The role of corticosterone in nevirapine-induced idiosyncratic drug-induced liver injury.

Nevirapine, an antiretroviral used in the treatment of HIV, is associated with idiosyncratic drug-induced liver injury (IDILI), a potentially life-threatening adverse drug reaction. Its usage has decreased due to this concern, but it is still widely used in lower-resource settings. In general, the mechanisms underlying idiosyncratic drug reactions (IDRs) are poorly understood, but evidence indicates that most are immune-mediated. There is very limited understanding of the early immune response following administration of drugs associated with IDRs, which likely occurs due to reactive metabolite formation. In this work, we aimed to characterize the links between covalent binding of nevirapine, the development of an early immune response, and the subsequent liver injury using a mouse model. We describe initial attempts to characterize an early immune response to nevirapine followed by the discovery that nevirapine induced the release of corticosterone. Corticosterone release was partially associated with the degree of drug covalent binding in the liver but was also likely mediated by additional mechanisms at higher drug doses. Transcriptomic analysis confirmed metabolic activation, glucocorticoid signaling, and decreased immune activation; GDF-15 also warrants further investigation as part of the immune response to nevirapine. Finally, glucocorticoid blockade preceding the first dose of nevirapine attenuated nevirapine-induced liver injury at 3 weeks, suggesting that acute glucocorticoid signaling is harmful in the context of nevirapine-induced liver injury. This work demonstrates that nevirapine induces acute corticosterone release, which contributes to delayed-onset liver injury. It also has implications for screening drug candidates for IDILI risk and preventing nevirapine-induced IDILI.

The development and characterization of a CRISPR/Cas9-mediated PD-1 functional knockout rat as a tool to study idiosyncratic drug reactions.

Idiosyncratic drug reactions are rare but serious adverse drug reactions unrelated to the known therapeutic properties of the drug and manifest in only a small percentage of the treated population. Animal models play an important role in advancing mechanistic studies examining idiosyncratic drug reactions. However, to be useful, they must possess similarities to those seen clinically. Although mice currently represent the dominant mammalian genetic model, rats are advantageous in many areas of pharmacologic study where their physiology can be examined in greater detail and is more akin to that seen in humans. In the area of immunology, this includes autoimmune responses and susceptibility to diabetes, in which rats more accurately mimic disease states in humans compared with mice. For example, oral nevirapine treatment can induce an immune-mediated skin rash in humans and rats, but not in mice due to the absence of the sulfotransferase required to form reactive metabolites of nevirapine within the skin. Using CRISPR-mediated gene editing, we developed a modified line of transgenic rats in which a segment of IgG-like ectodomain containing the core PD-1 interaction motif containing the native ligand and therapeutic antibody domain in exon 2 was deleted. Removal of this region critical for mediating PD-1/PD-L1 interactions resulted in animals with an increased immune response resulting in liver injury when treated with amodiaquine.

The role of CYP2B6 516G>T polymorphism on efavirenz/nevirapine toxicity. Implications on treatment outcomes: Lessons from Botswana.

ABSTRACT: The two non-nucleoside reverse transcriptase inhibitors (NNRTIs), efavirenz (EFV) and nevirapine (NVP), are currently the core antiretroviral drugs for treatment of HIV in sub-Saharan Africa including Botswana. The drugs are metabolized by Cytochrome P450 2B6 (CYP2B6) liver enzyme. The CYP2B6 gene that encodes for metabolism of these drugs is known to be highly polymorphic. One of the polymorphism in the CYP2B6 gene, 516G>T, particularly the 516T allele, is known to confer poor metabolism of EFV and NVP. This may lead to high levels of plasma drug concentrations and development of treatment toxicities, like central nervous system toxicities, and cutaneous and hepatic toxicities, for EFV and NVP, respectively. The CYP2B6 516G allele on the other hand is associated with an extensive metabolism of the two NNRTIs drugs. We sought to establish association between possible developments of NNRTIs toxicities with CYP2B6 516G>T variation in Botswana.A total of 316 peripheral blood mononuclear cells samples were used in a retrospective view. All the samples were from participants on EFV/NVP-containing regimen with known toxicity output. TaqMan Real-Time PCR approach was applied for assessing CYP2B6 516 allele variation in cases with treatment toxicity and those without. Analysis was performed by chi-square statistics and logistic regression analysis.The rate of poor metabolizers among participants with toxicity and those without toxicity was 18.4% and 15.1%, respectively. The CYP2B6 516 genotype distribution comparisons between the participants with toxicity and those without were not statistically different (chi-square = .326; P = .568).CYP2B6 516 variation was not associated with NNRTI toxicity. No other factors were associated with toxicity when considering age, baseline body mass index, baseline CD4, baseline HIV viral load and adherence. The results were discussed in the context of all the studies done in Botswana to date.

