Bcl-2 Regulates HIF-1α Protein Stabilization in Hypoxic Melanoma Cells via the Molecular Chaperone HSP90

Hypoxia-Inducible Factor 1 (HIF-1) is a transcription factor that is a critical mediator of the cellular response to hypoxia. Enhanced levels of HIF-1alpha, the oxygen-regulated subunit of HIF-1, is often associated with increased tumour angiogenesis, metastasis, therapeutic resistance and poor prognosis. It is in this context that we previously demonstrated that under hypoxia, bcl-2 protein promotes HIF-1/Vascular Endothelial Growth Factor (VEGF)-mediated tumour angiogenesis.By using human melanoma cell lines and their stable or transient derivative bcl-2 overexpressing cells, the current study identified HIF-1alpha protein stabilization as a key regulator for the induction of HIF-1 by bcl-2 under hypoxia. We also demonstrated that bcl-2-induced accumulation of HIF-1alpha protein during hypoxia was not due to an increased gene transcription or protein synthesis. In fact, it was related to a modulation of HIF-1alpha protein expression at a post-translational level, indeed its degradation rate was faster in the control lines than in bcl-2 transfectants. The bcl-2-induced HIF-1alpha stabilization in response to low oxygen tension conditions was achieved through the impairment of ubiquitin-dependent HIF-1alpha degradation involving the molecular chaperone HSP90, but it was not dependent on the prolyl hydroxylation of HIF-1alpha protein. We also showed that bcl-2, HIF-1alpha and HSP90 proteins form a tri-complex that may contribute to enhancing the stability of the HIF-1alpha protein in bcl-2 overexpressing clones under hypoxic conditions. Finally, by using genetic and pharmacological approaches we proved that HSP90 is involved in bcl-2-dependent stabilization of HIF-1alpha protein during hypoxia, and in particular the isoform HSP90beta is the main player in this phenomenon.We identified the stabilization of HIF-1alpha protein as a mechanism through which bcl-2 induces the activation of HIF-1 in hypoxic tumour cells involving the beta isoform of molecular chaperone HSP90

The effect of fluconazole on ritonavir and saquinavir pharmacokinetics in HIV-1-infected individuals

To study the effect of fluconazole on the steady-state pharmacokinetics of the protease inhibitors ritonavir and saquinavir in HIV-1-infected patients. Five subjects treated with saquinavir and three with ritonavir received the protease inhibitor alone (saquinavir 1200 mg three times daily, ritonavir 600 mg twice daily) on day 1, and the same protease inhibitor in combination with fluconazole (400 mg on day 2 and 200 mg on days 3 to 8). Pharmacokinetic parameters were determined on days 1 and 8. In the saquinavir group, the median increase in the area under the plasma concentration vs time curve was 50% from 1800 microg l(-1) h to 2700 microg l(-1) h (P = 0.04, median increase: 900 microg l(-1) h; 2.5 and 97.5 percentile: 500-1300), and 56% for the peak concentration in plasma (from 550 to 870 microg l(-1), P = 0.04; median increase: 320 microg l(-1) h, 2.5 and 97.5 percentile: 60-450 microg l(-1)). In the ritonavir group, there were no detectable changes in the pharmacokinetic parameters on addition of fluconazole. Because of the favourable safety profile of saquinavir, dose adjustments are probably not necessary with concomitant use of fluconazole, as is the case for ritonavi

Once-daily dosing of saquinavir and low-dose ritonavir in HIV-1-infected individuals: a pharmacokinetic pilot study

