Presence Of Ritonavir Research Articles

Renal Insufficiency Has No Effect on the Pharmacokinetics of Vicriviroc in a Ritonavir-Containing Regimen

Vicriviroc is a small-molecule CCR5 antagonist currently in development for the treatment of HIV in patients on a regimen containing a ritonavir-boosted protease inhibitor. As renal disease and renal dysfunction are prevalent in the HIV-infected population, patients with varying degrees of renal insufficiency may receive vicriviroc, which is metabolized by cytochrome P450 (CYP) 3A4. The present study therefore examined the impact of renal insufficiency on the pharmacokinetics and safety of vicriviroc alone and in the presence of ritonavir, a strong CYP3A4 inhibitor. This study was an open-label, randomized, two-treatment crossover trial conducted in HIV-negative subjects with haemodialysis-dependent end-stage renal disease (ESRD) and healthy subjects with normal renal function matched by age, height, bodyweight and sex. Subjects received a single dose of vicriviroc 75 mg alone in one period, and in another period they received a single dose of vicriviroc 15 mg after 4 days of ritonavir 100 mg once daily. Ritonavir treatment was then continued for an additional 13 days. The two trial periods were separated by an interval of at least 3 weeks. The primary endpoints were the log-transformed area under the plasma concentration-time curve (AUC) and the maximum plasma concentration (C(max)), and the 90% confidence intervals (CIs) of the mean differences between subjects with ESRD and matched healthy subjects. The protocol provided the option of dose modification and further study if the vicriviroc C(max) and AUC values were at least twice as high in subjects with ESRD compared with healthy subjects, or if warranted by other safety and tolerability observations. Twelve subjects (six with ESRD, six healthy) completed the study. When vicriviroc was administered alone, the mean vicriviroc C(max) and AUC ratio estimates (90% CI) for subjects with ESRD versus healthy subjects were 74% (53, 103) and 84% (49, 145), respectively. When ritonavir was added to the regimen, the ratio estimates (90% CI) were 81% (59, 111) and 134% (105, 171), respectively. Ritonavir plasma concentrations were substantially higher in subjects with ESRD than in healthy subjects. Treatment-emergent adverse events considered possibly or probably related to treatment occurred only during the ritonavir period of the study and in one healthy subject and two subjects with ESRD; all were of mild or moderate severity. ESRD had no clinically relevant impact on exposure of vicriviroc when vicriviroc was administered alone or in the presence of ritonavir. In this single-dose study, vicriviroc was well tolerated both by healthy subjects and by those with ESRD. Dose adjustment of vicriviroc is therefore not necessary in renally impaired populations.

Impact of Ritonavir, Atazanavir and Their Combination on the CYP3A4 Induction by Efavirenz in Primary Human Hepatocytes

Currently used combinations of anti-HIV drugs, known as Highly Active Antiretroviral Therapy (HAART), have considerably reduced the mortality in patients with AIDS. However, HAART medications such as efavirenz (EFV), atazanavir (ATV) and ritonavir (RTV) often cause adverse drug-drug interactions (DDIs) that result from changes in the expression and activity of drug metabolizing enzymes. Since EFV is most commonly used with ATV and RTV, the known CYP inhibitors, we evaluated the effects of combinations of these agents on the CYP3A4 induction by EFV. We determined the induction of CYP3A4 by EFV, RTV, ATV, EFV+RTV, EFV+ATV, EFV+RTV+ATV and rifampicin (RIF) employing primary human hepatocytes from 3 donors. Also, concentration dependent activation of human Pregnane X-receptor (hPXR) which is key transcriptional regulator of CYP3A4 by EFV, RIF and RTV was estimated in transiently transfected LS180 cells. CYP3A4 activity (testosterone-6beta-hydroxylation) was induced by EFV (3 fold) and RIF (4 fold), but was significantly suppressed in the presence of RTV and ATV. All treatments significantly induced the CYP3A4 transcripts (3-25 fold) as quantitated by RT-PCR. hPXR activation data in LS180 cells were consistent with the induction of transcripts and the estimated EC(50) values were 0.87 microM, 0.44 microM and 3.7 microM for RIF, RTV and EFV, respectively. However, in primary hepatocytes the net effect was suppression of EFV mediated CYP3A4 induction by RTV and ATV. This observation corresponds to the clinical observations of attenuated CYP3A4 induction by EFV induction in the presence of RTV and other protease inhibitors (PIs). Our results underscore the limitation of transcriptional activation assays in predicting the net outcome for compounds that exhibit complex interactions resulting from induction and inhibition of CYP enzymes.

