Nelfinavir Research Articles

Nelfinavir Induces Liposarcoma Apoptosis through Inhibition of Regulated Intramembrane Proteolysis of SREBP-1 and ATF6

We previously reported that nelfinavir (NFV) induces G(1) cell-cycle block and apoptosis selectively in liposarcoma cell lines due to increased SREBP-1 (sterol regulatory element binding protein-1) expression in the absence of increased transcription. We postulate that NFV interferes with regulated intramembrane proteolysis of SREBP-1 and ATF6 (activating transcription factor 6). Time-lapse, confocal microscopic studies show that NFV inhibits the nuclear translocation of full-length SREBP-1-EGFP and ATF6-EGFP fusion proteins. siRNA-mediated knockdown of site-1 protease (S1P) and/or site-2 protease (S2P) leads to inhibition of SREBP-1 intracellular trafficking to the nucleus and reduces liposarcoma cell proliferation. Treatment of LiSa-2 liposarcoma cells with 3,4-dichloroisocoumarin, a serine protease inhibitor of S1P, did not affect SREBP-1 processing. In contrast, 1,10-phenanthroline, an S2P-specific inhibitor, reproduces the molecular and biological phenotypes observed in NFV-treated cells, which implicates S2P as a target of NFV. In vivo evaluation of NFV in a murine liposarcoma xenograft model leads to inhibition of tumor growth without significant toxicity. NFV-induced upregulation of SREBP-1 and ATF6 results from inhibition of S2P, which together with S1P mediates regulated intramembrane proteolysis from their precursor to their transcriptionally active forms. The resulting endoplasmic reticulum (ER) stress and concurrent inhibition of the unfolded protein response induce caspase-mediated apoptosis. These results provide new insight into the mechanism of NFV-mediated induction of ER stress and cell death in liposarcomas and are the first to report targeting S2P for cancer therapy.

Complex Drug Interactions of HIV Protease Inhibitors 1: Inactivation, Induction, and Inhibition of Cytochrome P450 3A by Ritonavir or Nelfinavir

Conflicting drug-drug interaction (DDI) studies with the HIV protease inhibitors (PIs) suggest net induction or inhibition of intestinal or hepatic CYP3A. As part of a larger DDI study in healthy volunteers, we determined the effect of extended administration of two PIs, ritonavir (RTV) or nelfinavir (NFV), or the induction-positive control rifampin on intestinal and hepatic CYP3A activity as measured by midazolam (MDZ) disposition after a 14-day treatment with the PI in either staggered (MDZ ∼12 h after PI) or simultaneous (MDZ and PI coadministered) manner. Oral and intravenous MDZ areas under the plasma concentration-time curves were significantly increased by RTV or NFV and were decreased by rifampin. Irrespective of method of administration, RTV decreased net intestinal and hepatic CYP3A activity, whereas NFV decreased hepatic but not intestinal CYP3A activity. The magnitude of these DDIs was more accurately predicted using PI CYP3A inactivation parameters generated in sandwich-cultured human hepatocytes rather than human liver microsomes.

LC, LC–MS/TOF and MS n studies for the identification and characterization of degradation products of nelfinavir mesylate

The objective of the present investigation was to separate, identify and characterize the major degradation products (DPs) of nelfinavir mesylate generated under hydrolytic, oxidative, photolytic and thermal stress conditions as advised in International Conference on Harmonization (ICH) guideline Q1A(R2). The drug was found to degrade under acidic, basic, oxidative and photolytic stress, while it was stable in neutral and thermal stress conditions. A total of three degradation products were formed, which were separated on a C-18 column employing a gradient HPLC method. A complete mass fragmentation pathway of the drug was first established with the help of multi-stage (MS n ) and MS/TOF accurate mass studies. Then stressed samples were subjected to LC–MS/TOF studies, which provided their fragmentation pattern and accurate masses. The mass spectral data were employed to characterize the DPs and assign structures to them. The total information was also used to establish the degradation pathway of the drug. The degradation products were identified as 3-hydroxy- N-((2 R,3 R)-3-hydroxy-1-(phenylthio)butan-2-yl)-2-methylbenzamide and (3 S,4a S,8a S)- N- tert-butyl-2-((2 R,3 R)-2-hydroxy-3-(3-hydroxy-2-methylbenzamido)-4-(phenylsulfinyl)butyl)decahydroisoquinoline-3-carboxamide.

