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Highlights: [•] Epistatic fitness interactions can be learned from other drugs that target the same protein. [•] Longitudinal fitness landscape can predict the failing genotype. [•] Fitness and genetic barrier to resistance are predictive for treatment outcome. [ABSTRACT FROM AUTHOR]

Abstract: Indinavir is a protease inhibitor used in the treatment of HIV infection. However, it has limited efficacy in eradicating the virus in the brain due to efflux by P-glycoprotein (P-gp) expressed at the blood–brain barrier (BBB). The objective of this work was to develop an o/w lipid nanoemulsion (LNE) of indinavir using Tween 80 as co-emulsifier to improve its brain specific delivery. LNEs were prepared with different compositions and were characterized for globule size, polydispersity index, zeta potential and in vitro drug release. Five formulations were then evaluated for drug content, entrapment efficiency and stability after which brain uptake studies were carried out using fluorescent labeled LNEs and pharmacokinetic (PK) and tissue distribution studies were conducted after intravenous administration in mice. Brain uptake of indinavir was shown to be improved for a 1% Tween 80 containing formulation (F5) compared to a formulation containing 0.3% cholesterol (F2). In PK studies, the brain level of indinavir subsequent to administration of F5 was significantly (P<0.05) higher than produced by administration of a drug solution (2.44-fold) or a control nanoemulsion (F1) (1.48-fold) or formulation F2 (1.6-fold). The increased brain specific accumulation of indinavir from F5 is probably due to enhanced low density lipoprotein-mediated endocytosis and P-gp inhibition by Tween 80 at the BBB. These results suggest Tween 80 containing LNEs could provide a simple but effective means of delivering indinavir to brain. [Copyright &y& Elsevier]

Purpose. The results of an analysis of suspected antiviral-associated adverse drug reactions (ADRs) in Italy over a 22-year period are presented. Methods. A case/non-case analysis was conducted using ADR reports compiled in the nationwide spontaneous-reporting database through September 2010. All reported events included in the analysis were evaluated and coded by drug safety experts; causality assessments were performed according to the algorithm of Naranjo et al. The association between an adverse reaction and antiviral use was assessed by estimating the reporting odds ratio (ROR), with 95% confidence interval (CI), as a measure of disproportionality. Results. Overall, 863 reports of suspected ADRs involving antivirals and 42,430 reports of adverse reactions to other drugs were identified; of those events, 3.3% and 64.3% were determined to be definite or probable ADRs, respectively, and an additional 32.4% were deemed possibly drug related. Several ADRs were disproportionately associated with antivirals relative to other drugs: renal colic (ROR, 25.5; 95% CI, 13.3-49.0), lactic acidosis (ROR, 18.6; 95% CI, 9.2-37.7), depression (ROR, 18.0; 95% CI, 11.6-27.9), anemia (ROR, 15.9; 95% CI, 12.3-20.4), hallucination (ROR, 4.3; 95% CI, 2.7-7.1), neutropenia (ROR, 4.1; 95% CI, 2.9-5.8), acute renal failure (ROR, 3.9; 95% CI, 2.3-6.4), fever (ROR, 3.8; 95% CI, 2.8- 5.1), hyperpyrexia (ROR, 2.9; 95% CI, 1.7-4.9), and asthenia (ROR, 1.8; 95% CI, 1.2-2.8). Conclusion. Analysis of data from a large Italian database showed that, among antiviral agents, the ribavirin-interferon combination, acyclovir, valacyclovir, indinavir, and zidovudine accounted for the most serious hematologic, neuropsychiatric, and renal ADRs. [ABSTRACT FROM AUTHOR]

Abstract: Food can have a significant impact on the pharmacokinetics of orally administered drugs, as it may affect drug solubility as well as permeability. Since fed state conditions cannot easily be implemented in the presently available permeability tools, including the frequently used Caco-2 system, exploring food effects during drug development can be quite challenging. In this study, we investigated the effect of fasted and fed state conditions on the intestinal absorption of the HIV protease inhibitor indinavir using simulated and human intestinal fluids in the in situ intestinal perfusion technique in mice. Although the solubility of indinavir was 6-fold higher in fed state human intestinal fluids (FeHIF) as compared to fasted state HIF (FaHIF), the intestinal permeation of indinavir was 22-fold lower in FeHIF as compared to FaHIF. Dialysis experiments showed that only a small fraction of indinavir is accessible for absorption in FeHIF due to micellar entrapment, possibly explaining its low intestinal permeation. The presence of ritonavir, a known P-gp inhibitor, increased the intestinal permeation of indinavir by 2-fold in FaHIF, while there was no increase when using FeHIF. These data confirm that drug–food interactions form a complex interplay between solubility and permeability effects. The use of HIF in in situ intestinal perfusions holds great promise for biorelevant absorption evaluation as it allows to directly explore this complex solubility/permeability interplay on drug absorption. [Copyright &y& Elsevier]

Cross-talk between insulin and the renin angiotensin system signaling system shows that angiotensin 2 (A2) negatively modulates insulin signaling by stimulating multiple serine phosphorylation events in the early stages of the insulin-signaling cascade; however, the biological actions of A2 on insulin sensitivity remain controversial. Preservation of glucose transporter 4 (GLUT4) expression during hypertension has been shown to prevent the increased vascular reactivity associated with hypertension. This study tested the hypothesis that GLUT4 contributes to the renal actions of A2. In the euvolemic anesthetized rat, acute infusion of the GLUT4 antagonist, indinavir (1 mg/kg/minute), enhanced an A2-induced increase in mean arterial blood pressure (MABP) (P , 0.01), but attenuated an A2-induced increase in medullary blood flow (MBF) and glomerular filtration rate (P , 0.01). Insulin, a GLUT4 activator (20 mU/kg/minute and 40 mU/kg/minute), decreased basal MABP and urine volume (P , 0.05), but it increased MBF, and these effects were reversed and blunted by indinavir. Subchronic indinavir treatment (80 mg/kg/day orally for 15 days) did not affect A2-induced changes in MABP, cortical blood flow, and MBF, but significantly decreased basal MBF (P , 0.01) and global kidney perfusion (P , 0.05). We concluded that acute but not subchronic inhibition of GLUT4 alters A2-induced changes in systemic and renal hemodynamics by attenuating A2-induced increase in MBF and glomerular filtration rate. [ABSTRACT FROM AUTHOR]