To investigate the steady-state pharmacokinetics of a once-daily dosing regimen of saquinavir soft gelatin capsules in combination with a low dose of ritonavir in HIV-1-infected individuals. Open-label, multi-dose, pharmacokinetic pilot study. Seven HIV-1-infected individuals who were treated with saquinavir hard gelatin capsules 400 mg twice daily + ritonavir liquid formulation 400 mg twice daily were switched to saquinavir soft gelatin formulation 1600 mg once daily in combination with ritonavir liquid formulation 200 mg once daily (day 0). Patients were instructed to ingest saquinavir and ritonavir simultaneously in the morning and with a meal. Steady-state pharmacokinetics of saquinavir and ritonavir were assessed during a 24 h dosing interval after 2 weeks of continued therapy (day 14). Plasma saquinavir and ritonavir concentrations were measured using a validated high performance liquid chromatography assay. In addition, plasma HIV-1 RNA, and fasting total cholesterol, high-density lipoprotein, low-density lipoprotein, and triglyceride levels were measured on days 0 and 14. A non-compartmental pharmacokinetic method was used to calculate the area under the plasma concentration versus time curve (AUC[0-24h]), the maximum and trough plasma concentrations (Cmax and Cmin), the time to reach Cmax (Tmax), the elimination half-life (t1/2), the apparent clearance (Cl/F), and the apparent volume of distribution (V/F). Median (range) values of the pharmacokinetic parameters for saquinavir after 2 weeks of treatment were: AUC[0-24h], 19,802h* ng/ml (3720-74,016); Cmax, 2936 ng/ml (573-6848); Cmin, 84 ng/ml (11-854); Tmax, 3.5 h (3.0-4.0), t1/2, 6.8 h (4.6-10.2); Cl/F, 81 l/h (22-430); V/F, 1189 l (215-3086). Ritonavir concentrations were always below the 90% effective concentration of 2100 ng/ml (median Cmax, 1323 ng/ml; range, 692-1528 ng/ml). No significant changes were observed for total serum cholesterol, high-density lipoprotein, and low-density lipoprotein levels between days 0 and 14 (P > or = 0.24). In six out of seven patients the fasting serum triglyceride levels were lower 2 weeks after the treatment switch (median decrease was 32%, P = 0.03). No significant changes in plasma HIV-1 RNA concentrations were observed between days 0 and 14. The regimen was generally well tolerated. This pharmacokinetic study indicates that the combination of 1600 mg of saquinavir (soft gelatin capsules) and 200 mg of ritonavir (liquid formulation) in a once-daily dosing regimen generally results in therapeutic plasma concentrations of saquinavir. Due to the large interindividual variation in saquinavir exposure, the monitoring of saquinavir concentrations in plasma is warranted. These pharmacokinetic findings rationalize the further clinical evaluation of once-daily dosing of this combination of protease inhibitor

Comparison of the plasma pharmacokinetics and renal clearance of didanosine during once and twice daily dosing in HIV-1 infected individuals

OBJECTIVE: To compare the plasma pharmacokinetics of didanosine during once daily (qd) and twice daily (bid) dosing. DESIGN: Open-label, randomized, cross-over study. METHODS: HIV-1 infected patients who used didanosine were randomized to either a qd or a bid dosing regimen of didanosine. The total daily dose of didanosine was identical in both regimens. Seven days after the start of the study, the pharmacokinetic profile of didanosine in plasma and urine was assessed during an 8-h period. The next day, the patient was switched to the opposite dosing regimen, and after another 7 days, the study was concluded by again assessing the plasma and urine pharmacokinetics of didanosine during 8 h. RESULTS: A total of 19 patients completed the study. The pharmacokinetics of didanosine in plasma (with maximum plasma concentration adjusted for dose) and urine were not significantly different in the qd and bid dosing regimen (P > 0.28 for all parameters). CONCLUSION: We conclude that qd dosing of didanosine leads to a similar exposure in plasma as bid dosing (using the same total daily dose). Since qd dosing may lead to improved compliance of patients to regimens containing didanosine, these results provide a rationale for prescribing didanosine in a qd regimen, and is reassuring when we realize that the drug is being administered in a qd dosing regimen on a large scale in clinical practic

Limited sampling strategies for the estimation of the systemic exposure to the HIV-1 nonnucleoside reverse transcriptase inhibitor nevirapine