Inulin solid dispersion technology to improve the absorption of the BCS Class IV drug TMC240

TMC240 is a very poorly soluble and poorly permeating HIV protease inhibitor. In order to enhance its oral bioavailability, a fast dissolving inulin-based solid dispersion tablet was developed. During the dissolution test in water (0.5% or 1.0% SLS), this tablet released at least 80% of TMC240 within 30 min, while a tablet with the same composition, but manufactured as physical mixture, released only 6% after 2 h. In a subsequent single-dose study in dogs (200 mg of TMC240), plasma concentrations of TMC240 remained below the lower limit of quantification (<1.00 ng/mL) in all animals ( n = 3 per tested formulation), except in one dog receiving the inulin solid dispersion tablet ( C max = 1.8 ng/mL, AUC 0-7 h = 3.0 ng h/mL). In the latter treatment group, ritonavir co-administration (10 mg/kg b.i.d.) increased TMC240 exposure more than 30-fold (mean AUC 0-7 h = 108 ng h/mL; F rel = 3588%). Exposure was also 16-fold higher than after TMC240 administration as PEG400 suspension in the presence of ritonavir (AUC 0-7 h = 6.7 ng h/mL). The current data demonstrate that a solid dispersion of TMC240 in an inulin matrix allows considerable improvement in the release of poorly water-soluble TMC240, both in vitro in the presence of a surfactant and in vivo upon oral administration.

Improved bioavailability of darunavir by use of κ-carrageenan versus microcrystalline cellulose as pelletisation aid

The aim of this study was to produce pellet formulations containing a high drug load (80%) of the poorly soluble HIV-protease inhibitor darunavir, using wet extrusion/spheronisation with κ-carrageenan or microcrystalline cellulose (MCC) as pelletisation aid. Drug release was assessed in vitro by a standardized paddle-dissolution test and in vivo by a single-dose pharmacokinetic study in dogs. Mean dissolution time (MDT) was 78.2 ± 3.5 h from MCC pellets (1301 ± 301 μm) and 6.1 ± 0.7 min from κ-carrageenan pellets (966 ± 136 μm). In contrast to κ-carrageenan pellets, MCC pellets did not disintegrate and showed a diffusion-controlled drug release. In line with the in vitro findings, the darunavir peak plasma levels and exposure after the administration of a 300 mg dose were more than 60-fold higher when formulated with κ-carrageenan pellets when compared with MCC pellets, and 10-fold higher after co-administration with 10 mg/kg of ritonavir. The relative bioavailability of darunavir versus the reference tablet ( F rel) was 155% with κ-carrageenan pellets and 2% with MCC pellets without ritonavir, while 78% and 9%, respectively, in presence of ritonavir. In conclusion, when compared with MCC pellets, the bioavailability of darunavir was substantially improved in κ-carrageenan pellets, likely due to their better disintegration behavior.

Minimal Effects of Ritonavir and Efavirenz on the Pharmacokinetics of Raltegravir

Raltegravir is a novel human immunodeficiency virus type 1 (HIV-1) integrase strand transfer inhibitor with potent in vitro activity against HIV-1 (95% inhibitory concentration = 31 nM in 50% human serum). The possible effects of ritonavir and efavirenz on raltegravir pharmacokinetics were separately examined. Two clinical studies of healthy subjects were conducted: for ritonavir plus raltegravir, period 1, 400 mg raltegravir; period 2, 100 mg ritonavir every 12 h for 16 days with 400 mg raltegravir on day 14; for efavirenz plus raltegravir, period 1, 400 mg raltegravir; period 2, 600 mg efavirenz once daily for 14 days with 400 mg raltegravir on day 12. In the presence of ritonavir, raltegravir pharmacokinetics were weakly affected: the plasma concentration at 12 h (C(12 h)) geometric mean ratio (GMR) (90% confidence interval [CI]) was 0.99 (0.70, 1.40), area under the concentration-time curve from zero to infinity (AUC(0-infinity)) was 0.84 (0.70, 1.01), and maximum concentration of drug in serum (C(max)) was 0.76 (0.55, 1.04). In the presence of efavirenz, raltegravir pharmacokinetics were moderately to weakly reduced: C(12 h) GMR (90% CI) was 0.79 (0.49, 1.28); AUC(0-infinity) was 0.64 (0.52, 0.80); and C(max) was 0.64 (0.41, 0.98). There were no substantial differences in the time to maximum concentration of drug in plasma or the half-life. Plasma concentrations of raltegravir were not substantially affected by ritonavir. Though plasma concentrations of raltegravir were moderately to weakly reduced by efavirenz, the degree of this reduction was not clinically meaningful. No dose adjustment is required for raltegravir with coadministration with ritonavir or efavirenz.

