Systems pharmacology modeling of drug-induced hyperbilirubinemia: Differentiating hepatotoxicity and inhibition of enzymes/transporters.

Elevations in serum bilirubin during drug treatment may indicate global liver dysfunction and a high risk of liver failure. However, drugs also can increase serum bilirubin in the absence of hepatic injury by inhibiting specific enzymes/transporters. We constructed a mechanistic model of bilirubin disposition based on known functional polymorphisms in bilirubin metabolism/transport. Using physiologically based pharmacokinetic (PBPK) model-predicted drug exposure and enzyme/transporter inhibition constants determined in vitro, our model correctly predicted indinavir-mediated hyperbilirubinemia in humans and rats. Nelfinavir was predicted not to cause hyperbilirubinemia, consistent with clinical observations. We next examined a new drug candidate that caused both elevations in serum bilirubin and biochemical evidence of liver injury in rats. Simulations suggest that bilirubin elevation primarily resulted from inhibition of transporters rather than global liver dysfunction. We conclude that mechanistic modeling of bilirubin can help elucidate underlying mechanisms of drug-induced hyperbilirubinemia, and thereby distinguish benign from clinically important elevations in serum bilirubin.

Efavirenz Causes Oxidative Stress, Endoplasmic Reticulum Stress, and Autophagy in Endothelial Cells.

The non-nucleoside reverse transcriptase inhibitor efavirenz is a widely prescribed antiretroviral drug used in combined antiretroviral therapy. Despite being an essential and life-saving medication, the required lifelong use of HIV drugs has been associated with a variety of adverse effects, including disturbances in lipid metabolism and increased cardiovascular risk. Efavirenz belongs to those HIV drugs for which cardiovascular and endothelial dysfunctions have been reported. It is here shown that elevated concentrations of efavirenz can inhibit endothelial meshwork formation on extracellular matrix gels by normal and immortalized human umbilical vein cells. This inhibition was associated with an increase in oxidative stress markers, endoplasmic reticulum (ER) stress markers, and autophagy. Induction of ER stress occurred at pharmacologically relevant concentrations of efavirenz and resulted in reduced proliferation and cell viability of endothelial cells, which worsened in the presence of elevated efavirenz concentrations. In combination with the HIV protease inhibitor nelfinavir, both oxidative stress and ER stress became elevated in endothelial cells. These data indicate that pharmacologically relevant concentrations of efavirenz can impair cell viability of endothelial cells and that these effects may be aggravated by either elevated concentrations of efavirenz or by a combined use of efavirenz with other oxidative stress-inducing medications.

Induction of suicidal erythrocyte death by nelfinavir.

The HIV protease inhibitor, nelfinavir, primarily used for the treatment of HIV infections, has later been shown to be effective in various infectious diseases including malaria. Nelfinavir may trigger mitochondria-independent cell death. Erythrocytes may undergo eryptosis, a mitochondria-independent suicidal cell death characterized by cell shrinkage and phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include oxidative stress and increase of cytosolic Ca2+-activity ([Ca2+]i). During malaria, accelerated death of infected erythrocytes may decrease parasitemia and thus favorably influence the clinical course of the disease. In the present study, phosphatidylserine abundance at the cell surface was estimated from annexin V binding, cell volume from forward scatter, reactive oxidant species (ROS) from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, and [Ca2+]i from Fluo3-fluorescence. A 48 h treatment of human erythrocytes with nelfinavir significantly increased the percentage of annexin-V-binding cells (≥5µg/mL), significantly decreased forward scatter (≥2.5µg/mL), significantly increased ROS abundance (10 µg/mL), and significantly increased [Ca2+]i (≥5 µg/mL). The up-regulation of annexin-V-binding following nelfinavir treatment was significantly blunted, but not abolished by either addition of the antioxidant N-acetylcysteine (1 mM) or removal of extracellular Ca2+. In conclusion, exposure of erythrocytes to nelfinavir induces oxidative stress and Ca2+ entry, thus leading to suicidal erythrocyte death characterized by erythrocyte shrinkage and erythrocyte membrane scrambling.

Nelfinavir and nelfinavir analogs block site-2 protease cleavage to inhibit castration-resistant prostate cancer.