Resistant mechanism against nelfinavir of subtype C human immunodeficiency virus type 1 proteases

Human immunodeficiency virus type 1 protease (HIV-1 PR) is one of the major targets of anti-AIDS drug discovery, and the subtype C HIV-1 is infecting more and more humans. In this work, we executed computational simulations of subtype B (labeled B(WT)) HIV-1 PR, D30N mutant B (labeled B(D30N)) HIV-1 PR, subtype C (labeled C(Ref)) HIV-1 PR, D30N mutant C (labeled C(D30N)) HIV-1 PR, and D30N/N83T mutant C (labeled C(D30N/N83T)) HIV-1 PR with drug nelfinavir (NFV), aiming at clarifying (1) the resistant mechanism against NFV due to D30N mutation in subtype C HIV-1 PR; (2) the reason that the emergence rate of N83T mutation in C(D30N) HIV-1 PR is higher than that in B(D30N) HIV-1 PR; (3) the affinity of NFV with C(D30N/N83T) HIV-1 PR is higher than B(D30N) and C(D30N) HIV-1 PRs. The results indicate: (1) D30N mutation in subtype C HIV-1 PR reduces the hydrogen bond between the 30th residue and NFV, and the binding free energy contributions of some residues decrease; (2) the hydrogen bonds between the 83th/83′th residue and the 34th/34′th residue exist in both B(D30N) and C(D30N/N83T) HIV-1 PRs, while they disappear in C(D30N) HIV-1 PR. Meanwhile, the binding free energy contribution of N30 in C(D30N) is lower than that in B(D30N) and C(D30N/N83T); (3) N83T mutation makes some residues dislocate, and the contributions of these residues to binding free energy in C(D30N/N83T) increase comparing to those in B(D30N) and C(D30N). Our findings suggest that despite the nonactive site mutations, the polymorphisms regulate the emergence rates of these drug-resistant mutants.

Evaluation of HIV protease and nucleoside reverse transcriptase inhibitors on proliferation, necrosis, apoptosis in intestinal epithelial cells and electrolyte and water transport and epithelial barrier function in mice

BackgroundProtease inhibitors (PI's) and reverse transcriptase drugs are important components of highly active antiretroviral therapy (HAART) for treating human acquired immunodeficiency syndrome (AIDS). Long-term clinical therapeutic efficacy and treatment compliance of these agents have been limited by undesirable side-effects, such as diarrhea. This study aims to investigate the effects of selected antiretroviral agents on intestinal histopathology and function in vivo and on cell proliferation and death in vitro.MethodsSelected antiretroviral drugs were given orally over 7 days, to Swiss mice, as follows: 100 mg/kg of nelfinavir (NFV), indinavir (IDV), didanosine (DDI) or 50 mg/kg of zidovudine (AZT). Intestinal permeability measured by lactulose and mannitol assays; net water and electrolyte transport, in perfused intestinal segments; and small intestinal morphology and cell apoptosis were assessed in treated and control mice. In vitro cell proliferation was evaluated using the WST-1 reagent and apoptosis and necrosis by flow cytometry analysis.ResultsNFV, IDV, AZT and DDI caused significant reductions in duodenal and in jejunal villus length (p < 0.05). IDV and AZT increased crypt depth in the duodenum and AZT increased crypt depth in the jejunum. NFV, AZT and DDI significantly decreased ileal crypt depth. All selected antiretroviral drugs significantly increased net water secretion and electrolyte secretion, except for DDI, which did not alter water or chloride secretion. Additionally, only NFV significantly increased mannitol and lactulose absorption. NFV and IDV caused a significant reduction in cell proliferation in vitro at both 24 h and 48 h. DDI and AZT did not alter cell proliferation. There was a significant increase in apoptosis rates in IEC-6 cells after 24 h with 70 ug/mL of NFV (control: 4.7% vs NFV: 22%) while IDV, AZT and DDI did not show any significant changes in apoptosis compared to the control group. In jejunal sections, IDV and NFV significantly increased the number of TUNEL positive cells.ConclusionThe PI's, NFV and IDV, increased cell apoptosis in vivo, water and electrolyte secretion and intestinal permeability and decreased villus length and cell proliferation. NFV was the only drug tested that increased cell apoptosis in vitro. The nucleoside reverse transcriptase inhibitors, AZT and DDI, did not affect cell apoptosis or proliferation. These findings may partly explain the intestinal side-effects associated with PI's.