Histopathological changes in Oreochromis mossambicus (Peters, 1852) ovaries after a chronic exposure to a mixture of the HIV drug nevirapine and the antibiotics sulfamethoxazole and trimethoprim.

The burden of the human immunodeficiency virus and acquired immunodeficiency syndrome (HIV/AIDS) infection has transformed the African continent into a major consumer of antiretrovirals (ARVs) drugs. In addition to HIV burden, the African continent has also a high incidence of tuberculosis (TB) and has been experiencing recurring outbreaks of several other viral, bacterial, and parasitic epidemic diseases. The novel severe acute respiratory syndrome coronavirus 2 (SARS-COV-2 or Covid-19) pandemic outbreak is adding to the continent's infectious diseases burden as experts are predicting that it will be here for a long time. One of the consequences of these infectious diseases is that antiviral and antibiotic compounds have become some of the most consumed pharmaceuticals on the continent. Many of these drugs have been frequently detected in surface waters across Africa. There is limited information available on the adverse effects of the mixtures of different types of pharmaceuticals in African aquatic environments on fish reproduction. The present study investigated the effects of the ARV drug nevirapine (NVP - 1.48 and 3.74 µg/L) and its mixture with the antibiotic sulfamethoxazole (3.68 µg/L) and trimethoprim (0.87 µg/L) on O. mossambicus gonads using histopathological endpoints as biomarkers. The fish (n = 52) were exposed for 30 days in a static renewal system. Female O. mossambicus exposed to nevirapine (3.74 µg/L) and to NVP - antibiotic mixture recorded higher ovary indices. Statistically significant differences were found in female ovary indices between the fish exposed to NVP (3.74 µg/L) and the control fish (p = 0.002) as well as between the fish exposed to the NVP - antibiotic mixture and the control fish (p = 0.009). The main observed histopathological changes in the ovaries were increased vitellogenic oocyte atresia and vacuolation of the interstitial tissue in the fish exposed to NVP - antibiotic mixture. It is evident that the presence of NVP - antibiotics mixture in water triggered the observed histopathology in female fish ovaries. The detected abnormal high rate of atretic oocytes could result in impaired fish reproduction.

A review of the potential mechanisms of neuronal toxicity associated with antiretroviral drugs.

Highly active antiretroviral treatment has led to unprecedented efficacy and tolerability in people living with HIV. This effect was also observed in the central nervous system with the nowadays uncommon observation of dementias; yet in more recent works milder forms are still reported in 20-30% of optimally treated individuals. The idea of a subclinical neuronal toxicity induced by antiretrovirals has been proposed and was somehow supported by the late-emerging effects associated with efavirenz use. In this manuscript we are reviewing all the potential mechanisms by which antiretroviral drugs have been associated with in vitro, ex vivo, or in vivo toxicity to cells pertaining to the central nervous system (neurons, astrocytes, oligodendrocytes, and endothelial cells). These include direct or indirect effects and pathological pathways such as amyloid deposition, damage to small cerebral vessels, and impairment in neurotransmission. The aim of this review is therefore to provide a detailed description of the available literature in order to guide further clinical research for improving patients' neurocognition and quality of life.

Anti-HIV drugs promote ß-amyloid deposition and impair learning and memory in BALB/c mice.