Darunavir: Pharmacokinetics and Drug Interactions

Darunavir (TMC114) is a new HIV protease inhibitor that has demonstrated substantial antiretroviral activity against wild-type HIV-1 virus and multidrug-resistant strains. Darunavir inhibits and is primarily metabolized by cytochrome P450 3A (CYP3A) isoenzymes and is coadministered with low-dose ritonavir (darunavir/r); ritonavir is an inhibitor of CYP3A isoenzymes and pharmacologically enhances darunavir, resulting in increased plasma concentrations and allowing for a lower daily dose. The t1/2 (terminal elimination half-life) of darunavir is 15 h in the presence of ritonavir. An extensive darunavir/r drug-drug interaction programme has been undertaken, covering a wide range of therapeutic areas. Studies conducted in HIV-negative healthy volunteers and in HIV-infected patients show that the potential for interactions is well characterized and the interactions are manageable. For most drugs investigated, no dose adjustments of darunavir/r or the co-administered drug are required. This article reviews all the pharmacokinetic and drug-drug interaction studies conducted to date for darunavir/r, providing guidance on how to co-administer darunavir/r with many other antiretroviral or non-antiretroviral medications commonly used in HIV-infected individuals.

Inhibition of placental P-glycoprotein: impact on indinavir transfer to the foetus.

We have shown previously using the dually perfused isolated human placenta model that the maternal to foetal transfer of the antiviral protease inhibitor drug indinavir is substantially lower than the transfer in the opposite direction. This finding is not consistent with passive diffusion and indicates that a carrier-mediated mechanism is involved in retarding the movement in the maternal to foetal direction. The efflux transporter P-gp located in the apical membrane domain of the placental trophoblast cells has been implicated as the likely cause of the differential bi-directional transport. The present study also utilizes the human perfused human isolated placenta to investigate the possible inhibitory effects of the P-gp inhibitor PSC833 and the P-gp substrate/inhibitor ritonavir on the maternal to foetal transfer clearance of indinavir. The studies, which were conducted such that each placenta served as its own control, demonstrated a statistically significant increase in the maternal to foetal transfer of indinavir in the presence of PSC833 but not in the presence of ritonavir, a protease inhibitor that is often used in combination with other protease inhibitors in dual therapy. The lack of effect of ritonavir is most likely related to the relatively low inhibitory activity at the clinically relevant concentration used in this study. To investigate the effect of P-gp inhibition on the maternal to foetal transfer of indinavir. Term human placentae (n = 12) were from non-HIV infected women. Maternal to foetal transfer of indinavir was examined in the absence and presence of P-gp inhibitors PSC833 (n = 7) or ritonavir (n = 5), in the perfused human placenta. Antipyrine and [(3)H]-vinblastine were included as markers of passive diffusion and P-gp transport, respectively. These markers and indinavir were added to maternal perfusate at 0 min; PSC833 or ritonavir was added at 25 min. Steady-state maternal to foetal transfer clearance was calculated during control and inhibitor phases. Indinavir and vinblastine clearances were normalized to antipyrine clearance (clearance index). Indinavir clearance index increased between the control (0.25 +/- 0.03) and PSC833 phases (0.37 +/- 0.14) (95% CI of the difference -0.23, -0.002). Vinblastine clearance index increased from (0.25 +/- 0.08) to (0.34 +/- 0.06) in the control and PSC833 phases, respectively (95% CI of difference -0.14, -0.05). Indinavir clearance index was unchanged between control (0.34 +/- 0.14) and ritonavir phases (0.39 +/- 0.13) (95% CI of the difference -0.19, 0.08). Vinblastine clearance index increased from (0.24 +/- 0.12) to (0.32 +/- 0.12) in the control and ritonavir phases, respectively (95% CI of the difference -0.15, -0.009). Maternal to foetal transfer clearance of indinavir and vinblastine increased following P-gp inhibition. The potential role for co-administration of P-gp inhibitors with PIs to reduce perinatal HIV transmission warrants further investigation.