Objective: To demonstrate that our assay is sensitive capable to detect 10 HIV infected cells per well and that is feasible to quantify HIV-1 resistance in seropositive patients. Method: To compare 3 different phenotypic assays by quantifying their sensitivities, first we co-cultivated HeLa-CD4-LTR/β-gal cells with infected PBMCs at different concentrations diluted 1:2 from 4,000 cells per well in order to elucidate the minimum quantity of cells needed for the assay to be detected as positive. On day one in a 96 well plate we seeded HeLa-CD4-LTR/β-gal (5 x 104) cells in 50 HeLa-CD4-LTR/β-gal (5 x 104) of DMEM. 24 h later we added the following antiretrovirals Abacavir, Didanosina, Lamivudina, Tenofovir, Efavirenz, Nevirapina, Enfurtivide, Amprenavir, Atazanavir, Indinavir, aphic.org.mx Lopinavir, Nelfinavir, Ritonavir, y Saquinavir . To obtain dose response we diluted 1:2 the drugs. Then we added the four patients' PBMCs infected with HIV-1, then it is incubated at 37 °C for 24 h then the plates are readed after the addition of luciferasa to the 96 well plate at day 3. Results: HIV infection was quantified as very sensitive, rapid and specific. We obtained the IC50 for HIVwt OR wild type and VIHRV or resistant virus. Our method was more sensitive than the MT-2 assay (by 200 fold) and more sensitive than Blue cells (by 30 fold). The assay quantified the infection by PBMC+ cells from seropositive patients being able to classify the sensitivities and HIV resistance. In four patients was detected resistance to protease inhibitors, light resistance to NRTI, moderate resistance to NNRTI, sensitive to Enfurtivide and two patients sensitive to Lamivudine. Conclusions: In Mexico there is no antivirogram for HIV-1. The assay that we tested is a Bioluminiscent assay that detect resistant virus and is capable to quantify the IC50 of HIV-1 with precision. It is a valuable tool for the physician to be guided to prescript efficient antiretrovirals and not to prescribe a drug that is not efficient and is expensive, toxic and with secondary effects for the seropositive patient [ABSTRACT FROM AUTHOR]

Abstract: Indinavir sulphate was subjected to forced degradation under hydrolysis (acidic, basic and neutral), oxidation, photolysis and thermal stress as prescribed by ICH guidelines. It was degraded under acidic, basic, neutral and oxidative stress conditions, while it was stable under other conditions. After degradation total eight degradation products were formed. The degradation products were identified and their separation was accomplished on Waters XTerra® C18 column (250mm×4.6mm i.d., 5μm) using 20mM ammonium actate:acetonitrile as (50:50, v/v) mobile phase in an isocratic elution mode by LC. The method was extended to LC–MS/MS for characterization of the degradation products and the fragmentation pathways were proposed. The proposed structures of degradation products were also confirmed by HRMS studies. No previous reports were found in the literature regarding the characterization of degradation products of indinavir sulphate. [Copyright &y& Elsevier]

In this work, we report the benchmark binding energies of the seven complexes within the L7 data set, six host-guest complexes from the S12L data set, a C 60 dimer, the DNA-ellipticine intercalation complex, and the largest system of the study, the HIV-indinavir system, which contained 343 atoms or 139 heavy atoms. The high-quality values reported were obtained via a focal point method that relies on the canonical form of second-order Møller-Plesset theory and the domain-based local pair natural orbital scheme for the coupled cluster with single double and perturbative triple excitations [DLPNO-CCSD(T)] extrapolated to the complete basis set (CBS) limit. The results in this work not only corroborate but also improve upon some previous benchmark values for large noncovalent complexes albeit at a relatively steep cost. Although local CCSD(T) and the largely successful fixed-node diffusion Monte Carlo (FN-DMC) have been shown to generally agree for small- to medium-size systems, a discrepancy in their reported binding energy values arises for large complexes, where the magnitude of the disagreement is a definite cause for concern. For example, the largest deviation in the L7 data set was 2.8 kcal/mol (∼10%) on the low end in C3GC. Such a deviation only grows worse in the S12L set, which showed a difference of up to 10.4 kcal/mol (∼25%) by a conservative estimation in buckycatcher-C 60 . The DNA-ellipticine complex also generated a disagreement of 4.4 kcal/mol (∼10%) between both state-of-the-art methods. The disagreement between local CCSD(T) and FN-DMC in large noncovalent complexes shows that it is urgently needed to have the canonical CCSD(T), the Monte Carlo CCSD(T), or the full configuration interaction quantum Monte Carlo approaches available to large systems on the hundred-atom scale to solve this dilemma. In addition, the performances of cheaper popular computational methods were assessed for the studied complexes with respect to DLPNO-CCSD(T)/CBS. r 2 SCAN-3c, B97M-V, and PBE0+D4 work well in large noncovalent complexes in this work, and GFN2-xTB performs well in π-π stacking complexes. B97M-V is the most reliable computationally efficient approach to predicting noncovalent interactions for large complexes, being the only one to have binding errors within the so-called 1 kcal/mol "chemical accuracy". The benchmark interaction energies of these host-guest complexes, molecular materials, and biological systems with electronic and medicinal implications provide crucial reference data for the improvement of current and future lower-cost methods.