OBJECTIVES: Growing evidence suggested that antiretroviral (ARV) drugs may promote amyloid beta (Aß) accumulation in HIV-1-infected brain and the persistence of HIV-associated neurocognitive disorders (HANDs). It has also been shown that lipid peroxidation upregulates ß-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) expression and subsequently promotes Aß peptide production. In the present study, we examined whether chronic exposure to the anti-HIV drugs tenofovir disoproxil fumarate (TDF) and nevirapine induces lipid peroxidation thereby promoting BACE1 and Aß generation and consequently impair cognitive function in mice. METHODS: TDF or nevirapine was orally administered to female BALB/c mice once a day for 8 weeks. On the 7th week of treatment, spatial learning and memory were assessed using the Morris water maze test. The levels of lipid peroxidation, BACE1, amyloid ß 1-42 (Aß1-42) and Aß deposits were measured in the hippocampal tissue upon completion of treatment. RESULTS: Chronic administration of nevirapine induced spatial learning and memory impairment in the Morris water maze test, whereas TDF did not have an effect. TDF and nevirapine administration increased hippocampal lipid peroxidation and Aß1-42 concentration. Nevirapine further upregulated BACE1 expression and Aß deposits. CONCLUSION: Our results suggest that chronic exposure to TDF and nevirapine contributes to hippocampal lipid peroxidation and Aß accumulation, respectively, as well as spatial learning and memory deficits in mice even in the absence of HIV infection. These findings further support a possible link between ARV drug toxicity, Aß accumulation and the persistence of HANDs.

Prevalence of ABCC3-1767G/A polymorphism among patients with antiretroviral-associated hepatotoxicity.

BACKGROUND: Plasma concentrations of antiretrovirals (ARVs) regimens have considerably varied in individuals of human immunodeficiency virus (HIV) because of variations in the expression of drug-metabolizing and transporter genes. Transporter genes play an important role in the disposition of drugs. Polymorphism in transporter gene (ABCC3) affects the MRP3 expression and varies the treatment outcome. METHOD: We examined the polymorphism of ABCC3-1767G/A gene in a total of 165 HIV patients (out of 165 HIV patients, 34 were with and 131 were without hepatotoxicity) and 156 healthy individuals using the polymerase chain reaction-restriction fragment length polymorphism method. RESULTS: In univariate analysis, we found a decreased prevalence of ABCC3 1767GA, 1767GA+AA genotypes, and 1767A allele in patients with hepatotoxicity as compared to patients without hepatotoxicity (23.5% vs. 28.2% and 23.5% vs. 30.53%; 11.76% vs. 16.41%), while a higher prevalence of 1767AA genotype was observed in HIV patients in comparison with healthy controls (2.3% vs. 1.3%, odds ratio [OR] = 1.71, 95% confidence interval [CI]: 0.23-15.03, p = .89). The frequency of ABCC3-1767AA genotype was dispersed higher in individuals with early and advanced HIV disease stage in comparison with healthy controls (5.3% vs. 1.3%, OR = 4.73, p = .70; 8.9% vs. 1.3%, OR = 1.89, p = .91). A higher occurrence of ABCC3-1767AA genotype was found in tobacco using HIV patients without hepatotoxicity compared with nonusers (4.7% vs. 1.1%, OR = 4.28, p = .52). The distribution of ABCC3-1767GA genotype was higher in nevirapine receiving HIV patients irrespective of their hepatotoxicity status as compared to nonusers (30.4% vs. 9.1%, OR = 3.34, p = .22; 29.4% vs. 16.7%, OR = 1.69, p = .77). In multivariate analysis, HIV patients receiving nevirapine and with hepatotoxicity was found to have a significant risk for severity of hepatotoxicity (OR = 4.56, 95% CI: 1.60-12.99, p = .004). CONCLUSION: ABCC3 1767G/A polymorphism was not significantly associated with susceptibility to ARV-associated hepatotoxicity, although ABCC3 1767AA genotype designated a risk for acquisition of hepatotoxicity and advancement of the disease. Nevirapine usage emerged as an independent risk factor for hepatotoxicity severity.

A personally guided tour on some of our data with the Ames assay-A tribute to Professor Bruce Ames.

In contributing to this Special Issue of Mutation Research dedicated to Professor Bruce N. Ames in recognition of his 90th birthday in December 2018, we intend to portray the importance not only of the Ames Salmonella/mammalian-microsome mutagenicity assay in some of our studies over the years, but also the importance of the insight that Bruce Ames brought to the field of genetic toxicology.