Nelfinavir and its analogs inhibit proliferation and induce apoptosis of castration-resistant prostate cancer through inhibition of site-2 protease (S2P) activity, which leads to suppression of regulated intramembrane proteolysis. Western blotting in nelfinavir and its analog treated cells confirms accumulation of precursor SREBP-1 and ATF6. Nelfinavir and its analogs inhibit human homolog M. jannaschii S2P cleavage of an artificial protein substrate CED-9 in an in vitro proteolysis assay in a dose-dependent manner. Nelfinavir and its analogs are more potent inhibitors of S2P cleavage activity than 1,10-phenanthroline, a metalloprotease-specific inhibitor. Further, cluster analysis of gene expression from treated DU145 and PC3 cell lines demonstrate a close similarity of nelfinavir, its analogs, and 1,10-phenanthroline. These results show nelfinavir and its analogs inhibit castration-resistant prostate cancer proliferation by blocking regulated intramembrane proteolysis through suppression of S2P cleavage activity. This leads to accumulation of precursor SREBP-1 and ATF6, and development of insufficient reserves of their transcriptionally-active forms. The present results validate S2P and regulated intramembrane proteolysis as novel therapeutic targets for castration-resistant prostate cancer therapeutics. A clinical trial of nelfinavir or its analogs should be developed for castration-resistant prostate cancer.

Human immunodeficiency virus protease inhibitors interact with ATP binding cassette transporter 4/multidrug resistance protein 4: a basis for unanticipated enhanced cytotoxicity.

Human immunodeficiency virus (HIV) pharmacotherapy, by combining different drug classes such as nucleoside analogs and HIV protease inhibitors (PIs), has increased HIV-patient life expectancy. Consequently, among these patients, an increase in non-HIV-associated cancers has produced a patient cohort requiring both HIV and cancer chemotherapy. We hypothesized that multidrug resistance protein 4/ATP binding cassette transporter 4 (MRP4/ABCC4), a widely expressed transporter of nucleoside-based antiviral medications as well as cancer therapeutics might interact with PIs. Among the PIs evaluated (nelfinavir, ritonavir, amprenavir, saquinavir, and indinavir), only nelfinavir both effectively stimulated MRP4 ATPase activity and inhibited substrate-stimulated ATPase activity. Saos2 and human embryonic kidney 293 cells engineered to overexpress MRP4 were then used to assess transport and cytotoxicity. MRP4 expression reduced intracellular accumulation of nelfinavir and consequently conferred survival advantage to nelfinavir cytotoxicity. Nelfinavir blocked Mrp4-mediated export, which is consistent with its ability to increase the sensitivity of MRP4-expressing cells to methotrexate. In contrast, targeted inactivation of Abcc4/Mrp4 in mouse cells specifically enhanced nelfinavir and 9-(2-phosphonylmethoxyethyl) adenine cytotoxicity. These results suggest that nelfinavir is both an inhibitor and substrate of MRP4. Because nelfinavir is a new MRP4/ABCC4 substrate, we developed a MRP4/ABCC4 pharmacophore model, which showed that the nelfinavir binding site is shared with chemotherapeutic substrates such as adefovir and methotrexate. Our studies reveal, for the first time, that nelfinavir, a potent and cytotoxic PI, is both a substrate and inhibitor of MRP4. These findings suggest that HIV-infected cancer patients receiving nelfinavir might experience both enhanced antitumor efficacy and unexpected adverse toxicity given the role of MRP4/ABCC4 in exporting nucleoside-based antiretroviral medications and cancer chemotherapeutics.

Toxicology and carcinogenesis studies of mixtures of 3'-azido-3'-deoxythymidine (AZT), lamivudine (3TC), nevirapine (NVP), and nelfinavir mesylate (NFV) (Cas Nos. 30516-87-1, 134678-17-4, 129618-40-2, 159989-65-8) in B6C3F1 Mice (transplacental exposure studies).