Estimating the individualized HIV-1 genetic barrier to resistance using a nelfinavir fitness landscape

BackgroundFailure on Highly Active Anti-Retroviral Treatment is often accompanied with development of antiviral resistance to one or more drugs included in the treatment. In general, the virus is more likely to develop resistance to drugs with a lower genetic barrier. Previously, we developed a method to reverse engineer, from clinical sequence data, a fitness landscape experienced by HIV-1 under nelfinavir (NFV) treatment. By simulation of evolution over this landscape, the individualized genetic barrier to NFV resistance may be estimated for an isolate.ResultsWe investigated the association of estimated genetic barrier with risk of development of NFV resistance at virological failure, in 201 patients that were predicted fully susceptible to NFV at baseline, and found that a higher estimated genetic barrier was indeed associated with lower odds for development of resistance at failure (OR 0.62 (0.45 - 0.94), per additional mutation needed, p = .02).ConclusionsThus, variation in individualized genetic barrier to NFV resistance may impact effective treatment options available after treatment failure. If similar results apply for other drugs, then estimated genetic barrier may be a new clinical tool for choice of treatment regimen, which allows consideration of available treatment options after virological failure.

The Effect of Clade-Specific Sequence Polymorphisms on HIV-1 Protease Activity and Inhibitor Resistance Pathways

The majority of HIV-1 infections around the world result from non-B clade HIV-1 strains. The CRF01_AE (AE) strain is seen principally in Southeast Asia. AE protease differs by approximately 10% in amino acid sequence from clade B protease and carries several naturally occurring polymorphisms that are associated with drug resistance in clade B. AE protease has been observed to develop resistance through a nonactive-site N88S mutation in response to nelfinavir (NFV) therapy, whereas clade B protease develops both the active-site mutation D30N and the nonactive-site mutation N88D. Structural and biochemical studies were carried out with wild-type and NFV-resistant clade B and AE protease variants. The relationship between clade-specific sequence variations and pathways to inhibitor resistance was also assessed. AE protease has a lower catalytic turnover rate than clade B protease, and it also has weaker affinity for both NFV and darunavir (DRV). This weaker affinity may lead to the nonactive-site N88S variant in AE, which exhibits significantly decreased affinity for both NFV and DRV. The D30N/N88D mutations in clade B resulted in a significant loss of affinity for NFV and, to a lesser extent, for DRV. A comparison of crystal structures of AE protease shows significant structural rearrangement in the flap hinge region compared with those of clade B protease and suggests insights into the alternative pathways to NFV resistance. In combination, our studies show that sequence polymorphisms within clades can alter protease activity and inhibitor binding and are capable of altering the pathway to inhibitor resistance.