Hatching success and survival of fish early life stages in a chronic exposure to nevirapine: a case study of the Mozambique tilapia.

The anti-retroviral nevirapine has been detected in surface waters throughout South Africa and its effects on non-target aquatic animals are still unknown. The aim was to investigate the potential effects of nevirapine on the hatching success and survival of Oreochromis mossambicus early life stages through a chronic exposure. The exposer started with newly fertilized O. mossambicus eggs and concluded 30 days after hatching. Environmental relevant concentration of nevirapine (1.48 µg/l) was used in a static renewal system and a controlled environment (27 ± 1°C; 14:10 day/night cycle). The main endpoints assessed included hatching success and survival; a morphological assessment was also done on whole individual on day 1 and 30 post-hatching to identify any physical abnormality. Nevirapine had no noticeable effects on the hatching success and survival of O. mossambicus larvae; no statistically significant differences were observed between the control and the nevirapine exposed fish (p > 0.05).

Effects of human sulfotransferases on the cytotoxicity of 12-hydroxynevirapine.

Nevirapine, a non-nucleoside reverse transcriptase inhibitor used for the treatment of AIDS, can cause serious skin rashes and hepatotoxicity. Previous studies have indicated that the benzylic sulfate 12-sulfoxynevirapine, the formation of which is catalyzed by human sulfotransferases (SULTs), may play a causative role in these toxicities. To characterize better the role of 12-sulfoxynevirapine in nevirapine-induced cytotoxicity, the ability of 12 expressed human SULT isoforms to conjugate 12-hydroxynevirapine was assessed. Of the 12 human SULTs, no detectable 12-sulfoxynevirapine was observed with SULT1A3, SULT1C2, SULT1C3, SULT2B1, SULT4A1, or SULT6B1. As determined by the Vmax/Km ratio, SULT2A1 had the highest overall 12-hydoxynevirapine sulfonation activity; lower activities were observed with SULT1A1, SULT1A2, SULT1B1, SULT1C4, and SULT1E1. Incubation of 12-sulfoxynevirapine with glutathione and cysteine led to adduct formation; lower yields were obtained with deoxynucleosides. 12-Hydroxynevirapine was more cytotoxic than nevirapine to TK6, TK6/SULT vector, and TK6/SULT2A1 cells. With nevirapine, there was no difference in cytotoxicity among the three cell lines, whereas with 12-hydroxynevirapine, TK6/SULT2A1 cells were more resistant than TK6 and TK6/SULT vector cells. Co-incubation of 12-hydroxynevirapine with the competitive SULT2A1 substrate dehydroepiandrosterone decreased the level of 12-sulfoxynevirapine and increased the cytotoxicity in TK6/SULT2A1 cells. These data demonstrate that although 12-sulfoxynevirapine reacts with nucleophiles to form adducts, sulfonation of 12-hydroxynevirapine decreases the cytotoxicity of 12-hydroxynevirapine in TK6 cells.

Nevirapine-induced liver lipid-SER inclusions and other ultrastructural aberrations.

Nevirapine (NVP) therapy is associated with a high risk of serious liver injury and skin rash. Treatment of Brown Norway rats with NVP causes an immune-mediated skin rash. Even though NVP does not cause serious liver injury in wildtype animals, incubation of hepatocytes with NVP leads to the release of presumably danger-associated molecular pattern molecules (DAMPs), which activate macrophages. In this study, we examined the liver biopsies of Brown Norway rats treated with NVP to determine the histologic correlate to the release of DAMPs by hepatocytes. In vivo, debris from necrotic hepatocytes and endothelial cells were present in the liver sinusoids, a condition that can trigger an immune response. In addition to mitochondrial, hepatocytic, and endothelial damage, the drug induced large hepatocytic inclusions composed of lipid droplets surrounded by concentric whorls of smooth endoplasmic reticulum (SER) cisternae-lipid-SER (LSER) inclusions, which were deposited in the sinusoids. NVP is lipid soluble, and these LSER inclusions may be sinks of NVP or its metabolites. LSERs are deposited in the blood stream where they may be picked up by lymph nodes and contribute to initiation of an immune response leading to serious liver injury or skin rash. LSERs migration from liver to the blood stream may signify a novel mechanism of drug exocytosis.