BACKGROUND: Antiretroviral drugs are used to treat patients positive for the human immunovirus HIV-1, and increasingly treatments include a combination of such drugs. The noninfected children of women who are pregnant and receiving such treatment may also be exposed to the drugs by transplacental exposure. We studied the long-term effects of such transplacental exposure in mice by exposing pregnant mice to combinations of four such antiretroviral drugs for seven days and then observing their pups for two years following birth. The four drugs studied were 3'-azido-3'-deoxythymidine (AZT), lamivudine (3TC), nevirapine (NVP), and nelfinavir mesylate (NFV). METHODS: Four different sets of exposure studies were performed: exposure to AZT; to AZT plus 3TC; to AZT, 3TC, and NVP; or to AZT, 3TC, and NFV. In each of these studies, groups of pregnant females were given one of three concentrations of the drug combinations seven times though a tube directly into their stomachs, and after birth their pups were maintained with no further exposure for two years. The offspring of another group of pregnant females not treated with the drugs served as controls. At the end of the study, tissues from more than 40 sites were examined for every animal. RESULTS: Survival of pups whose mothers were exposed to AZT or AZT plus 3TC was similar to their controls, while the survival rates for offspring of mice exposed to AZT, 3TC, and NVP or AZT, 3TC, and NFP were lower than for controls. In most cases the body weights of pups from mothers exposed were slightly less than those of the controls. There were slight increases in the incidences of thyroid gland tumors and skin tumors in the female pups of mothers exposed to AZT alone and of lung tumors in female pups of mothers exposed to AZT plus 3TC. For offspring of mothers exposed to AZT, 3TC, and NVP there were increased incidences of skin tumors in both male and female pups, and more so in the males. CONCLUSIONS: We conclude that exposure to the combination of AZT, 3TC, and NVP during pregnancy caused an increase in skin tumors in the male offspring and possibly also to the female offspring. Exposure to AZT alone during pregnancy may have been related to thyroid gland or skin tumors in female offspring, and exposure to AZT plus 3TC may have been related to lung tumors in female offspring.

Synergistic effects of nelfinavir and bortezomib on proteotoxic death of NSCLC and multiple myeloma cells.

Exploiting protein homeostasis is a new therapeutic approach in cancer. Nelfinavir (NFV) is an HIV protease inhibitor that induces endoplasmic reticulum (ER) stress in cancer cells. Under conditions of ER stress, misfolded proteins are transported from the ER back to the cytosol for subsequent degradation by the ubiquitin-proteasome system. Bortezomib (BZ) is a proteasome inhibitor and interferes with degradation of misfolded proteins. Here, we show that NFV and BZ enhance proteotoxicity in non-small cell lung cancer (NSCLC) and multiple myeloma (MM) cells. The combination synergistically inhibited cell proliferation and induced cell death. Activating transcription factor (ATF)3 and CCAAT-enhancer binding protein homologous protein (CHOP), markers of ER stress, were rapidly increased, and their siRNA-mediated knockdown inhibited cell death. Knockdown of double-stranded RNA activated protein kinase-like ER kinase, a signal transducer in ER stress, significantly decreased apoptosis. Pretreatment with the protein synthesis inhibitor, cycloheximide, decreased levels of ubiquitinated proteins, ATF3, CHOP, and the overall total cell death, suggesting that inhibition of protein synthesis increases cell survival by relieving proteotoxic stress. The NFV/BZ combination inhibited the growth of NSCLC xenografts, which correlated with the induction of markers of ER stress and apoptosis. Collectively, these data show that NFV and BZ enhance proteotoxicity in NSCLC and MM cells, and suggest that this combination could tip the precarious balance of protein homeostasis in cancer cells for therapeutic gain.

Therapeutic effects of Nigella sativa on chronic HAART-induced hyperinsulinemia in rats.

Prolonged use of highly active antiretroviral therapy (HAART) is associated with insulin resistance in HIV-1-positive patients. Small animal models that recapitulate the long-term effects of HAART may facilitate the identification of therapeutic agents to suppress these side effects. We investigated the protective effects of black seed oil (BSO) from Nigella sativa in Sprague-Dawley rats treated with a daily HAART regimen for 7 months. The antiretroviral drugs, consisting of nelfinavir (200 mg/kg), zidovudine (50 mg/kg), and efavirenz (20 mg/kg), were mixed with diet with or without BSO (400 microL/kg) supplementation. Significant increases in insulin and C-peptide levels were observed in HAART-treated groups, and concomitant BSO treatment reduced this hyperinsulinemia. Interestingly, HAART-treated rats showed reduced size of pancreatic islets that was not seen in BSO-exposed rats. In vitro studies showed that nelfinavir, alone and in combination with HAART, induced oxidative stress and decreased glucose-induced insulin production in INS-1 cells. Suppressed insulin production was restored in cells coexposed to either BSO or thymoquinone. Our findings demonstrated that chronic HAART may increase serum insulin levels by dysregulating both insulin production by beta cells and insulin action at the periphery. These deleterious effects may be prevented by dietary supplementation with BSO.