Understanding the HIV-1 protease nelfinavir resistance mutation D30N in subtypes B and C through molecular dynamics simulations

A major concern in the antiretroviral (ARV) treatment of HIV infections with protease inhibitors (PI) is the emergence of resistance, which results from the selection of distinct mutations within the viral protease (PR) gene. Among patients who do not respond to treatment with the PI nelfinavir (NFV), the D30N mutation is often observed. However, several reports have shown that D30N emerges with different frequencies in distinct HIV-1 genetic forms or subtypes. In the present work, we analyzed the binding of NFV and the Gag substrate CA/p2 to PR from HIV-1 subtypes B and C through molecular dynamics (MD) simulations. The wild-type and drug-resistant D30N mutants were investigated in both subtypes. The compensatory mutations N83T and N88D, observed in vitro and in vivo when subtype C acquires D30N, were also studied. D30N appears to facilitate conformational changes in subtype B PR, but not in that from subtype C, and this could be associated with disestablishment of an α-helical region of the PR. Furthermore, the total contact areas of NFV or the CA/p2 substrate with the mutant PR correlated with changes in the resistance patterns and replicative capacity. Finally, we observed in our MD simulations that mutant PR proteins show different patterns for hydrophobic/van der Waals contact. These findings suggest that different molecular mechanisms contribute to resistance, and we propose that a single mutation has distinct impacts on different HIV-1 subtypes.

Increased Levels of Platelet Microparticles in HIV-Infected Patients With Good Response to Highly Active Antiretroviral Therapy

Increased Levels of Platelet Microparticles in HIV-Infected Patients With Good Response to Highly Active Antiretroviral Therapy

Phase I, pharmacokinetic study of temsirolimus in combination with nelfinavir in patients with solid tumors.

2572 Background: Temsirolimus (CCI-779, TEM) is a selective inhibitor of mTOR with anticancer activity in a limited number of cancer types. A potential mechanism of drug resistance of TEM is feedback activation of the PI3k signaling pathway. Dual inhibition of PI3 kinase and mTOR may overcome this resistance. Nelfinavir (NFV), a human immunodeficiency protease inhibitor and a strong CYP3A4 inhibitor, has PI3kinase-inhibiting activity. In this phase I dose-escalation study TEM and NFV were combined (start dose of weekly 10 mg IV TEM infusion and daily 1,500 mg oral NFV in a 3-week cycle). Methods: Standard dose escalation was used in cohorts of 3-6 patients (pts). PK profiles of TEM, its active metabolite sirolimus (SIR), and NFV were obtained in each pt after the first administration and after combination of TEM and NFV (D1, D4, and D11of Cycle 1). Peripheral blood mononuclear cells and, where possible, tumor material of pts were obtained to determine the PD effects. Results: Pretreated pts received NFV at dose level 1 (1,500 mg, 7 pts) and dosel evel 0 (1,000 mg, 3 pts) in combination with 10mg TEM. Pts (6 females, 4 males, median age: 49 years [24-66], WHO 0-2, main tumor types: pancreas, glioblastoma, sarcoma, and galbladder) received up to 6 cycles. The most common reason for treatment discontinuation was disease progression. DLT was seen in 2 pts at dose level 1 (1,500 mg NFV and 10 mg TEM): grade 3 mucositis and nausea. Ulcerative mucositis was the most common toxicity (all grades) (7/10pts), then nausea (5/10 pts) and diarrhea (5/10pts). Neither CRs nor PRs were observed. TEM plasma exposure alone and in combination with NFV did not change (mean AUC0-24 1,435 vs. 1,250 nghr/mL, respectively), while SIR plasma exposure increased when TEM was combined with NFV (mean AUC0-24 678 vs. 1,050 respectively, p<0.05). NFV showed a high interpatient variability alone as well as in combination with TEM (CV= 55 and 71%, respectively). Pharmacodynamic investigations are ongoing. Conclusions: Nelfinavir 1,000 mg/day and temsirolimus 10 mg/wk can be safely combined. DLT was mucositis and nausea. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Wyeth

Phase I study of nelfinavir (NFV) in liposarcoma (LPS).