Supernatant from Hepatocyte Cultures with Drugs That Cause Idiosyncratic Liver Injury Activates Macrophage Inflammasomes.

There is increasing evidence that most idiosyncratic drug-induced liver injury (IDILI) is immune mediated, and in most cases, reactive metabolites appear to be responsible for the induction of this immune response. Reactive metabolites can cause cell damage with the release of damage-associated molecular patterns (DAMPs), which is thought to be involved in immune activation. Presumably, the reason that the liver is a common target of idiosyncratic drug reactions is because it is the major site of drug metabolism and reactive metabolite formation. Inflammasomes can be activated by DAMPs, and this may be a common mechanism by which DAMPs initiate an immune response. In this study, we tested the ability of drugs to induce the release of DAMPs that activate inflammasomes. The drugs tested were amodiaquine and nevirapine; both are associated with significant incidences of severe IDILI. The hepatocytes were a human hepatocarcinoma functional liver cell-4 (FLC-4) cell line. For the detection of inflammasome activation, we used the human macrophage cell line, THP-1 cells. We found that the supernatant from the incubation of both drugs with FLC-4 cells for 7 days led to increased caspase-1 activity and production of IL-1ß by THP-1 cells. However, amodiaquine alone also directly activated THP-1 cells. This is presumably because the myeloperoxidase in THP-1 cells can bioactivate amodiaquine to a reactive metabolite. In contrast, nevirapine requires cytochromes P450 for reactive metabolite formation and therefore required incubation with hepatocytes. These results support the hypothesis that reactive metabolites of drugs can cause the release of DAMPs, which in turn can activate inflammasomes. Inflammasome activation may be an important step in the activation of the immune system by drugs, which in some patients can lead to IDILI. Our in vitro model is simple and convenient for evaluating inflammasome activation, and this may be a method to screen drugs for IDILI risk.

Comparison of antioxidant effects of kolaviron and vitamin C interventions on testicular structures following nevirapine administration in Sprague-Dawley rats / Comparación de los efectos antioxidantes en la intervención con kolaviron y vitamina C sobre las estructuras testiculares después de la administración de nevirapina en ratas Sprague-Dawley

Testicular toxicity has been implicated in highly active anti-retroviral therapy (HAART) treatment. Hence there is need to identify an effective antioxidant product that can alleviate testicular necrosis due to HAART administration. Forty eight adult male Sprague-Dawley rats were used in this study. The animals were divided into eight (8) groups: A-H (n= 6). Group A animals received normal saline as the control; Group B was given Nevirapine (Nv); Group C was given Kolaviron (Kv); Group D was given vitamin C; Group E was given Nv and Kv; Group F was given Nv and Vitamin C; Group G was given Nv for 56 d and Kv for 28 d serving as a withdrawal group; Group H was given corn oil. Nv, Kv and Vit. C were given at 1.54, 200 and 250 (mg·kg)/bw respectively while all administrations were through oral gavage. The body weights were taken every other day. Thereafter, they were anaesthetized with halothane. The testes were excised, weighed, fixed in Bouin's fluid and stained with H&E while the epididymes removed for semen fluid analyses. The results showed a significant (P<0.05) decrease in sperm motility in group E (Nevirapine + kolaviron) when compared with group F (Nevirapine + Vitamin C) while Sperm count was not significantly different (P>0.05) across the groups. The testicular histoarchitectural studies revealed indistinct spermatogonia, necrotic interstititial endocrine cells in the altered interstitial space, fragmented spermatids, atrophy of mature spermatocytes, degenerated germ cells, obliterated seminiferous tubules lumen, undifferentiated spermatogonia and cellular debris in the somniferous tubules lumen of nevirapine administered group but normal across the other groups. In the testis, there were no significant reduction in SOD, Catalase and GPx activities but a significant decrease in GST activity (P<0.001) when group E was compared with group F. In conclusion, vitamin C presents a better remediation in nevirapine induced spermiotoxicity compared to kolaviron in Sprague-Dawley rats.

