is currently available to combat this virus, how it has been utilized in the pregnant population, and what data have been made available about how these treatments affect fetal development and the neonate.

<p> Malaria, Human Immunodeficiency Virus (HIV) and tuberculosis (TB) are global health problems having their worst situation in sub-Saharan Africa. Consequently, concomitant use of antimalarial, antiretroviral and antitubercular drugs may be needed, resulting in a potential risk of drug-drug interactions.</p><p>Cytochrome P-450 (CYP) enzyme induction/inhibition may lead to drug-drug interactions and can be detected by probe drugs. An analytical method was developed for the quantitation of mephenytoin, CYP2B6 and CYP2C19 probe, and its metabolites. </p><p>Induction/inhibition of principal CYP enzymes by the antimalarials; artemisinin, dihydroartemisinin, arteether, artemether and artesunate, was evaluated using the 4-hour plasma concentration ratios of probe drugs and their metabolites along with modelling the population pharmacokinetics of S-mephenytoin and its metabolites. The extent of change in enzymatic activities was different among the antimalarials, with artemisinin having strongest capacity for induction and inhibition, consequently, the strongest potential risk for drug-drug interactions. </p><p>Drug-drug interactions between the antitubercular rifampicin and the antiretrovirals nevirapine and lopinavir were assessed, in TB/HIV patients, by developing population pharmacokinetic models. Rifampicin increased nevirapine oral clearance. Simulations suggested that increasing the nevirapine dose to 300 mg twice daily when co-administered with rifampicin, would result in nevirapine concentrations above subtherapeutic levels, with minimum exposure above the recommended maximum concentration. Lopinavir is co-formulated with ritonavir in the ratio of 4:1. In children, increasing ritonavir dose four times did not completely compensate the enhancement of lopinavir oral clearance caused by rifampicin. However, the predicted lopinavir trough concentration was above the recommended minimum therapeutic concentration.</p><p>The work presented in this thesis followed an investigation line though not done for a particular drug. First the CYP enzymes involved in the interaction are identified. Afterwards, the expected drug-drug interaction is investigated where the potentially interacting drugs are concomitantly administered and an adjustment in the dose regimen is proposed that is subsequently evaluated.</p>

Malaria, Human Immunodeficiency Virus (HIV) and tuberculosis (TB) are global health problems having their worst situation in sub-Saharan Africa. Consequently, concomitant use of antimalarial, antiretroviral and antitubercular drugs may be needed, resulting in a potential risk of drug-drug interactions. Cytochrome P-450 (CYP) enzyme induction/inhibition may lead to drug-drug interactions and can be detected by probe drugs. An analytical method was developed for the quantitation of mephenytoin, CYP2B6 and CYP2C19 probe, and its metabolites. Induction/inhibition of principal CYP enzymes by the antimalarials; artemisinin, dihydroartemisinin, arteether, artemether and artesunate, was evaluated using the 4-hour plasma concentration ratios of probe drugs and their metabolites along with modelling the population pharmacokinetics of S-mephenytoin and its metabolites. The extent of change in enzymatic activities was different among the antimalarials, with artemisinin having strongest capacity for induction and inhibition, consequently, the strongest potential risk for drug-drug interactions. Drug-drug interactions between the antitubercular rifampicin and the antiretrovirals nevirapine and lopinavir were assessed, in TB/HIV patients, by developing population pharmacokinetic models. Rifampicin increased nevirapine oral clearance. Simulations suggested that increasing the nevirapine dose to 300 mg twice daily when co-administered with rifampicin, would result in nevirapine concentrations above subtherapeutic levels, with minimum exposure above the recommended maximum concentration. Lopinavir is co-formulated with ritonavir in the ratio of 4:1. In children, increasing ritonavir dose four times did not completely compensate the enhancement of lopinavir oral clearance caused by rifampicin. However, the predicted lopinavir trough concentration was above the recommended minimum therapeutic concentration. The work presented in this thesis followed an investigation line though not done for a particular drug. First the CYP enzymes involved in the interaction are identified. Afterwards, the expected drug-drug interaction is investigated where the potentially interacting drugs are concomitantly administered and an adjustment in the dose regimen is proposed that is subsequently evaluated.