e13538 Background: LPS's are the most common type of soft-tissue sarcoma. Chemotherapy for recurrent LPS is only modestly active. We began investigating the HIV protease inhibitor (PI), NFV, for LPS therapy because of its association with the HIV PI-induced Lipodystrophy Syndrome. We hypothesized that adipocytic tumors respond similarly to NFV as normal adipocytes. We report the results of a phase I study of NFV for LPS. Methods: Patients (pts) with recurrent, surgically unresectable LPS were eligible. There was no limit to prior chemotherapy regimens. Additional eligibility included: Age ≥ 18 y, ECOG 0-2, and normal organ function. The starting dose of NFV was 1,250 mg BID (level 1). Doses were escalated in cohorts of 3 up to a maximal dose of 4,250 mg BID (level 5). A cycle was defined as 28 days. Steady-state pharmacokinetics (PK) for NFV and its primary active metabolite, M8, were determined on Day 1 Cycle 2 at levels 4 (3,000 mg BID) and 5. Results: 20 pts (13 M; 7 F) were enrolled (age range 37-81y; median age 64y). 1 pt at level 1 experienced transient, grade 3 pancreatitis after 1 wk of NFV, and was inevaluable for response. No other dose-limiting toxicities were observed. The length of NFV treatment ranged from 0.6 to 13.5 mos (median 3 mos). Response data include: 1 PR (unconfirmed), 5 SD on fist evaluation after 3 cycles, 13 PD, and 1 inevaluable. PK data are available from six subjects at level 4 and three at level 5. Interestingly, mean peak plasma levels and AUCs for NFV were higher at the 3,000 (7.3 mg/L; 60.9 mg/L × hr) vs. 4,250 mg BID dose level (6.3 mg/L; 37.7 mg/L × hr). Additionally, time to peak plasma concentration of NFV and M8 occurred later (4-6 h) with 3,000 mg vs. 2 h for the 4,250 mg dose. The mean ratio of M8:NFV AUCs for both dose levels was ∼1:3. Peak plasma concentrations were within the range where anticancer activity was demonstrated in vitro. Conclusions: The PK data demonstrate auto-induction of NFV clearance at the doses studied, although the mechanism remains unclear. M8 metabolite is present at ∼1/3 the level of the parent NFV, and may also contribute to the anticancer activity seen. In accordance with the phase I results, a phase II study of NFV for LPS at 3,000 mg BID is ongoing to better define its activity. Supported by R01FD003006. No significant financial relationships to disclose.

HIV-1 Protease Codon 36 Polymorphisms and Differential Development of Resistance to Nelfinavir, Lopinavir, and Atazanavir in Different HIV-1 Subtypes

The amino acid at position 36 of the HIV-1 protease differs among various viral subtypes, in that methionine is usually found in subtype B viruses but isoleucine is common in other subtypes. This polymorphism is associated with higher rates of treatment failure involving protease inhibitors (PIs) in non-subtype B-infected patients. To investigate this, we generated genetically homogeneous wild-type viruses from subtype B, subtype C, and CRF02_AG full-length molecular clones and showed that subtype C and CRF02_AG I36 viruses exhibited higher levels of resistance to various PIs than their respective M36 counterparts, while the opposite was observed for subtype B viruses. Selections for resistance with each variant were performed with nelfinavir (NFV), lopinavir (LPV), and atazanavir (ATV). Sequence analysis of the protease gene at week 35 revealed that the major NFV resistance mutation D30N emerged in NFV-selected subtype B viruses and in I36 subtype C viruses, despite polymorphic variation. A unique mutational pattern developed in subtype C M36 viruses selected with NFV or ATV. The presence of I47A in LPV-selected I36 CRF02_AG virus conferred higher-level resistance than L76V in LPV-selected M36 CRF02_AG virus. Phenotypic analysis revealed a >1,000-fold increase in NFV resistance in I36 subtype C NFV-selected virus with no apparent impact on viral replication capacity. Thus, the position 36 polymorphism in the HIV-1 protease appears to have a differential effect on both drug susceptibility and the viral replication capacity, depending on both the viral subtype and the drug being evaluated.