La toxicidad testicular ha sido implicada en la terapia antirretroviral altamente activa (TARAA). Por lo tanto existe la necesidad de identificar un producto antioxidante eficaz que pueda aliviar la necrosis testicular en la administración de la TARAA. Cuarenta y ocho ratas macho Sprague-Dawley adultas fueron utilizadas. Los animales se dividieron en ocho (8) grupos: AH (n= 6). Grupo A, animales recibieron solución salina normal como el control; Grupo B, recibió Nevirapina (Nv); Grupo C, recibió Kolaviron (Kv); Grupo D, recibió vitamina C; Grupo E, recibió Nv y Kv; Grupo F, recibió Nv y vitamina C; Grupo G, recibió Nv durante 56 d y Kv por 28 d como un grupo de retirada; Grupo H, recibió aceite de maíz. Nv, Kv y Vit. C se administraron en dosis de 1, 54, 200 y 250 (mg · kg) de peso corporal respectivamente; todas las administraciones fueron por sonda oral. Los pesos corporales se tomaron cada dos días. A partir de ese momento los animales fueron anestesiados con halotano. Los testículos fueron extirpados, pesados y fijados en solución de Bouin y teñidos con H&E, mientras que el epidídimo se retiró para analizar el semen. Los resultados mostraron un descenso (p<0,05) en la motilidad de los espermatozoides en el grupo E (Nevirapina + Kolaviron) en comparación con el grupo F (Nevirapina + vitamina C), mientras que el recuento espermático no mostró diferencias significativas (P>0,05) entre los grupos. El estudio de la histoarquitectura testicular reveló espermatogonias indiferenciadas, con células intersticiales necróticas en el espacio intersticial y espermátidas fragmentadas. Además, en el grupo que recibió Nevirapina mostró espermatocitos maduros atrofiados, degeneración de células germinales, lumen de los túbulos seminíferos obliterados, espermatogonias indiferenciadas y restos celulares en el lumen de los tubulos seminíferos. En el resto de los grupos los resultados fueron normales. En el testículo hubo una reducción significativa en las actividades de la superóxido dismutasa, catalasa y glutatión peroxidasa, pero una disminución significativa en la actividad glutatión S-transferasa (P <0,001) al comparar los grupo E y F.

Drug-induced Liver Fibrosis: Testing Nevirapine in a Viral-like Liver Setting Using Histopathology, MALDI IMS, and Gene Expression.

Nevirapine (NVP) is associated with hepatotoxicity in 1-5% of patients. In rodent studies, NVP has been shown to cause hepatic enzyme induction, centrilobular hypertrophy, and skin rash in various rat strains but not liver toxicity. In an effort to understand whether NVP is metabolized differently in a transiently inflamed liver and whether a heightened immune response alters NVP-induced hepatic responses, female brown Norway rats were dosed with either vehicle or NVP alone (75 mg/kg/day for 15 days) or galactosamine alone (single intraperitoneal [ip] injection on day 7 to mimic viral hepatitis) or a combination of NVP (75/100/150 mg/kg/day for 15 days) and galactosamine (single 750 mg/kg ip on day 7). Livers were collected at necropsy for histopathology, matrix-assisted laser desorption/ionization imaging mass spectrometry and gene expression. Eight days after galactosamine, hepatic fibrosis was noted in rats dosed with the combination of NVP and galactosamine. No fibrosis occurred with NVP alone or galactosamine alone. Gene expression data suggested a viral-like response initiated by galactosamine via RNA sensors leading to apoptosis, toll-like receptor, and dendritic cell responses. These were exacerbated by NVP-induced growth factor, retinol, apoptosis, and periostin effects. This finding supports clinical reports warning against exacerbation of fibrosis by NVP in patients with hepatitis C.

Transcriptional profiling suggests that Nevirapine and Ritonavir cause drug induced liver injury through distinct mechanisms in primary human hepatocytes.