The intense research on small molecule inhibitors of Human immunodeficiency virus (HIV)-protease (PR) has produced a diverse class of chemical scaffolds which includes clinically available HIV PR inhibitors (PRI). Till now, these inhibitors are insignificant for targeting proteolytic activity and few drug molecules on alterations can enhance the inhibition of PR enzyme. Here, we developed a method for screening of new hits from Cambridge structural database, based on binding mode of indinavir interaction participating atoms. Knowledge-based ligand screening technique approximately informs that new hits are also having same binding mode-like indinavir interaction patterns. Considering the importance of ligand fitting in binding pocket, we developed induced-fit models for each compound and we obtained accurate energy values in terms of binding and interaction energy. We found that newly search molecules are interacting better than known drug-indinavir and these new compounds are comparatively having better drug-like property. Finally, we demonstrated that pocket specific docking, energy utilization, interactions, and ADME for screened compounds are showing new hit compounds of indinavir are better HIV PRI and these new compounds can also show better activity in in vivo and in vitro conditions. [ABSTRACT FROM AUTHOR]

Tenofovir (TDF), atazanovir (ATAZ) and indinavir (IND) have been reported as possible risk factors for incident chronic kidney disease (CKD) in HIV-infected patients. We investigated the relationship between the duration of antiretroviral exposure and estimated glomerular filtration rate (eGFR) evolution in CKD patients. In a cohort of 1,750 HIV-infected patients, we identified 121 CKD patients with a mean follow-up of 44 ± 35 months. The relationship between mean eGFR at baseline, eGFR slope and time exposure to antiretroviral treatment as well as confounding factors were investigated using a joint modeling procedure. Seventy (58%), 30 (25%) and 33 patients (27%), with a mean age of 50.3 ± 11.7 years, mean eGFR at baseline of 53.0 ± 0.8 (ml/min/1.73 m2) and eGFR slope of 0.46 ± 0.07 ml/min/1.73 m2/year, were exposed to TDF, ATAZ and IND, respectively. In univariate analysis, hepatitis C virus infection, decreased nadir of log CD4 count, high blood pressure at baseline, angiotensin-converting enzyme inhibitor treatment and greater time exposure to TDF during follow-up were associated with a higher slope, whereas greater time exposure to IND was associated with a lower slope. In multivariate analysis, higher TDF time exposure was still significantly associated with eGFR decline, with a dose-effect relationship (slope ± standard error of the mean: 1.1 ± 0.1, 0.5 ± 0.1, -0.07 ± 0.08 and -0.87 ± 0.06 ml/min/1.73 m2/year for no time exposure, <34, 34-67 and ≥67%, respectively; trend test: p < 0.001), whereas the IND time exposure association was abolished. In HIV patients with CKD, a greater TDF time exposure was independently associated, in a graded manner, with a greater eGFR decline. Copyright © 2012 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Impaired renal function is of major concern in human immunodeficiency virus (HIV)-infected patients. Methods: We used a mixed effects linear regression model to determine estimated glomerular filtration rates (eGFRs) in a population-based cohort of incident Danish HIV patients and stratified on baseline eGFR (eGFRB) < 90 and ≥ 90 ml/min per 1.73 m2. Incidence rate ratios (IRRs) for chronic kidney disease (CKD) - 2 consecutive eGFR values < 60 ml/min per 1.73 m2 measured > 3 months apart - were estimated (time-updated Cox-regression model). Results: The effect of time with HIV on eGFR was small in both strata (− 0.09 (95% confidence interval (CI) − 0.27, 0.09) and − 0.46 (95% CI − 0.64, − 0.27) ml/min per 1.73 m2 per y). Treatment with tenofovir and indinavir was associated with lower eGFR in both strata: tenofovir − 2.00 (95% CI − 3.45, − 0.56) and − 1.94 (95% CI − 3.43, − 0.44) ml/min per 1.73 m2 and indinavir − 2.14 (95% CI − 3.63, − 0.64) and − 3.29 (95% CI − 5.25, − 1.32) ml/min per 1.73 m2. Nevirapine, atazanavir, and the combination of tenofovir and atazanavir were associated with lower eGFR in patients with eGFRB < 90 ml/min per 1.73 m2. Highly active antiretroviral therapy (HAART) and exposure to tenofovir and atazanavir in combination were associated with CKD in patients with eGFRB < 90 ml/min per 1.73 m2 (adjusted IRRs 6.08 (95% CI 2.76-13.41) and 26.75 (95% CI 9.54-75.05)). Conclusion: Tenofovir and indinavir reduce eGFR, while time with HIV only has a modest effect on this parameter. Low eGFRB is associated with an increased risk of CKD, especially when receiving HAART regimens containing the combination tenofovir/atazanavir. [ABSTRACT FROM AUTHOR]

A selective, sensitive and high-throughput ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS-MS) method has been developed and validated for the quantification of HIV-protease inhibitors ritonavir (RTV), lopinavir (LPV) and indinavir (IDV) in human plasma. Sample clean-up involved protein precipitation of both drugs and fluconazole used as internal standard from 100 µL human plasma. All the analytes were chromatographically separated on a Waters Acquity UPLC BEH C18 (2.1 × 50 mm, 1.7 µm particle size) analytical column using 0.1% formic acid and methanol (40:60, v/v) as the mobile phase. The parent → product ion transitions for ritonavir (m/z 721.40→ 296.10), lopinavir (m/z 629.40→ 447.40) and indinavir (m/z 614.4→ 421.0) IS (m/z 307.10 → 220.10) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring and positive ion mode. The method was validated over the concentration range of 30–15,000 ng/mL for LPV and IDV and 3–1500 ng/mL for RTV. The method was successfully applied to a pilot bioequivalence study in 36 healthy human subjects after oral administration of lopinavir 200 mg and ritonavir 50 mg tablet formulation under fasting conditions. [ABSTRACT FROM AUTHOR]

The article discusses various scientific considerations for pharmacoenhancers in antiretroviral therapy (ART). Topics include an overview of protease inhibitors (PI), which are an integral component of highly active ART, a historical perspective of PIs, such as ritonavir, and limitations associated with the use of ritonavir as a pharmacoenhancer. Scientific considerations include the identification of appropriate doses of pharmacoenhancer and drug-drug interactions.