Kaletra® (lopinavir e ritonavir) é uma combinação fixa de dois inibidores da protease do vírus da imunodeficiência humana (VIH) indicada para o tratamento da infecção pelo VIH em associação com outros anti-retrovirais. Esta classe de fármacos inibe a protease do VIH evitando a clivagem da poliproteina gag-pol, levando à produção de vírus imaturos, incapazes de infectar outras células. Este trabalho teve como objetivo estabelecer o perfil de dissolução in vitro baseado nos dados in vivo, avaliar a estabilidade térmica do lopinavir e do ritonavir cápsulas, determinar a cinética de reação, isolar e identificar os principais produtos de degradação do ritonavir. Método por CLAE indicativo de estabilidade foi desenvolvido e validado para a quantificação do lopinavir e do ritonavir cápsulas. Método alternativo por espectrometria derivada também foi desenvolvido. Os resultados demonstraram uma correlação nível A entre o perfil de dissolução in vitro e a absorção in vivo nas condições propostas. O método por CLAE para a quantificação desta associação demonstrou ser específico, linear exato, preciso e robusto. O teste t demonstrou que houve diferença significativa entre o método CLAE e UV derivada. Os estudos de estabilidade térmica demonstraram que o lopinavir foi estável nas condições testadas enquanto que o ritonavir foi sensível ao calor, seguindo uma cinética de degradação de primeira ordem. De acordo com os resultados da RMN os principais produtos de degradação do ritonavir são os compostos de fórmula molecular C33H45N5O5S e C25H33N3O6. / Kaletra® (lopinavir and ritonavir) is a combination of two human immunodeficiency virus (HIV) protease inhibitors indicated as part of a multi drug therapy along with other antiretroviral agents. This class of drugs inhibits the HIV protease preventing cleavage of the gag-pol polyprotein, reducing the probability of viral particles reaching a mature, infectious state. The objective of this work was: to establish the in vitro dissolution profile based on in vivo data; to determine their thermal stability in the dosage form; to determine their kinetics of degradation and to isolate and identify their major products of degradation. A stability indicating method was developed and validated for simultaneous quantitation of both drugs in Kaletra®, using liquid chromatography. An alternative method using UV-derivative spectrometry was also proposed. The result showed a level A correlation between the in vitro dissolution profile and in vivo absorption under the proposed conditions. The LC method was fully validated and have shown to be stability indicating. The UV-derivative method showed not to be equivalent (t-test) to the LC method. Thermal stability studies have shown that lopinavir was very stable under the conditions tested while ritonavir was sensitive to heat, following a first order degradation kinetic. According to NMR results ritonavir the main degradation products have the molecular formula C33H45N5O5S and C25H33N3O6.

Kaletra® (lopinavir e ritonavir) é uma combinação fixa de dois inibidores da protease do vírus da imunodeficiência humana (VIH) indicada para o tratamento da infecção pelo VIH em associação com outros anti-retrovirais. Esta classe de fármacos inibe a protease do VIH evitando a clivagem da poliproteina gag-pol, levando à produção de vírus imaturos, incapazes de infectar outras células. Este trabalho teve como objetivo estabelecer o perfil de dissolução in vitro baseado nos dados in vivo, avaliar a estabilidade térmica do lopinavir e do ritonavir cápsulas, determinar a cinética de reação, isolar e identificar os principais produtos de degradação do ritonavir. Método por CLAE indicativo de estabilidade foi desenvolvido e validado para a quantificação do lopinavir e do ritonavir cápsulas. Método alternativo por espectrometria derivada também foi desenvolvido. Os resultados demonstraram uma correlação nível A entre o perfil de dissolução in vitro e a absorção in vivo nas condições propostas. O método por CLAE para a quantificação desta associação demonstrou ser específico, linear exato, preciso e robusto. O teste t demonstrou que houve diferença significativa entre o método CLAE e UV derivada. Os estudos de estabilidade térmica demonstraram que o lopinavir foi estável nas condições testadas enquanto que o ritonavir foi sensível ao calor, seguindo uma cinética de degradação de primeira ordem. De acordo com os resultados da RMN os principais produtos de degradação do ritonavir são os compostos de fórmula molecular C33H45N5O5S e C25H33N3O6. / Kaletra® (lopinavir and ritonavir) is a combination of two human immunodeficiency virus (HIV) protease inhibitors indicated as part of a multi drug therapy along with other antiretroviral agents. This class of drugs inhibits the HIV protease preventing cleavage of the gag-pol polyprotein, reducing the probability of viral particles reaching a mature, infectious state. The objective of this work was: to establish the in vitro dissolution profile based on in vivo data; to determine their thermal stability in the dosage form; to determine their kinetics of degradation and to isolate and identify their major products of degradation. A stability indicating method was developed and validated for simultaneous quantitation of both drugs in Kaletra®, using liquid chromatography. An alternative method using UV-derivative spectrometry was also proposed. The result showed a level A correlation between the in vitro dissolution profile and in vivo absorption under the proposed conditions. The LC method was fully validated and have shown to be stability indicating. The UV-derivative method showed not to be equivalent (t-test) to the LC method. Thermal stability studies have shown that lopinavir was very stable under the conditions tested while ritonavir was sensitive to heat, following a first order degradation kinetic. According to NMR results ritonavir the main degradation products have the molecular formula C33H45N5O5S and C25H33N3O6.