HIV-1 protease inhibitor induced oxidative stress suppresses glucose stimulated insulin release: protection with thymoquinone.

The highly active anti-retroviral therapy (HAART) regimen has considerably reduced the mortality rate in HIV-1 positive patients. However, long-term exposure to HAART is associated with a metabolic syndrome manifesting cardiovascular dysfunction, lipodystrophy, and insulin resistance syndrome (IRS). The inclusion of HIV-1 protease inhibitors (PIs) in HAART has been linked to the induction of IRS. Although several molecular mechanisms of PI-induced effects on insulin action have been postulated, the deleterious effects of PIs on insulin production by pancreatic beta-cells have not been fully investigated and therapeutic strategies to ameliorate insulin dysregulation at this level have not been targeted. The present study showed that exposure to several different PIs, nelfinavir (5-10 microM), saquinavir (5-10 microM) and atazanavir (8-20 microM), decreases glucose stimulated insulin secretion from rat pancreatic beta-cells (INS-1). Nelfinavir significantly increased reactive oxygen species (ROS) generation and suppressed cytosolic, but not mitochondrial superoxide dismutase (SOD) levels. Nelfinvair also decreased both glutathione and ATP and increased UCP2 levels in these cells. Simultaneous treatment with thymoquinone (TQ) (2.5 microM), an active ingredient of black seed oil, significantly inhibited the effect of nelfinavir on augmented ROS production and suppressed SOD levels. Both TQ and black seed oil exposure increased glucose stimulated insulin secretion and ameliorated the suppressive effect of nelfinavir. The present findings imply a direct role of ROS in PI induced deleterious effects on pancreatic beta-cells. Our findings also suggest that TQ may be used as a potential therapeutic agent to normalize the dysregulated insulin production observed in HAART treated patients.

Safety and pharmacokinetics of nelfinavir coadministered with zidovudine and lamivudine in infants during the first 6 weeks of life.

The safety and pharmacokinetics of nelfinavir coadministered with zidovudine and lamivudine were studied in 26 infants during the first 6 weeks of life. Cohort 1 infants (n = 7) received 10 mg/kg 3 times a day, and cohort 2 infants (n = 19) received 40 mg/kg twice a day. Two cohort 1 infants at week 1 and none at week 6 exceeded the target 24-hour area under the curve (AUC) of 30 mug.h/mL, equal to the 10th percentile of the AUC for adults receiving standard nelfinavir dosing. In cohort 2, the median 24-hour AUC was 38 mug.h/mL at both time points, with considerable variability among the infants. Three of 11 cohort 2 infants at week 1 and 4 of 11 at week 6 did not meet the AUC target. Median nelfinavir oral clearance was 2.1 L/h/kg at weeks 1 and 6. The median ratio of the plasma concentrations of the nelfinavir metabolite M8 to unchanged nelfinavir increased from 0.16 (range: 0-0.38) during week 1 to 0.56 (range: 0.4-1.47) during week 6 (P < 0.01). There were no significant differences in any of the other pharmacokinetic parameters when week 1 and week 6 results were compared. Few adverse events were attributed to nelfinavir. These data suggest that nelfinavir is well tolerated in infants at these doses, but exposure was frequently less than that seen in adults taking standard nelfinavir dosing. Further investigations of larger doses, such as 75 mg/kg twice a day, should be undertaken.

Immunotoxicity evaluation of nelfinavir in rats.