Several observational studies have identified tenofovir as an independent risk factor for kidney impairment. Conversely, randomized trials have only demonstrated minor tenofovir-related changes in kidney function, but these studies included patients with normal kidney function and with low underling risk for progression of their renal function, had limited size, and limited follow-up. Several potential mechanisms of tenofovir nephrotoxicity are known. Atazanavir can, equally to indinavir, cause urolithiasis, but both drugs have also been associated with chronic kidney disease (CKD) and fast declining eGFR in persons without clinical symptoms of urolithiasis, especially when the plasma drug concentration is boosted by concomitant ritonavir use. In 2012, only a minority of HIV-positive individuals are affected by drug-induced nephrotoxicity. However, in the future, the clinical impact and hence the requirement for more research in this area will likely increase due to ageing and continued cART exposure of the HIV-positive population. [ABSTRACT FROM AUTHOR]

Purpose: To assess the effects of venlafaxine extended-release (XR) capsules and desvenlafaxine extended-release (XR) tablets upon indinavir pharmacokinetic properties when co-administrated to healthy volunteers. Methods: This was an open-label, two-period, fixed-dose study conducted at the clinical research unit located on a university campus. Twenty-four healthy volunteers enrolled in the study (mean age 28.3 ± 8.0 years). Each subject received a single dose of indinavir 800 mg on day 1. Subsequently, subjects were then randomly assigned to either the venlafaxine XR group ( N = 12) or the desvenlafaxine XR group ( N = 12). Starting on day 2, venlafaxine XR was dosed at 37.5 mg/day for 4 days and increased to 75 mg/day for 6 days. Desvenlafaxine XR was dosed at 50 mg/day for 10 days. On day 12, indivanvir 800 mg was co-administered to both the venlafaxine XR and the desvenlafaxine XR groups. The pharmacokinetics of indinavir were determined both before and at the end of antidepressant dosing. Plasma indinavir, venlafaxine, and desvenlafaxine concentrations were assayed by high-performance liquid chromatography with ultra-violet (UV) detection. Indinavir pharmacokinetic parameters were calculated by noncompartmental analysis using validated computer software. Results: Venlafaxine XR and desvenlafaxine XR did not produce any significant changes in indinavir disposition. Both antidepressants were well tolerated by the subjects with only minor adverse side effects. Conclusions: No pharmacokinetic drug-drug interaction was demonstrated between venlafaxine XR and indinavir or between desvenlafaxine XR and indinvair. The lack of interaction could be due to the venlafaxine and desvenlafaxine extended-release formulation. [ABSTRACT FROM AUTHOR]

Objectives. We studied the effect of antiretroviral therapy (ART) on the quality of life (QOL) of Cubans with HIV/AIDS. Methods. We conducted a cross-sectional study including administration of the Medical Outcomes Study-HIV Health Survey Questionnaire to a representative sample of the 1592 Cubans receiving ART in 2004. For univariate analyses, we compared mean HIV scale scores. We used logistic regression models to estimate the association between role function and year of diagnosis, between pain and sex, and between health transition and region of diagnosis,with adjustment for demographics, ART regimen, and clinical status. Results. There were 354 participants (73 women, 281 men). Scores for all functional activities showed means higher than 80 out of 100. Pain interfered more in women than in men (73.2 vs 81.9; P = .01). When HIV diagnosis occurred after 2001, the probability of experiencing difficulties performing work (odds ratio [OR] = 4.42; 95% CI = 1.83, 10.73) and pain (OR = 1.70; 95% CI = 1.01, 2.88) increased compared with earlier diagnosis. People treated with indinavir showed a greater perception of general health (58.9 vs 52.4; P = .045) and greater health improvement (78.6 vs 67.8; P = .002). Conclusions. Although Cubans receiving ART are maintaining a high QOL, we observed significant differences by sex and time of diagnosis. QOL assessment can serve as a health outcome and may allow identification of QOL reductions potentially related to ART side effects. [ABSTRACT FROM AUTHOR]

Abstract: A comparative study with three conventional extraction techniques namely protein precipitation (PP), liquid–liquid extraction (LLE) and solid phase extraction (SPE) has been demonstrated to assess the magnitude of matrix interference by post-column analyte infusion and post extraction analyte spiking for the determination of atazanavir from human plasma. Severe ion suppression observed in PP and to a lesser extent in LLE was circumvented by SPE on LiChrosep Sequence extraction cartridge. Based on these observations a selective, rugged and high throughput SPE-LC–MS/MS method has been developed for reliable determination of atazanavir in human plasma. The chromatographic separation was achieved on a Hypersil Gold C18 (50mm×4.6mm, 5μm) analytical column using 5mM ammonium formate in water:methanol (10:90, v/v) as the mobile phase under isocratic conditions. The method was validated over a wide dynamic concentration range of 10–6000ng/mL. The mean relative recovery and absolute matrix effect across quality controls were 84.9 and 93.2%, respectively. The precision value for relative matrix effect between eight different lots of plasma, expressed as %CV of the slopes of the calibration lines was 2.41. The stability of atazanavir under different storage conditions varied from −8.4 to 5.4%. The method was successfully applied to a bioequivalence study of 300mg atazanavir capsule formulation in 24 healthy Indian males under fasting condition. [Copyright &y& Elsevier]

Sophora flavescens is a Chinese medicinal herb used for the treatment of gastrointestinal hemorrhage, skin diseases, pyretic stranguria and viral hepatitis. In this study the herb-drug interactions between S. flavescens and indinavir, a protease inhibitor for HIV treatment, were evaluated in rats. Concomitant oral administration of Sophora extract (0.158 g/kg or 0.63 g/kg, p.o.) and indinavir (40 mg/kg, p.o.) in rats twice a day for 7 days resulted in a dose-dependent decrease of plasma indinavir concentrations, with 55%-83% decrease in AUC0-∞ and 38%-78% reduction in Cmax. The CL (Clearance)/F (fraction of dose available in the systemic circulation) increased up to 7.4-fold in Sophora-treated rats. Oxymatrine treatment (45 mg/ kg, p.o.) also decreased indinavir concentrations, while the ethyl acetate fraction of Sophora extract had no effect. Urinary indinavir (24-h) was reduced, while the fraction of indinavir in faeces was increased after Sophora treatment. Compared to the controls, multiple dosing of Sophora extract elevated both mRNA and protein levels of P-gp in the small intestine and liver. In addition, Sophora treatment increased intestinal and hepatic mRNA expression of CYP3A1, but had less effect on CYP3A2 expression. Although protein levels of CYP3A1 and CYP3A2 were not altered by Sophora treatment, hepatic CYP3A activity increased in the Sophora-treated rats. All available data demonstrated that Sophora flavescens reduced plasma indinavir concentration after multiple concomitant doses, possibly through hepatic CYP3A activity and induction of intestinal and hepatic P-gp. The animal study would be useful for predicting potential interactions between natural products and oral pharmaceutics and understanding the mechanisms prior to human studies. Results in the current study suggest that patients using indinavir might be cautioned in the use of S. flavescens extract or Sophora-derived products. [ABSTRACT FROM AUTHOR]