Kaletra® (lopinavir e ritonavir) é uma combinação fixa de dois inibidores da protease do vírus da imunodeficiência humana (VIH) indicada para o tratamento da infecção pelo VIH em associação com outros anti-retrovirais. Esta classe de fármacos inibe a protease do VIH evitando a clivagem da poliproteina gag-pol, levando à produção de vírus imaturos, incapazes de infectar outras células. Este trabalho teve como objetivo estabelecer o perfil de dissolução in vitro baseado nos dados in vivo, avaliar a estabilidade térmica do lopinavir e do ritonavir cápsulas, determinar a cinética de reação, isolar e identificar os principais produtos de degradação do ritonavir. Método por CLAE indicativo de estabilidade foi desenvolvido e validado para a quantificação do lopinavir e do ritonavir cápsulas. Método alternativo por espectrometria derivada também foi desenvolvido. Os resultados demonstraram uma correlação nível A entre o perfil de dissolução in vitro e a absorção in vivo nas condições propostas. O método por CLAE para a quantificação desta associação demonstrou ser específico, linear exato, preciso e robusto. O teste t demonstrou que houve diferença significativa entre o método CLAE e UV derivada. Os estudos de estabilidade térmica demonstraram que o lopinavir foi estável nas condições testadas enquanto que o ritonavir foi sensível ao calor, seguindo uma cinética de degradação de primeira ordem. De acordo com os resultados da RMN os principais produtos de degradação do ritonavir são os compostos de fórmula molecular C33H45N5O5S e C25H33N3O6. / Kaletra® (lopinavir and ritonavir) is a combination of two human immunodeficiency virus (HIV) protease inhibitors indicated as part of a multi drug therapy along with other antiretroviral agents. This class of drugs inhibits the HIV protease preventing cleavage of the gag-pol polyprotein, reducing the probability of viral particles reaching a mature, infectious state. The objective of this work was: to establish the in vitro dissolution profile based on in vivo data; to determine their thermal stability in the dosage form; to determine their kinetics of degradation and to isolate and identify their major products of degradation. A stability indicating method was developed and validated for simultaneous quantitation of both drugs in Kaletra®, using liquid chromatography. An alternative method using UV-derivative spectrometry was also proposed. The result showed a level A correlation between the in vitro dissolution profile and in vivo absorption under the proposed conditions. The LC method was fully validated and have shown to be stability indicating. The UV-derivative method showed not to be equivalent (t-test) to the LC method. Thermal stability studies have shown that lopinavir was very stable under the conditions tested while ritonavir was sensitive to heat, following a first order degradation kinetic. According to NMR results ritonavir the main degradation products have the molecular formula C33H45N5O5S and C25H33N3O6.

<p>In the past few decades, invasive mycosis has become a growing threat to global health, afflicting millions of people and claiming the lives of more than 1.5 million patients every year. Moreover, the economic burden of mycotic infections has become increasingly exhausting especially with the recent increases in the number of the high-risk population, the immunocompromised individuals. In the USA, the cost incurred by mycotic infections was estimated to be of more than $7.2 billion only in 2017. Of particular concern, <i>Candida</i> species are the most common fungal pathogens that infect humans, resulting in considerable morbidities and mortality rates that often exceed 50%. Unfortunately, the antifungal drug discovery is currently unable to keep pace with the urgent demand for more effective therapeutic options. Further complicating the situation is the recent emergence of multidrug-resistant species such as <i>Candida</i> <i>auris</i>, triggering outbreaks of deadly Candidemia across the globe. Given the risks inherent to the traditional de-novo drug discovery, combinatorial therapeutics stands out as a promising tool to hamper drug resistance and extend the clinical utility of the existing drugs. In this study, we assembled and screened ~3147 FDA-approved drugs and clinical molecules against fluconazole-resistant <i>C. albicans</i> and <i>C. auris</i> isolates, for the aim of restoring the antifungal activity of azole antifungals against drug-resistant <i>Candida </i>species. The screen revealed five promising hits: pitavastatin (antihyperlipidemic), ospemifene (estrogen receptor modulator), sulfa antibacterial drugs, lopinavir (antiviral), and aprepitant (antiemetic).</p> <p>All identified hits demonstrated variable azole chemosensitizing activities depending on the tested <i>Candida</i> species and the azole drug. Pitavastatin displayed broad-spectrum synergistic interactions with both fluconazole and voriconazole against isolates of <i>C. albicans</i>, <i>C. glabrata</i>, and <i>C. auris</i>. Ospemifene was able to interact synergistically with itraconazole against multiple fungal isolates including <i>Candida</i>, <i>Cryptococcus</i>, and <i>Aspergillus</i> species. Sulfa drugs displayed potent synergistic activities with different azoles against <i>C. albicans</i>, however, a limited efficacy was observed against efflux-hyperactive isolates such as <i>C. auris</i>. On the other hand, both lopinavir and aprepitant exerted potent and broad-spectrum synergistic activities with itraconazole and were effective against multiple <i>Candida</i> species including <i>C. albicans</i>, <i>C. auris</i>, <i>C. glabrata</i>, <i>C. krusie</i>, <i>C. tropicalis</i>, and <i>C. parapsilosis</i>. Furthermore, using <i>Caenorhabditis elegans</i> as an infection model, all drug combinations significantly reduced the fungal burden in the infected nematodes and significantly prolonged their survival as compared to single-drug treatments. Multiple phenotypic and molecular assays indicted that the identified hit compounds use distinct mechanisms to enhance the antifungal activity of azole drugs. These mechanisms include efflux pump inhibition, interference with the folate biosynthesis and disturbance of iron homeostasis. Taken together, this study reveals novel and potent azole chemosensitizing agents effective against multiple azole-resistant isolates and opens the door for more investigations to assess their clinical potential in human medicine as promising antifungal adjuvants.</p>