The objective of these investigations was to determine whether exposure to the HIV-1 protease inhibitor nelfinavir compromises immune function in Sprague Dawley rats. Animals (20/sex per group) were exposed orally for 1 or 6 months to nelfinavir at doses of 0 (1% carboxymethylcellulose vehicle), 100, 300 or 1000 mg/kg per day. Animals were observed daily for morbidity/mortality and for clinical signs of toxicity. Body weights were recorded weekly (weeks 1-14) and then monthly thereafter and at study termination. At termination (1 month or 6 months; 10/sex per group), serum was collected and retained for toxicokinetic analysis. The spleen, thymus and liver were removed from each animal and weighed; thymuses and liver were discarded after weighing. Spleens were prepared and immunophenotyping, natural killer (NK) cell activity, and proliferative responses to mitogenic stimuli (e.g., concanavalin A, Salmonella typhimurium) were evaluated. There were no treatment-related effects on immune cell populations (absolute or percent values) or in proliferative responses. At the 1-month interval, a decrease in NK cell activity (0.45-fold control) was noted in male rats at 100 and 1000 mg/kg per day but not at the middle dose of 300 mg/kg per day. Female rats at 1 month were noted for an increase in NK cell activity (1.4-fold control) at 100 mg/kg per day, but there was no difference in the NK response between vehicle-treated animals and those exposed to higher doses of nelfinavir. No effects on NK activity were noted in female animals after 6 months of nelfinavir treatment. Assay difficulties prevented evaluation of male rats at the 6-month interval. Taken together, the absence of a dose-response effect for NK activity in male rats treated for 1 month, the lack of suppressive effects in females treated for either 1 or 6 months, and the unchanged splenic NK cell numbers in nelfinavir-treated animals at both 1 and 6 months suggest that the decreased NK activity noted in male rats at 1 month is not biologically relevant. It was therefore concluded that, under the experimental conditions used, oral treatment with nelfinavir for 1 or 6 months at doses up to 1000 mg/ kg per day is not immunosuppressive in rats. C8hr values following nelfinavir treatment at 1000 mg/kg per day for 6 months were between 1- and 2.7-fold the reported Cmax values in humans.

Safety of nelfinavir use during pregnancy. An experimental approach in rats.

This experimental study aimed to evaluate the safety of nelfinavir when administered in normal up to high doses during the entire period of rat pregnancy. The renal and liver compartments of both mothers and fetuses were studied. For this purpose, three groups of pregnant rats were treated with nelfinavir (E1 = 40 mg/kg; E2 = 120 mg/kg; E3 = 360 mg/kg; no. = 10 in every group) from "zero" up to the 20th day of gestation. These doses were divided into two daily administrations by gavage. Controls (no. = 10) received distilled water in the same schedule. At term-pregnancy, the rats were deeply anesthesized and blood samples were collected for alanine and aspartate aminotransferases, creatinine and urea determinations. Fragments of maternal and fetal livers and kidneys were taken and processed for histopathological study. In all groups blood transaminases were within the normal limits, as were the levels of creatinine and urea, thus indicating that the treatment with nelfinavir during the entire gestation was essentially devoid of liver or kidney effects which could result in altered metabolic parameters. Morphological (light microscopy) studies revealed that no significant effects of the drug could be detected regarding either maternal or fetal organs of the E1 and E2 groups. However, the maternal hepatocytes in the E3 group showed heterochromatic nuclei. In addition, there was some fatty infiltration, congested sinusoids and portal dilatation. It is concluded that only doses of nelfinavir used during the entire gestation in doses well above the usual human doses could be considered to be potentially hepatotoxic for the pregnant rat.

Increase in thyroid follicular cell tumors in nelfinavir-treated rats observed in a 2-year carcinogenicity study is consistent with a rat-specific mechanism of thyroid neoplasia.