Aim: Assess possible adverse effects of the chronic use of indinavir during pregnancy in a rat model. Methods: 40 pregnant EOM-1 albino rats were randomly allocated into four groups of 10 animals each: a control (Ctr) group (without any handling) and three experimental groups (Exp 1, Exp 2 e Exp 3) which received indinavir 9, 27 e 81 mg/kg, respectively). Rats were treated by gavage once daily. The treatment period extended from day 0 until the 20th day of pregnancy. Body weights were recorded on days 0, 7, 14 and 20. At term, the rats were sacrificed, and the implantation sites, number of live and dead fetuses and placentas, resorptions, fetal and placental weights were recorded. The fetuses were evaluated for external abnormalities under a stereomicroscope. Results: Weight gain during pregnancy did not differ significantly between the groups. Average weight gains between the 7th and 20th day were 7.95-42.70 g; 7.22-45.27 g; 7.12-46.26 g and 8.05-42.29 g in groups Ctr, Exp 1, Exp 2 and Exp 3, respectively. All other parameters assessed did not differ significantly between groups. Conclusions: Chronic use of various dosages of indinavir during pregnancy was not associated significant adverse outcomes. [ABSTRACT FROM AUTHOR]

Background: Given that HIV-protease inhibitors (HIV-PIs) are substrates/inhibitors of the multidrug transporter ABCB1, can induce ABCB1 expression, and are used in combination with doxorubicin for AIDS-Kaposi's Sarcoma (KS) treatment, the role that ABCB1 plays in mediating multidrug resistance (MDR) in a fully transformed KS cell line (SLK) was explored.Methods: The KS cells were exposed to both acute and chronic treatments of physiological concentrations of different HIV-PIs (indinavir, nelfinavir, atazanavir, ritonavir, or lopinavir), alone or together with doxorubicin. The ABCB1 mRNA and protein expression levels were then assessed by qRT-PCR and western blotting, flow cytometry, and immunofluorescence.Results: Chronic treatment of SLK cells with one of the five HIV-PIs alone or together resulted in increased resistance to doxorubicin. Co-treatment with one of the HIV-PIs in combination with doxorubicin resulted in a synergistic increase in resistance to doxorubicin, and the degree of resistance was found to correlate with the expression of ABCB1. The SLK cells were also revealed to be cross-resistant to the structurally unrelated drug paclitaxel.Conclusion: These studies suggest that ABCB1 is primarily responsible for mediating MDR in SLK cells selected with either HIV-PIs alone or in combination with doxorubicin. Therefore, the roles that ABCB1 and drug cocktails play in mediating MDR in KS in vivo should be evaluated. [ABSTRACT FROM AUTHOR]

utofluorescence of the mandible and femur bones taken from newborn rats (7-, 14- and 28-day old) was studied. Endogenous fluorophores were excited with 231 nm, 291 nm, 340 nm and 360 nm wavelengths. Modifications in content and microenvironment of both noncolagenous and collagenous constituents of bone tissue as well as metabolic coenzymes during the bone formation with age were reflected in fluorescence emission spectra. The increase of emission from peptide bonds and tryptophan residues was noted with rat age while for collagen and metabolic coenzymes at the first 2 weeks only. After maternal administration of indinavir the changes in fluorescence intensity and shifts in position of peak maximum were found. The distinct drop of emission from peptide bonds and tryptophan residues in studied bones was detected. In the case of collagen and metabolic coenzymes the red shift of peak maximum was revealed. Fluorescence spectroscopy could be used to follow bone development in newborn rats and effect of maternal indinavir administration on offspring. [ABSTRACT FROM AUTHOR]

Abstract: The era of highly active antiretroviral therapy (HAART) has controlled AIDS and its related disorders considerably; however, the prevalence of HIV-1-associated neurocognitive disorders has been on the rise in the post-HAART era. In view of these developments, we investigated whether a HAART drug combination of 3′-azido-2′,3′-deoxythymidine (AZT) and indinavir (IDV) can alter the functionality of the blood–brain barrier (BBB) endothelial cells, thereby exacerbating this condition. The viability of hCMEC/D3 cells (in vitro model of BBB) that were exposed to these drugs was significantly reduced after 72h treatment, in a dose-dependent manner. Reactive oxygen species were highly elevated after the exposure, indicating that mechanisms that induce oxidative stress were involved. Measures of oxidative stress parameters, such as glutathione and malondialdehyde, were altered in the treated groups. Loss of mitochondrial membrane potential, as assessed by fluorescence microscopy and decreased levels of ATP, indicated that cytotoxicity was mediated through mitochondrial dysfunction. Furthermore, AZT+IDV treatment caused apoptosis in endothelial cells, as assessed by the expression of cytochrome c and procaspase-3 proteins. Pretreatment with the thiol antioxidant N-acetylcysteine amide reversed some of the pro-oxidant effects of AZT+IDV. Results from our in vitro studies indicate that the AZT+IDV combination may affect the BBB in HIV-infected individuals treated with HAART drugs. [Copyright &y& Elsevier]

The aim of our present work was to develop indinavir O/W submicron lipid emulsions (SLEs) loaded with lipoamino acids for specific delivery to brain. Tetradecyl aspartic acid (A) and decyl glutamic acid (G) loaded stable SLEs of indinavir having a mean size range of 210-220 nm and average zeta potential of −23.54 ± 1.2 mV were developed using homogenization and ultrasonication. The cumulative % drug release from different SLEs varied in between 26% and 85%. The formulations, SLE, SLE-A3, and SLE-G3 were stable to the centrifugal stress, dilution stress, and storage at RT. The total drug content and entrapment efficiency were determined by HPLC method. During pharmacokinetic studies in male Wistar rats there was no significant difference in the serum levels of indinavir for SLE, SLE-A3 and SLE-G3 formulations at all time points. In tissue distribution studies, the therapeutic availability (TA) of indinavir in brain and kidneys for SLE-A3 were 4.27- and 2.66-fold whereas for SLE-G3 were 2.94 and 2.12 times, respectively, higher than that of indinavir solution. But when compared with that of SLE, in brain tissue the levels of indinavir from SLE-G3 and SLE-A3 varied in between 2.5- and 3.38-fold. While in case of the kidney, it was between 1.23- and 1.54-fold only. However, the TA is not significantly different in tissues like the heart, liver, and spleen. Thus, brain-specific delivery of indinavir was improved by including tetradecyl aspartic acid and decyl glutamic acid in submicron lipid emulsions. [ABSTRACT FROM AUTHOR]