Non‐medically supervised treatment interruptions among participants in a universally accessible antiretroviral therapy programme

We examined clinical outcomes, patient characteristics and trends over time of non-medically supervised treatment interruptions (TIs) from a free-of-charge antiretroviral therapy (ART) programme in British Columbia (BC), Canada. Data from ART-naïve individuals > or =18 years old who initiated triple combination highly active antiretroviral therapy (HAART) between January 2000 and June 2006 were analysed. Participants having > or =3 month gap in HAART coverage were defined as having a TI. Cox proportional hazards modelling was used to examine factors associated with TIs and to examine factors associated with resumption of treatment. A total of 1707 participants were study eligible and 643 (37.7%) experienced TIs. TIs within 1 year of ART initiation decreased from 29% of individuals in 2000 to 19% in 2006 (P<0.001). TIs were independently associated with a history of injection drug use (IDU) (P=0.02), higher baseline CD4 cell counts (P<0.001), hepatitis C co-infection (P<0.001) and the use of nelfinavir (NFV) (P=0.04) or zidovudine (ZDV)/lamivudine (3TC) (P=0.009) in the primary HAART regimen. Male gender (P<0.001), older age (P<0.001), AIDS at baseline (P=0.008) and having a physician who had prescribed HAART to fewer patients (P=0.03) were protective against TIs. Four hundred and eighty-eight (71.9%) participants eventually restarted ART with male patients and those who developed an AIDS-defining illness prior to their TI more likely to restart therapy. Higher CD4 cell counts at the time of TI and unknown hepatitis C status were associated with a reduced likelihood of restarting ART. Treatment interruptions were associated with younger, less ill, female and IDU participants. Most participants with interruptions eventually restarted therapy. Interruptions occurred less frequently in recent years.

Stress degradation studies of nelfinavir mesylate by Fourier transform infrared spectroscopy

Nelfinavir mesylate is the first nonpeptidic protease inhibitor available in pediatric formulation. In the present paper the stability of nelfinavir mesylate under different stress conditions is evaluated using Fourier transform infrared spectroscopy. The drug is subjected to thermal degradation, photodegradation, acid hydrolysis, base hydrolysis and oxidation as per ICH guidelines. Differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), X-ray diffraction (XRD) and high performance liquid chromatography (HPLC) are carried out to support the implementation of infrared spectroscopy for the stability studies of nelfinavir mesylate. Significant changes are observed in the IR spectra collected after exposing the drug to thermal radiations, acid and base hydrolysis and oxidative degradation. No change is observed in the spectra of the drug after exposing it to sunlight indicating the good photostability of nelfinavir mesylate. The results of infrared spectroscopy agree well with that of other complementary techniques as DSC, TGA, XRD and HPLC.

Mitochondrial T9098C Sequence Change in the MTATP6 Gene and Development of Severe Mitochondrial Disease After In Utero Antiretroviral Prophylaxis

Mitochondrial toxicity is a well-recognized adverse effect of nucleoside reverse transcriptase inhibitor therapy for human immunodeficiency virus (HIV) infection. Transient lactic acidosis is often observed in children born after in utero antiretroviral prophylaxis against mother-to-child transmission of HIV. However, the extent and the mechanism of in utero adverse effects are largely unknown. We describe a 4-year-old girl who presented with manifestations of severe mitochondrial disease, specifically, developmental and growth delay, hypotonia, lactic acidosis, congenital cataracts, and pancreatitis. Her HIV-positive mother was receiving lamivudine, zidovudine, and nelfinavir mesylate during her pregnancy. The child tested HIV negative after birth. She was found to have a homoplastic T9098C sequence change in the mitochondrial gene coding for adenosine 5'-triphosphate synthase 6 (MTATP6) that was previously reported as a mitochondrial polymorphism. This polymorphism is in the MTATP6 gene-coding sequence and leads to the replacement of a nonpolar amino acid with a polar amino acid. Because of the typical clinical manifestations of mitochondrial disorder and because of the nature of the mitochondrial sequence change, the observed polymorphism likely predisposed this patient to develop severe antiretroviral-associated mitochondrial disease. Mitochondrial sequence alterations may be important factors in mitochondrial toxicity associated with antiretroviral treatment. Mitochondrial sequencing may be warranted in cases of persistent lactic acidosis after antiretroviral prophylaxis to further study this association.