Drug induced liver injury (DILI), a major cause of pre- and post-approval failure, is challenging to predict pre-clinically due to varied underlying direct and indirect mechanisms. Nevirapine, a non-nucleoside reverse transcriptase inhibitor (NNRTI) and Ritonavir, a protease inhibitor, are antiviral drugs that cause clinical DILI with different phenotypes via different mechanisms. Assessing DILI in vitro in hepatocyte cultures typically requires drug exposures significantly higher than clinical plasma Cmax concentrations, making clinical interpretations of mechanistic pathway changes challenging. We previously described a system that uses liver-derived hemodynamic blood flow and transport parameters to restore primary human hepatocyte biology, and drug responses at concentrations relevant to in vivo or clinical exposure levels. Using this system, primary hepatocytes from 5 human donors were exposed to concentrations approximating clinical therapeutic and supra-therapeutic levels of Nevirapine (11.3 and 175.0 µM) and Ritonavir (3.5 and 62.4 µM) for 48 h. Whole genome transcriptomics was performed by RNAseq along with functional assays for metabolic activity and function. We observed effects at both doses, but a greater number of genes were differentially expressed with higher probability at the toxic concentrations. At the toxic doses, both drugs showed direct cholestatic potential with Nevirapine increasing bile synthesis and Ritonavir inhibiting bile acid transport. Clear differences in antigen presentation were noted, with marked activation of MHC Class I by Nevirapine and suppression by Ritonavir. This suggests CD8+ T cell involvement for Nevirapine and possibly NK Killer cells for Ritonavir. Both compounds induced several drug metabolizing genes (including CYP2B6, CYP3A4 and UGT1A1), mediated by CAR activation in Nevirapine and PXR in Ritonavir. Unlike Ritonavir, Nevirapine did not increase fatty acid synthesis or activate the respiratory electron chain with simultaneous mitochondrial uncoupling supporting clinical reports of a lower propensity for steatosis. This in vitro study offers insights into the disparate direct and immune-mediated toxicity mechanisms underlying Nevirapine and Ritonavir toxicity in the clinic.

The Combination of Anti-CTLA-4 and PD1-/- Mice Unmasks the Potential of Isoniazid and Nevirapine To Cause Liver Injury.

Our laboratory recently reported what we believe is the first valid animal model of idiosyncratic drug-induced liver injury (IDILI) by treating PD1-/- mice with an anti-CTLA-4 antibody and amodiaquine (AQ). PD1 and CTLA-4 are important immune checkpoint receptors that are involved in inducing immune tolerance. This model was able to produce significant liver injury that looks very similar to the liver injury seen in humans. Although this model was shown to work with AQ, the question becomes whether blocking immune tolerance would unmask the potential of other drugs to cause IDILI. In this study, we tested isoniazid and nevirapine, both drugs with significant histories of causing IDILI in humans even though they do not cause significant injury in animals with doses that result in therapeutic blood levels. Both drugs in combination with these immune checkpoint inhibitors caused mild but significant delayed onset liver injury, which is similar to the mild injury that they can cause in humans. INH-induced liver injury in this model was associated with an increase in NK cells, while NVP-induced liver injury was associated with a greater increase in CD8 T cells. Although the liver injury caused by these drugs in this model was mild, these results suggest that impairing immune tolerance may be a general method for unmasking the potential of drugs to cause IDILI and therefore provide a screening tool for drug development.

Human sulfotransferase 1A1-dependent mutagenicity of 12-hydroxy-nevirapine: the missing link?

Nevirapine (NVP) is a frequently used anti-HIV drug. Despite its efficacy, NVP has been associated with serious skin and liver injuries in exposed patients and with increased incidences of hepatoneoplasias in rodents. Current evidence supports the involvement of reactive metabolites in the skin and liver toxicities of NVP, formed by cytochrome P450-mediated oxidations and/or subsequent phase II sulfonation. However, to date, standard in vitro genotoxicity tests have provided no evidence that NVP is either mutagenic or clastogenic. The human sulfotransferase 1A1-dependent mutagenicity of 12-hydroxy-NVP, one of the major metabolites of NVP, is demonstrated here.

Foetal loss and enhanced fertility observed in mice treated with Zidovudine or Nevirapine.