It has been shown that the HIV protease inhibitors indinavir and lopinavir may have activity against the human papilloma virus (HPV) type 16 inhibiting HPV E6-mediated proteasomal degradation of p53 in cultured cervical carcinoma cells. However, their mode and site of action is unknown. HPV-negative C33A cervical carcinoma cells and the same cells stably transfected with E6 (C33AE6) were exposed to indinavir and lopinavir at concentrations of 1 mM and 30 μM, respectively. The intracellular distribution of metabolites and metabolic changes induced by these treatments were investigated by Raman microspectroscopic imaging combined with the analysis of cell fractionation products by liquid chromatography-mass spectrometry (LC-MS). A uniform cellular distribution of proteins was found in drug-treated cells irrespective of cell type. Indinavir was observed to co-localise with nucleic acid in the nucleus, but only in E6 expressing cells. Principal components analysis (PCA) score maps generated on the full Raman hypercube and the corresponding PCA loadings plots revealed that the majority of metabolic variations influenced by the drug exposure within the cells were associated with changes in nucleic acids. Analysis of cell fractionation products by LC-MS confirmed that the level of indinavir in nuclear extracts was approximately eight-fold greater than in the cytoplasm. These data demonstrate that indinavir undergoes enhanced nuclear accumulation in E6-expressing cells, which suggests that this is the most likely site of action for this compound against HPV. [ABSTRACT FROM AUTHOR]

We have previously demonstrated that moderate hyperbilirubinemia decreases blood pressure in ANG II-dependent hypertension through mechanisms that decrease oxidative stress and increase nitric oxide levels. Since decreases in renal hemodynamics play an important role in mediating the hypertensive actions of ANG II, the goal of the present study was to examine the effect of moderate hyperbilirubinemia on glomerular filtration rate (GFR) and renal blood flow (RBF) in a mouse model of ANG II hypertension. Mice were made moderately hyperbilirubinemic by two methods: indinavir or specific morpholino antisense oligonucleotides against UGT1A1, which is the enzyme responsible for the conjugation of bilirubin in the liver. GFR and RBF were measured in mice after implantation of an osmotic minipump delivering ANG II at a rate of 1 µg·kg-1·min-1. GFR was measured by continuous infusion of I125-labeled iothalamate on days 5 and 6 of ANG II infusion in conscious mice. RBF was measured on day 7 of ANG II infusion in anesthetized mice. Blood levels of unconjugated bilirubin were significantly increased in mice treated with indinavir or anti-UGT1A1 (P = 0.002). ANG II decreased GFR by 33% of control (n = 9, P = 0.004), and this was normalized by moderate hyperbilirubinemia (n = 6). Next, we examined the effect of moderate hyperbilirubinemia on RBF in ANG II-infused mice. ANG II infusion significantly decreased RBF by 22% (P = 0.037) of control, and this decrease was normalized by moderate hyperbilirubinemia (n = 6). These results indicate that improvement of renal hemodynamics may be one mechanism by which moderate hyperbilirubinemia lowers blood pressure in this model. [ABSTRACT FROM AUTHOR]

Abstract: Indinavir, as a protease inhibitor with a short biological half life, variable pH-dependent oral absorption, and extensive first-pass metabolism, presents a challenge with respect to its oral administration. The current work aims to formulate and characterize indinavir-bearing ethanolic liposomes (ethosomes), and to investigate their enhanced transdermal delivery potential. The prepared ethanolic liposomes were characterized to be spherical, unilamellar structures having low polydispersity, nanometric size range, and improved entrapment efficiency over other delivery formulations. Permeation studies of indinavir across human cadaver skin resulted in enhanced transdermal flux from ethanolic liposomes that was significantly (P < 0.05) greater than that with ethanolic drug solution, conventional liposomes, or plain drug solution. Additionally, the ethanolic liposomes showed the shortest lag time for indinavir, thus presenting a suitable approach for transdermal delivery of this protease inhibitor. From the Clinical Editor: This study characterizes indinavir bearing ethanolic liposomes (ethosomes), and investigate their enhanced transdermal delivery potential, demonstrating a potentially a suitable approach for transdermal delivery of this protease inhibitor for HIV treatment, which typically has been associated with limited bioavailability via the oral route. [ABSTRACT FROM AUTHOR]

Objective: To investigate the potential for a pharmacokinetic interaction between darunavir (DRV, TMC114, Prezista®), indinavir (IDV, Crixivan®) and low-dose ritonavir (RTV, Norvir®). Methods: In three 7-day sessions, 17 HIV-negative healthy volunteers received treatment A (DRV/r 400/100 mg b.i.d.), treatment B (IDV/r 800/100 mg b.i.d.) and treatment C (DRV/r 400/100 mg b.i.d. + IDV 800 mg b.i.d.). On day 7, full pharmacokinetic profiles of DRV, IDV and RTV were determined. Safety and tolerability were also assessed. Results: Based on the least-squares means ratios, the steady-state exposure (area under the curve, AUC12h) and plasma concentrations (Cmin and Cmax) of IDV were increased by 23, 125 and 8%, respectively, when DRV was co-administered. The co-administration of IDV with DRV/r resulted in increases of 24, 44 and 11% for, respectively, DRV AUC12h, Cmin and Cmax, compared with administration of DRV/r alone. Eight volunteers discontinued due to an adverse event. Overall, adverse events and laboratory abnormalities were more commonly reported during treatments including IDV. Conclusions: When used in combination with DRV/r, dose adjustment of IDV from 800 mg b.i.d. to 600 mg b.i.d. may be warranted in cases of intolerance. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Compartmental as well as noncompartmental analyses are used routinely in pharmacokinetic analysis. Materials and methods: Pharmacokinetic parameters of the anti-HIV agent, indinavir, have been determined in six male rats applying both the compartmental and the noncompartmental analysis. Results and discussion: A very slow declining phase was found in the indinavir plasma concentration profile using an extended sampling time period and applying a sensitive high-performance liquid chromatography assay method. This apparent terminal elimination phase can cause some significant errors when applying noncompartmental kinetic analysis to the data, with mean residence time (MRT) (544.2 ± 123.2 minutes), total systemic clearance (12.0 ± 2.1 mL/min/kg), and steady-state volume of distribution ( Vd (ss)) (6.4 ± 1.0 L/kg) being highly different from the results of compartmental kinetic analysis (MRT, Cltotal, and Vd (ss) values of 23.7 ± 5.9 minutes, 35.18 ± 5.1 mL/min/kg, and 0.84 ± 0.28 L/kg, respectively). The parameters estimated by our noncompartmental approach were also significantly different from the results of the same type of data analysis reported in the literature. Conclusion: The differences in parameter estimations, while being a result of the extended plasma sampling period, which is recommended in noncompartmental analysis, support the priority of applying the compartmental analysis approach in the similar cases with some pre-assumptions, mainly ignoring the final apparent terminal elimination phase(s), very deep tissue, which involves very low drug concentrations. [ABSTRACT FROM AUTHOR]