The carcinogenic potential of nelfinavir mesylate (nelfinavir) was evaluated in a 2-year oral (gavage) study on Sprague-Dawley rats at dose levels of 0 (control), 0 (vehicle control), 100, 300 and 1000 mg/kg per day. At the end of the treatment, increased incidences of thyroid follicular cell hyperplasia and neoplasms were observed at 300 (males) and 1000 mg/kg per day (both sexes). There were no other treatment-related effects and no tumors at other sites. Results from previous studies indicated a number of effects in the liver and thyroid, as well as metabolic profiles that suggested nelfinavir might cause thyroid hyperplasia/neoplasia secondary to hormone imbalance by altering thyroid hormone disposition. To investigate this hypothesis, the effects of nelfinavir on gene expression in rat hepatocytes and liver slices (in vitro), thyroxine plasma clearance, and thyroid gland function were evaluated. Compared to controls, gene expression analyses demonstrated an increased expression of glucuronyltransferase (UDPGT) and CYP450 3A1 in nelfinavir-treated rat hepatocytes and liver slices. In rats treated with nelfinavir (1000 mg/kg per day) for 4 weeks, liver weights and centrilobular hepatocellular hypertrophy were increased and minimal to mild diffuse thyroid follicular cell hypertrophy and follicular cell hyperplasia were evident in the thyroid gland. Thyroid-stimulating hormone (TSH) levels were significantly increased (three-fold), while tri-iodothyronine (T3)/tetra-iodothyronine (T4) and reverse T3(rT3) levels were unchanged, indicating that a compensated state to maintain homeostasis of T3/T4 had been achieved. Plasma 125I-thyroxine clearance was increased and the plasma thyroxine AUC0-48 was decreased (24%) compared to control. In conclusion, these data indicate that thyroid neoplasms observed in the nelfinavir-treated rats were secondary to thyroid hormone imbalance. Increased thyroxine clearance contributes to the effects of nelfinavir on thyroid gland function and is probably a result of UDPGT induction that leads to elevated TSH levels in the rat and eventual thyroid neoplasia. These results are consistent with a well-recognized rat-specific mechanism for thyroid neoplasms.

Risk of birth defects associated with nelfinavir exposure during pregnancy.

OBJECTIVE: The objective of this study was to examine the human teratogenic risk of the protease inhibitor, nelfinavir mesylate, used to treat human immunodeficiency virus. METHODS: This study used a subset of data from the Antiretroviral Pregnancy Registry, which was designed to monitor prenatal exposures to antiretroviral therapy and detect a potential increase in the risk of birth defects. The registry uses a prospective exposure-registration cohort design. All records of pregnant women exposed to nelfinavir, used alone or in combination, were extracted and analyzed. The prevalence of birth defects was compared with the Centers for Disease Control and Prevention's (CDC) population-based surveillance system. RESULTS: Through July 2002, the registry had monitored 915 live births exposed to nelfinavir. Among 301 first-trimester exposures, there were 9 birth defects, for a prevalence of 3% (95% confidence interval 1.4, 5.6). This rate is not significantly different from the CDC's system, which had a prevalence of 3.1 per 100 live births (95% confidence interval 3.1, 3.2; P =.99). There was no consistent pattern among reported birth defects. CONCLUSION: Adequate numbers of first-trimester exposures to nelfinavir have been monitored to detect a 2-fold increase in the prevalence of overall birth defects. No such increases have been detected when compared with the CDC rate. However, the numbers are not sufficient to detect any increased rate of specific defects. Although nelfinavir should only be used in pregnancy if the benefits outweigh the potential risks, the findings from this study should provide some assurance. LEVEL OF EVIDENCE: III

Acidosis láctica y terapia anti-VIH: una asociación infrecuente / Lactic acidosis and human immunodeficiency virus therapy: an infrequent association

La toxicidad mitocondrial es un efecto adverso poco frecuente del tratamiento con algunos antirretrovirales que, entre otros síntomas, se manifiesta por acidosis láctica. Describimos el caso de una paciente con infección por el virus de la inmunodeficiencia humana (VIH) que había recibido tratamiento con antirretrovirales del tipo inhibidores de la transcriptasa inversa e inhibidores de la proteasa durante un año y que ingresó en la unidad de cuidados intensivos (UCI) por un cuadro de shock e insuficiencia renal aguda oligúrica. En los diferentes estudios realizados, destacaba una acidosis láctica grave. A pesar de la retirada del tratamiento con antirretrovirales la paciente evolucionó hacia el fracaso multiorgánico (respiratorio, hemodinámico, renal y hematológico), y falleció a las 20 horas del ingreso (AU)

Absence of embryo-fetal toxicity in rats or rabbits following oral dosing with nelfinavir.