The objective of this study was to develop and validate a limited sampling strategy (LSS) that allows accurate and precise estimation of the area under the plasma concentration versus time curve (AUC) of nevirapine, when used in the licensed dosage of 200 mg twice daily. Because nevirapine has a long plasma elimination half-life and the plasma concentration shows little variation within the 12-hour dosing interval, the authors also wanted to explore whether a time frame exists for which a single-sample LSS can be applied. Twenty HIV-1-infected individuals receiving steady-state treatment with nevirapine (200 mg twice daily) were enrolled. For the development of the LSS, 10 patients were randomly selected from the study population (index set). The pharmacokinetic results from the other 10 patients (validation set) were used for prospective validation of the proposed LSS. Blood samples were obtained before and 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 10, and 12 hours after ingestion. The relationship between the nevirapine concentration at each of the designated time points and the AUC 12h was evaluated by univariate and multivariate linear regression analysis. At each of the sampling times, a strong correlation was observed between the nevirapine concentration and the corresponding AUC 12h (r > 0.97). This allows for a single-sample LSS, using any time point during the dosing interval. When a single equation is preferred, the concentration of nevirapine in a random sample drawn 2 to 4 hours after ingestion of nevirapine (C 2-4h; in microg/mL) can be used for accurate estimation of the AUC 12h (in h x microg/mL) by using the equation AUC 12h (h x microg/mL) = 11.699 (h) x C 2-4h (microg/mL) - 4.381 (h x microg/mL). Validation of this equation resulted in a predicted AUC 12h that was nonbiased and very precise. These data show that the nevirapine concentration at each time point during the dosing interval can be used for accurate estimation of the AUC 12h. Even more practical, a sample obtained at any time between 2 and 4 hours after ingestion of nevirapine can be used. The authors therefore conclude that less intensive sampling (i.e., a single sample) can readily be used to assess the AUC 12h of nevirapine when used in a dosage of 200 mg twice dail

Saliva as an alternative body fluid for therapeutic drug monitoring of the nonnucleoside reverse transcription inhibitor nevirapine

The objective of this study was to evaluate the applicability of saliva as an alternative body fluid for therapeutic drug monitoring of nevirapine. The pharmacokinetics of nevirapine in plasma and saliva during a dosing interval was assessed in HIV-1-infected patients taking nevirapine (200 mg twice daily) to explore the relation between the concentration of nevirapine in plasma and saliva. To validate the anticipated relationship prospectively, single, paired plasma and saliva samples were obtained from nevirapine-treated HIV-1-infected outpatients. The plasma nevirapine concentration was strongly correlated with the salivary concentration. The mean saliva/plasma concentration ratio was 0.51 and was independent of the time after ingestion. Salivary nevirapine concentrations were used to estimate the corresponding plasma concentrations for 31 outpatients. Compared with the true plasma concentrations, the estimated concentrations were biased by -4.2%, with a precision of 13.3%. These data show a strong correlation between the salivary and plasma concentrations of nevirapine at a dosage of 200 mg twice daily. This relation has been validated prospectively, and the prediction of plasma concentrations was accurate and precise. Therefore, the authors conclude that saliva can be a useful body fluid for therapeutic drug monitoring of nevirapin

Quantitative determination of efavirenz (DMP 266), a novel non-nucleoside reverse transcriptase inhibitor, in human plasma using isocratic reversed-phase high-performance liquid chromatography with ultraviolet detection