Abstract: The xenotropic murine leukemia virus-related virus (XMRV) is a human retrovirus, recently isolated from tissues of prostate cancer patients with impaired RNase L activity. In this study, we evaluated 10 licensed anti-HIV-1 compounds for their activity against XMRV, including protease inhibitors (PI), nucleoside reverse transcriptase (RT) inhibitors (NRTI), non-nucleoside RT inhibitors (NNRTI) and an integrase inhibitor. No PI affected XMRV production; even high concentrations of Ritonavir failed to inhibit the maturation of XMRV Gag polyproteins. Among the NRTI, NNRTI and integrase inhibitors used in this study, only AZT blocked XMRV infection and replication through inhibition of viral reverse transcription. This sensitivity of XMRV to AZT may be explained by the modest homology in the motif D sequences of HIV-1 and XMRV reverse transcriptases. If XMRV becomes established as an etiological agent for prostate cancer or other diseases, AZT may be useful for preventing or treating XMRV infections in humans. [Copyright &y& Elsevier]

As a result of high inter-patient variability, and efficacy–concentration and toxicity–concentration relationships, optimization of HIV-protease inhibitor (PI) doses based on plasma concentrations could be beneficial. During a 48-week open prospective non-randomized interventional study of 115 protease inhibitor-naïve patients initiating an indinavir/ritonavir- or lopinavir/ritonavir-, or nelfinavir-containing therapy, protease inhibitor dose was modified when plasma trough concentrations ( Ctrough) at weeks 2, 8, 16 and 24 were outside predefined optimal concentration ranges. Failure of the strategy was defined as the proportions of patients with HIV-RNA above 200 copies/mL from weeks 24 to 48 and/or experiencing grades 2, 3 or 4 PI-related adverse events during the study; proportion of patients with last Ctrough measurement outside the concentration range was determined at each visit. Virological failure and/or occurrence of adverse event were observed in 37/94 assessable patients (39%; 95% CI: 29.4–50.0). In the on-treatment analysis, failure of the strategy was noted in 16% of indinavir/r- or lopinavir/r-treated patients (8/51; 95% CI: 7.0–28.6; virological failure: 2; adverse event: 6) but in 44% of nelfinavir-treated patients (11/25; 95% CI: 24.4–65.1; virological failure: 10; adverse event: 1); Ctrough concentrations outside the range were less frequent at the last measurement than at W2 (41% vs. 66%; P < 0.05), with proportions of 35% for indinavir/r- or lopinavir/r-treated patients, but 57% for nelfinavir-treated patients. The proposed strategy of therapeutic drug monitoring may be beneficial to indinavir/r- and lopinavir/r-treated patients, but failed to move concentrations into the predefined range and to produce the expected virological success for nelfinavir-treated patients. [ABSTRACT FROM AUTHOR]

Objectives The aim of the study was to investigate the prevalence and aetiology of chronic kidney disease (CKD) and trends in estimated glomerular filtration rate (eGFR) in HIV-infected patients. Methods Ascertainment and review of CKD cases among patients attending King's College and Brighton Hospitals, UK were carried out. CKD was defined as eGFR <60 mL/min for ≥3 months. Longitudinal eGFR slopes were produced to examine trends in renal function before, during and after exposure to indinavir (IDV) or tenofovir (TFV). Results CKD prevalence was 2.4%. While HIV-associated nephropathy accounted for 62% of CKD in black patients, 95% of CKD in white/other patients was associated with diabetes mellitus, hypertension, atherosclerosis and/or drug toxicity. Exposure to IDV or TFV was associated with an accelerated decline in renal function (4.6-fold and 3.7-fold, respectively) in patients with CKD. In patients initiating IDV, age ≥50 years increased the odds of CKD [odds ratio (OR) 4.9], while in patients initiating TFV, age ≥50 years (OR 5.4) and eGFR 60–75 mL/min (OR 17.2) were associated with developing CKD. Conclusion This study highlights the importance of metabolic and vascular disease to the burden of CKD in an ageing HIV-infected cohort. In patients who developed CKD, treatment with IDV or TFV was associated with an accelerated decline in renal function. [ABSTRACT FROM AUTHOR]