The potential for nelfinavir mesylate (VIRACEPT) to induce maternal and embryo-fetal toxicity was evaluated in rats and rabbits following oral administration. The drug was administered by gavage to rats at doses of 200, 500, or 1000mg/kg/day on days 6-17 of gestation and to rabbits at doses of 200, 400, or 1000mg/kg/day on days 7-20 of gestation. Dams and does were euthanized on GD20 and 29, respectively, and the offspring were weighed and examined for external, visceral, and skeletal alterations. Maximum plasma nelfinavir concentrations (C(max)) in rats were comparable to C(max) values in humans and were 3- to 6-fold higher than the reported human trough levels, while plasma nelfinavir levels in rabbits were approximately 0.13-0.17x the human C(max) and 0.25-0.5x the human trough. In rats, no treatment-related maternal or embryo-fetal toxicity was observed at any dose level and the NOAEL for both maternal and fetal toxicity was considered to be 1000mg/kg/day. Two rabbits in the 400mg/kg/day group died prior to scheduled termination. Because no deaths occurred in the high dose group and there were no other treatment-related signs of clinical toxicity in any dose group, these deaths were considered unrelated to nelfinavir. Group mean body weight loss in rabbits was observed at 1000mg/kg/day on gestation days 7-10. Food consumption was also reduced in this treatment group throughout the dosing period. There were no treatment-related findings in other maternal or fetal parameters. Thus, the no-observed-adverse-effect-level (NOAEL) for maternal toxicity in the rabbit was considered to be 400mg/kg/day (based on maternal body weight loss in the high dose group), while the NOAEL for embryo-fetal toxicity in the rabbit was considered to be 1000mg/kg/day. Thus, under the conditions of this study, nelfinavir was not considered to be toxic to the rat or rabbit conceptus.

Absence of reproductive and developmental toxicity in rats following oral dosing with nelfinavir.

The potential for nelfinavir mesylate (VIRACEPT) to produce reproductive toxicity was evaluated in rats administered oral doses of 200, 500, or 1000mg/kg/day. In the fertility and early embryonic development to implantation study, male rats were treated beginning 28 days prior to mating until necropsy and females for 2 weeks prior to mating and through gestation day (GD) 7. In the pre- and postnatal development study, pregnant rats were treated from GD 6 through lactation day (LD) 20. Selected F(1) pups from this study were evaluated in sensory and behavioral tests and were subsequently mated. Pregnant F(1) females were euthanized on GD 20 and their F(2) fetuses were examined. F(1) animals were not directly dosed with the drug. No treatment-related effects were observed on any male reproductive parameters. Administration of nelfinavir did not produce adverse effects on fertility, pregnancy, embryo-fetal development, parturition, or lactation in the F(0) generation. Similarly, no adverse effects of nelfinavir treatment were observed on pre- and postnatal growth, development, reproductive performance and embryo-fetal development in the F(1) offspring. Based on the results of this study, the no-observed-adverse-effect-level (NOAEL) for developmental and reproductive toxicity in rats was considered to be 1000mg/kg/day, the highest dose tested.

Interaction of anti-HIV protease inhibitors with the multidrug transporter P-glycoprotein (P-gp) in human cultured cells.

The anti-HIV protease inhibitors represent a new class of agents for treatment of HIV infection. Saquinavir, ritonavir, indinavir, and nelfinavir are the first drugs approved in this class and significantly reduce HIV RNA copy number with minimal adverse effects. They are all substrates of cytochrome P450 3A4, and are incompletely bioavailable. The drug transporting protein, P-glycoprotein (P-gp), which is highly expressed in the intestinal mucosa, could be responsible for the low oral bioavailability of these and other drugs which are substrates for this transporter. To determine whether these protease inhibitors are modulators of P-gp, we studied them in cell lines which do and do not express P-gp. Saquinavir, ritonavir and nelfinavir significantly inhibited the efflux of [3H]paclitaxel and [3H]vinblastine in P-gp-positive cells, resulting in an increase in intracellular accumulation of these drugs. However, similar concentrations of indinavir did not affect the accumulation of these anticancer agents. In photoaffinity labeling studies, saquinavir and ritonavir displaced [3H]azidopine, a substrate for P-gp, in a dose-dependent manner. These data suggest that saquinavir, ritonavir, and nelfinavir are inhibitors and possibly substrates of P-gp. Because saquinavir has a low bioavailability, its interaction with P-gp may be involved in limiting its absorption.