BACKGROUND: Health concerns for HIV-infected persons on antiretroviral therapy (ART) have moved from morbidity to the challenges of long-term ART. We investigated the effect of Zidovudine or Nevirapine on reproductive capacity across two mouse generations. METHODS: A prospective mouse study with drugs administered through one spermatogenic cycle. Mouse groups (16 males and 10 females) were given Zidovudine or Nevirapine for 56 days. Males were mated to untreated virgin females to determine dominant lethal effects. Twenty females (10 treated and 10 untreated) mated with the treated males per dose and gave birth to the F1 generation. Parental mice were withdrawn from drugs for one spermatogenic cycle and mated to the same dams to ascertain if effects are reversible. The F1 generation were exposed for another 56 days and mated to produce the F2 generation. RESULTS: Foetal loss was indicated in the dominant lethal assay as early as four weeks into drug administration to the males. At the first mating of the parental generation to produce the F1 generation, births from 10 dams/dose when the 'father-only' was exposed to Zidovudine (10, 100 and 250 mg/kg) was 3, 2 and 1 while it was 7, 1 and 4 respectively when 'both-parents' were exposed. Similarly births from the parental generation first mating when the 'father-only' was exposed to Nevirapine (5, 50 and 150 mg/kg) was 2, 2 and 0 while it was 6, 5 and 9 respectively when 'both-parents' were exposed. However, fertility was not significantly different neither by dose nor by the parental exposure. The F1 mice mated to produce the F2 generation recorded only one birth. CONCLUSION: The dominant lethal analysis showed foetal loss occurred when the "fathers-only" were treated while fertility was enhanced when "both-parents" were on therapy at the time of mating.

Effect of antiretroviral drugs on the DNA damage in mice.

In order to investigate the effects of two non-nucleoside reverse transcriptase inhibitors (NNRTIs) on the DNA damage in vivo, nevirapine (NVP; 3.3 mg/kg), efavirenz (EFV; 10 mg/kg) or saline were administered orally. Acute effects were analyzed 24 h after the administration of a single NNRTI dose, and subchronic effects 24 h after the last dose. Peripheral blood, brain, heart and liver samples were subjected to genotoxicity analyses and polychromatic erythrocytes from the bone marrow to micronucleus test. The micronucleus test did not reveal any significant differences between animals from the acute or subchronic groups. Comet assay showed that acute and subchronic NNRTI treatment did not cause any significant DNA damage in heart, liver or peripheral blood cells. However, increased damage indexes and frequencies were observed in the brain of mice, subchronically treated with EFV. This result suggests for the first time that this drug might induce genotoxicity in the brain.

Differential responses of human hepatocytes to the non-nucleoside HIV-1 reverse transcriptase inhibitor nevirapine.

Nevirapine is a non-nucleoside reverse transcriptase (RT) inhibitor used for the treatment of AIDS and the prevention of mother-to-child transmission of HIV-1. Despite its therapeutic benefits, treatment with nevirapine has been associated with significant incidences of liver and dermal toxicity. The present study examined the effects of nevirapine on cell growth and death in human hepatocyte HepG2 cells and THLE2 cells and the possible pathways involved in these effects. The concentrations of nevirapine inhibiting 50% cell growth were similar for both cell lines. Nevirapine (0-250 µM) treatment caused a slight increase in the amount of lactate dehydrogenase released into the medium. Apoptotic cell death did not contribute to the decrease in viable cells. Exposing of HepG2 cells to nevirapine caused G2/M phase arrest, and the activity of senescence-associated ß-galactosidase was not altered. In THLE2 cells, the percentage of cells in G1/G0 phase was increased and cellular senescence was induced in a concentration-dependent manner. Endogenous non-telomeric RT activity was not detected in either cell line. Western blot analysis indicated lower levels of p53 and phospho-p53 (ser15) in HepG2 cells as compared to THLE2 cells; no significant changes in p53 or phospho-p53 (ser15) were noted with nevirapine treatment. These data demonstrate that nevirapine inhibits cell growth, induces cell cycle arrest at different phases, and has different effects on cellular senescence in HepG2 cells and THLE2 cells. The differential responses appear to be related to differences in the basal levels of p53 in the HepG2 cells and THLE2 cells.