Effects of obesity induced by high‐fat diet on the pharmacokinetics of nelfinavir, a HIV protease inhibitor, in laboratory rats

The effect of obesity induced by a high-fat diet on the pharmacokinetics (PK) of nelfinavir (NFV) was investigated, focusing on the change of distribution and elimination caused by dyslipidemia and hepatic steatosis.The plasma unbound fraction (f(u)) of NFV in obese rats (0.61+/-0.03%) was significantly lower than in the control (1.10+/-0.09%), caused by increasing the plasma triglyceride-rich lipoprotein level. After intravenous (i.v.) administration of NFV, the marked decrease of the distribution volume and slower total clearance (39.5% and 69.1% of the control, respectively) caused by the lower f(u) were the main reasons for the significantly higher area under the blood concentration versus time curve (AUC) in obese rats (145.3% of the control). The absorption of NFV after intraduodenal (i.d.) administration in obese rats was significantly greater than in the control (AUC; 170.4% of the control). The increased bile in obese rats was the main reason for the increasing absorption of NFV, and the lower expression of intestinal P-glycoprotein was also considered. On the other hand, although higher AUCs in obese rats were shown, unbound AUCs in the obese rats were slightly lower than in the control, namely, the plasma NFV concentration in obese rats to obtain the same pharmacological effect was higher than in the control, suggesting the difficulty of drug monitoring. These results suggest that it is necessary to pay further attention to therapeutic drug monitoring of NFV in patients manifesting metabolic syndrome, such as dyslipidemia and visceral fat accumulation, including hepatic steatosis.

Signaling pathways in adenoid cystic cancers: Implications for treatment

Adenoid cystic cancers (ACC) in the head and neck are rare yet present a clinical dilemma. Although 5 year survivals are excellent, their have a propensity for late recurrences. Most of these cancers are initially treated with surgery followed by radiation. When recurrences happen, treatment options are limited both by the morbidity and low efficacy of re-irradiation and repeated surgical resection. Reported response rates to chemotherapy are low and targeted therapies may be one option. We, therefore, investigated signaling pathways that may be active in adenoid cystic cancers. Tissues from the last 9 ACC patients resected at the University of Iowa were immunohistochemically stained with antibodies for EGFR, phosphorylated (P) Akt, and P-MAPK in order to molecularly characterize these tumors. An ACC cell line (ACC3) was also characterized by Western blot. We found that seven of the 9 tumor samples had strong expression of P-Akt and 5/9 had P-MAPK. None of them had EGFR expression. In the ACC3 cell line, similar data was found in that there was P-Akt and P-MAPK but no EGFR expression. We tested the HIV protease inhibitor nelfinavir (NFV) which has been shown to inhibit Akt signaling to see its effect on ACC3 cells. Both P-Akt and P-MAPK were inhibited with NFV in ACC3 cells and this resulted in growth inhibition and clonogenic death. In patients where re-irradiation or further surgery is not an option, a trial of NFV may be warranted.