Abstract: A growing body of evidence suggests HIV patients are at a greater risk for developing atherosclerosis. However, clinical investigations have generated conflicting results with regard to whether antiretrovirals are independently involved in the development of HIV-associated atherosclerosis. By administering antiretrovirals in an atherogenic mouse model, we determined whether two commonly prescribed antiretrovirals, the protease inhibitor indinavir and the nucleoside reverse transcriptase inhibitor AZT, can induce premature atherosclerosis. C57BL/6 mice were administered an atherogenic diet±AZT, indinavir, or AZT plus indinavir for 20 weeks. Aortic intima–media thickness (IMT) and cross-sectional area (CSA) were determined. Compared to controls, treatment with AZT, indinavir or AZT plus indinavir, significantly increased aortic IMT and CSA. This suggests that antiretrovirals can directly exacerbate atherogenesis, in the absence of interaction with a retroviral infection. To elucidate the role of oxidant injury in the drug-induced initiation of atherosclerosis, a separate group of mice were treated for 2 weeks with an atherogenic diet±AZT, indinavir or AZT plus indinavir. Aortic reactive oxygen species (ROS) production and glutathione/glutathione disulfide (GSH/GSSG) ratios, as well as plasma levels of 8-isoprostanes (8-iso-PGF2α) and lipids were determined. At 2 weeks, aortic ROS was increased and GSH/GSSG ratios were decreased in all antiretroviral treatment groups. Plasma 8-iso-PGF2α was increased in the AZT and AZT plus indinavir-treated groups. At 20 weeks, increased ROS production was maintained for the AZT and indinavir treatment groups, and increased 8-iso-PGF2α levels remained elevated in the AZT treatment group. Cholesterol levels were moderately elevated in the AZT and AZT plus indinavir-treated groups at 2 but not 20 weeks. Conversely, indinavir treatment increased plasma cholesterol at 20 but not 2 weeks. Thus, though effects on plasma lipid levels occurred, with effects of the individual antiretrovirals variable across the treatment period, there was consistent evidence of oxidant injury across both early and late time points. Together with the known metabolic abnormalities induced by antiretrovirals, drug-induced oxidant production may contribute to the development of antiretroviral-associated atherosclerosis. [Copyright &y& Elsevier]

Pharmacokinetic studies in mice traditionally require one animal per time point, resulting in dosing and euthanizing a large number of animals and producing suboptimal quality of pharmacokinetic data due to inter-animal variability and dosing error. These studies are time-consuming and labor-intensive. To improve the throughput and quality of pharmacokinetic evaluation in mice, we have developed a serial blood sampling methodology using the lateral saphenous vein puncture technique. Two marketed drugs, indinavir and rosuvastatin, were selected for this validation study because of their distinctly different physicochemical and pharmacokinetic properties. Each compound was dosed orally and intravenously in mice using both discrete and serial blood sampling methods. The pharmacokinetic results from serial bleeding are in excellent agreement with those from discrete sampling for both compounds. Compared to the discrete sampling, the serial sampling procedure is a more humane method, allowing for rapid and repeated sampling from the same site without the need for anesthesia. The application of this new method has led to a remarkable reduction in animal and compound usage, a significant increase in throughput and speed, and a drastic improvement in pharmacokinetic data quality. This approach is especially useful for the first-tier in vivo pharmacokinetic screening of discovery compounds. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:1877–1884, 2009 [ABSTRACT FROM AUTHOR]

Background/Aims: Gilbert’s syndrome is a frequent genetic conjugation abnormality associated with adverse drug effects. Genetic UDP glucuronosyltransferase (UGT)1A gene variants can influence gene transcription, inducibility and glucuronidation activity. Protease inhibitors used in human immunodeficiency virus (HIV) infection and chronic viral hepatitis can inhibit UGTs. Indinavir (IDV) can lead to hyperbilirubinemia in Gilbert’s syndrome (UGT1A1*28), which does not explain interindividual severity differences and may thus involve additional UGT1A variants. Methods: One hundred and twenty-five HIV patients receiving IDV and 427 healthy blood donors were genotyped for the presence of UGT1A1*28, UGT1A3 -66T/C, UGT1A7 -57T/G, UGT1A7N129K/R131K using Taqman 5′ nuclease assays. Results: Hyperbilirubinemia was observed in 42%. UGT1A1*28 frequencies did not differ between HIV patients and controls but were significantly higher in hyperbilirubinemic patients. The frequency of homozygous carriers of the 4 UGT1A marker haplotype increased with hyperbilirubinemia affecting all patients with bilirubin levels >85μmol/l. Conclusions: In IDV treatment the risk of severe hyperbilirubinemia is associated with genetic variants of the UGT1A3 and UGT1A7 genes in addition to Gilbert’s syndrome (UGT1A1*28). This haplotype is a useful predictor of protease inhibitor-induced side effects. [Copyright &y& Elsevier]

Abstract: Artificial neural networks (ANN) are biologically inspired computer programs designed to simulate the way in which the human brain processes the information. In the past few years, coupling of experimental design (ED) and ANN became useful tool in the method optimization. This paper presents the application of ED–ANN in analysis of chromatographic behavior of indinavir and its degradation products. According to preliminary study, full factorial design 24 was chosen to set input variables for network training. Experimental data (inputs) and results for retention factors from experiments (outputs) were used to train the ANN with aim to define correlation among variables. For networks training multi-layer perceptron (MLP) with back propagation (BP) algorithm was used. Network with the lowest root mean square (RMS) had 4–8–3 topology. Predicted data were in good agreement with experimental data (correlation was higher than 0.9713 for training set). Regression statistics confirmed good ability of trained network to predict compounds retention. [Copyright &y& Elsevier]

Retroviruses are a growing concern for the health of human beings, and one of the dangerous members of this family is the Human T-cell Leukemia Virus 1 (HTLV-1) virus. It has affected more than 20 million people so far, and since there are no registered treatments against it yet, urgent treatment solutions are needed. One of the most promising drug targets to fight this virus is the protease enzyme of the virus's protein machinery. In this study, by utilizing a computational method called Unaggregated Unbiased Molecular Dynamics (UUMD), we reconstructed the binding pathway of a HTLV-1 protease inhibitor, Indinavir, to find the details of the binding pathway, the influential residues, and also the stable states of the binding pathway. We achieved the native conformation of the inhibitor in 6 rounds, 360 replicas by performing over 4 micro-seconds of UMD simulations. We found 3 Intermediate states between the solvated state and the native conformation state in the binding pathway. We also discovered that aromatic residues such as Trp98 and Trp98′, catalytic residues Asp32 and Asp32′, and the flap region's residues have the most influential roles in the binding pathway and also have the most contribution to the total interaction energies. We believe that the details found in this study would be a great guide for developing new treatment solutions against the HTLV-1 virus by inhibiting the HTLV-1 protease. [Display omitted] • The HTLV-1 protease is a potent protein target for fighting the HTLV-1 virus. • Unraveling the binding pathway of an anti-HIV drug, Indinavir, to the HTLV-1 protease by Unaggregated Unbiased Molecular Dynamics simulation method. • Understanding the role of different regions of the HTLV-1 protease such as the Flap regions, and the aromatic residues such as Trp98 and Phe67 in the binding pathway. [ABSTRACT FROM AUTHOR]