Efavirenz is a novel non-nucleoside reverse transcriptase inhibitor for the treatment of HIV-1-infected individuals. A simple and rapid high-performance liquid chromatographic method for the quantification of efavirenz in human plasma suitable for therapeutic drug monitoring in plasma is described. Sample pretreatment consists of protein precipitation with acetonitrile and subsequent evaporation of the extract to concentrate the analyte. The drug is separated from endogenous compounds by isocratic reversed-phase high-performance liquid chromatography with ultraviolet detection at 246 nm. The method has been validated over the range of 10 to 10,000 ng/ml using a volume of 250 microl of plasma. The assay is linear over this concentration range as indicated by the F-test for lack of fit. Within- and between-day precisions are less than 4.3% for all quality control samples. The lower limit of quantitation is 10 ng/ml and the recovery of efavirenz from human plasma is 106.4% (+/- 1.8%). Frequently co-administered drugs did not interfere with the described methodology. Efavirenz is stable under various relevant storage conditions, for example when stored for 24 h at room temperature. This validated assay is suited for use in pharmacokinetic studies with efavirenz and can readily be implemented in the setting of a hospital laboratory for the monitoring of efavirenz concentration

Steady-state pharmacokinetics of twice-daily dosing of saquinavir plus ritonavir in HIV-1-infected individuals

To compare the steady state plasma pharmacokinetics of 1000 mg of saquinavir (SQV) in a soft-gel capsule (SGC) formulation in combination with 100 mg of ritonavir (RTV) (capsules) in a twice-daily dosing regimen in HIV-1-infected individuals with historical controls who used 400 mg of SQV in a hard-gel capsule (HGC) formulation in combination with 400 mg of RTV and to investigate the plasma pharmacokinetics of the 1000 mg/100 mg regimen after normal and high-fat breakfasts. Open-label, crossover, steady-state pharmacokinetic study. Six HIV-1-infected individuals who used either 1200 mg of SQV (SGC or HGC) three times daily or 400 mg twice daily in combination with 400 mg of RTV twice daily were included. Each patient was switched to 1000 mg of SQV SGC twice daily in combination with 100 mg of RTV twice daily. After 14 days, the patients came to the hospital for assessment of a pharmacokinetic profile during 12 hours. Patients were randomized to receive a high-fat (+/-45 g of fat) or normal (+/-20 g of fat) breakfast. After 7 days, a second pharmacokinetic profile was assessed after ingestion of the drugs with the alternate breakfast. A noncompartmental pharmacokinetic method was used to calculate the area under the plasma concentration versus time curve (AUC0-12h), the maximum plasma concentration (Cmax), the plasma trough concentration (C12h), and the elimination half-life in plasma (t1/2). The obtained pharmacokinetic parameters were compared with those of 12 patients using SQV HGC (400 mg twice daily) in combination with RTV (400 mg twice daily). The median values of the pharmacokinetic parameters for SQV SGC (1000 mg twice daily, normal breakfast) were: AUC0-12h, 18.84 h*mg/L; Cmax, 3.66 mg/L; C12h, 0.40 mg/L; and t1/2, 3.0 hours. The median values of the pharmacokinetic parameters for SQV HGC (400 mg twice daily, normal breakfast) were: AUC0-12h, 6.99 h*mg/L; Cmax, 1.28 mg/L; C12h, 0.23 mg/L; and t1/2, 3.9 hours. The exposure to SQV in the dosing regimen of 1000 mg twice daily in combination with 100 mg of RTV twice daily was significantly higher than the exposure to SQV in a dosing regimen of 400 mg twice daily in combination with 400 mg of RTV twice daily. The pharmacokinetic parameters of SQV SGC in the dosing regimen of 1000 mg twice daily in combination with 100 mg of RTV twice daily were not significantly different after ingestion of a high-fat or normal breakfast (p >.35). The combination of 1000 mg of SQV SGC twice daily and 100 mg of RTV twice daily resulted in a higher exposure to SQV compared with the exposure to SQV obtained when SQV is used in the 400 mg/400 mg twice-daily combination with RTV. In this small number of patients, no significant differences in exposure were seen after ingestion of either a normal or high-fat breakfast. From a pharmacokinetic perspective, the combination of 1000 mg of SQV SGC twice daily and 100 mg of RTV twice daily seems to be a good option for further clinical evaluatio