Elevated ethyl methanesulfonate (EMS) in nelfinavir mesylate (Viracept®, Roche): overview

Roche's protease inhibitor nelfinavir mesylate (Viracept®) produced between March 2007-June 2007 was found to contain elevated levels of ethyl methanesulfonate (EMS), a known mutagen (alkylator) – leading to a global recall of the drug. EMS levels in a daily dose (2,500 mg Viracept/day) were predicted not to exceed a dose of ~2.75 mg/day (~0.055 mg/kg/day based on 50 kg patient). As existing toxicology data on EMS did not permit an adequate patient risk assessment, a comprehensive animal toxicology evaluation of EMS was conducted. General toxicity of EMS was investigated in rats over 28 days. Two studies for DNA damage were performed in mice; chromosomal damage was assessed using a micronucleus assay and gene mutations were detected using the MutaMouse transgenic model. In addition, experiments designed to extrapolate animal exposure to humans were undertaken. A general toxicity study showed that the toxicity of EMS occurred only at doses ≥ 60 mg/kg/day, which is far above that received by patients. Studies for chromosomal damage and mutations in mice demonstrated a clear threshold effect with EMS at 25 mg/kg/day, under chronic dosing conditions. Exposure analysis (Cmax) demonstrated that ~370-fold higher levels of EMS than that ingested by patients, are needed to saturate known, highly conserved, error-free, mammalian DNA repair mechanisms for alkylation. In summary, animal studies suggested that patients who took nelfinavir mesylate with elevated levels of EMS are at no increased risk for carcinogenicity or teratogenicity over their background risk, since mutations are prerequisites for such downstream events. These findings are potentially relevant to >40 marketed drugs that are mesylate salts.

Pharmacokinetic study and comparative bioavailability of two nelfinavir tablet formulations in Iranian healthy volunteers after a low-dose administration

The objective of this study is to evaluate pharmacokinetic parameters, bioavailability of a potent HIV protease inhibitor, nelfinavir mesylate (NFV), following a single oral administration of 2 tablet formulations. A randomized, 2-way, crossover, bioequivalence study was conducted in 24 healthy male volunteers to compare 2 brands of nelfinavir 250 mg tablets, Nelfabiovir (Bakhtar Bioshimi, Iran) as test and Viracept (Roche, Germany) as reference product. Blood samples were collected at selected times during 12 h and plasma concentrations were determined with a sensitive and validated HPLC method involving a simple protein precipitation step. Individual pharmacokinetic parameters, t1/2, t1/2(abs), K, Ka, tmax, Cmax, Vd/F, Cl/F, AUC0-12 and AUC0- yen were determined from plasma concentration-time profiles for both formulations and were compared statistically. The analysis of variance (ANOVA) did not show any significant difference between the two formulations and 90% confidence intervals (CI) fell within the acceptable range, satisfying the bioequivalence criteria of the FDA. In vitro parameters of mean dissolution time (MDT) and time for 70% dissolution (T70) were also determined. There was no significant difference between these parameters for two dosage forms (p > 0.05). It was concluded that the two nelfinavir products are bioequivalent with respect to the rate and extent of absorption.

The impact of nelfinavir exposure on cancer development among a large cohort of HIV-infected patients.

Preclinical studies suggest that the antiretroviral agent, nelfinavir mesylate (NFV), may have antineoplastic properties. The relationship between NFV and cancer incidence among HIV-infected patients is unknown. We evaluated the impact of NFV on cancer development in a large cohort of HIV-infected persons with 108 cancer events during 13,421 person-years of follow-up. Using multivariate time-updated Cox proportional hazard models, the risk of cancer among those receiving NFV were compared to those on non-NFV antiretroviral regimens. The risk of cancer among those receiving NFV was similar to those on non-NFV antiretroviral regimens (hazard ratio 1.0, 95% confidence interval 0.5, 1.7, P = 0.90). We also examined AIDS-defining and non-AIDS-defining cancers separately and found no significant associations between NFV use and cancer risk. Antiretroviral use, with or without a protease inhibitor (PI) component, was associated with a reduced risk of AIDS-defining cancers compared with no antiretroviral therapy; however, the risk of cancer was the same among those using PI or PI-sparing regimens. Despite reports that NFV may have tumoricidal activity, we found no significant relationship between NFV or PI use compared with other antiretrovirals and the risk of developing cancer among a large cohort of HIV-infected persons.