Orosomucoid (α1-acid glycoprotein) plasma concentration and genetic variants: Effects on human immunodeficiency virus protease inhibitor clearance and cellular accumulation

Protease inhibitors are highly bound to orosomucoid (ORM) (alpha1-acid glycoprotein), an acute-phase plasma protein encoded by 2 polymorphic genes, which may modulate their disposition. Our objective was to determine the influence of ORM concentration and phenotype on indinavir, lopinavir, and nelfinavir apparent clearance (CL(app)) and cellular accumulation. Efavirenz, mainly bound to albumin, was included as a control drug. Plasma and cells samples were collected from 434 human immunodeficiency virus-infected patients. Total plasma and cellular drug concentrations and ORM concentrations and phenotypes were determined. Indinavir CL(app) was strongly influenced by ORM concentration (n = 36) (r2 = 0.47 [P = .00004]), particularly in the presence of ritonavir (r2 = 0.54 [P = .004]). Lopinavir CL(app) was weakly influenced by ORM concentration (n = 81) (r2 = 0.18 [P = .0001]). For both drugs, the ORM1 S variant concentration mainly explained this influence (r2 = 0.55 [P = .00004] and r2 = 0.23 [P = .0002], respectively). Indinavir CL(app) was significantly higher in F1F1 individuals than in F1S and SS patients (41.3, 23.4, and 10.3 L/h [P = .0004] without ritonavir and 21.1, 13.2, and 10.1 L/h [P = .05] with ritonavir, respectively). Lopinavir cellular exposure was not influenced by ORM abundance and phenotype. Finally, ORM concentration or phenotype did not influence nelfinavir (n = 153) or efavirenz (n = 198) pharmacokinetics. ORM concentration and phenotype modulate indinavir pharmacokinetics and, to a lesser extent, lopinavir pharmacokinetics but without influencing their cellular exposure. This confounding influence of ORM should be taken into account for appropriate interpretation of therapeutic drug monitoring results. Further studies are needed to investigate whether the measure of unbound drug plasma concentration gives more meaningful information than total drug concentration for indinavir and lopinavir.

Effects of atazanavir/ritonavir and lopinavir/ritonavir on glucose uptake and insulin sensitivity: demonstrable differences in vitro and clinically

The HIV protease inhibitor (PI) atazanavir does not impair insulin sensitivity acutely but ritonavir and lopinavir induce insulin resistance at therapeutic concentrations. To test the hypothesis that atazanavir combined with a lower dose of ritonavir would have significantly less effect on glucose metabolism than lopinavir/ritonavir in vitro and clinically. Glucose uptake was measured following insulin stimulation in differentiated human adipocytes in the presence of ritonavir (2 micromol/l) combined with either atazanavir or lopinavir (3-30 micromol/l). These data were examined clinically using the hyperinsulinemic euglycemic clamp and oral glucose tolerance testing (OGTT) in 26 healthy HIV-negative men treated with atazanavir/ritonavir (300/100 mg once daily) and lopinavir/ritonavir (400/100 mg twice daily) for 10 days in a randomized cross-over study. Atazanavir inhibited glucose uptake in vitro significantly less than lopinavir and ritonavir at all concentrations. Ritonavir (2 micromol/l) combined with either atazanavir or lopinavir (3-30 micromol/l) did not further inhibit glucose uptake. During euglycemic clamp, there was no significant change from baseline insulin sensitivity with atazanavir/ritonavir (P = 0.132), while insulin sensitivity significantly decreased with lopinavir/ritonavir from the baseline (-25%; P < 0.001) and from that seen with atazanavir/ritonavir (-18%; P = 0.023). During OGTT, the HOMA insulin resistance index significantly increased from baseline at 120 min with atazanavir/ritonavir and at 150 min with lopinavir/ritonavir. The area under the curve of glucose increased significantly with lopinavir/ritonavir but not with atazanavir/ritonavir. Both glucose uptake in vitro and clinical insulin sensitivity in healthy volunteers demonstrate differential effects on glucose metabolism by the combination PI atazanavir/ritonavir and lopinavir/ritonavir.

Effect of low-dose ritonavir (100 mg twice daily) on the activity of cytochrome P450 2D6 in healthy volunteers

In the treatment of human immunodeficiency virus infection, the protease inhibitor ritonavir is used in a low dose (100 mg twice daily) to inhibit cytochrome P450 (CYP) 3A4 and thereby increase plasma concentrations of coadministered protease inhibitors. When applied in a therapeutic dose (600 mg twice daily), ritonavir also inhibits CYP2D6. The effect of low-dose ritonavir on CYP2D6 is unknown and was investigated in this study. This was a 1-arm, 2-period, fixed-order study in 13 healthy male volunteers who were extensive metabolizers of CYP2D6. The first period examined baseline CYP2D6 activity by evaluating the pharmacokinetics of a single dose of desipramine and by metabolic phenotyping with dextromethorphan. During the second period, participants took ritonavir, 100 mg twice daily, for 2 weeks, followed by repeat assessment of desipramine pharmacokinetics and the dextromethorphan metabolic phenotype in the presence of ritonavir. Low-dose ritonavir (100 mg twice daily) significantly increased the exposure to single-dose desipramine, as reflected in a geometric mean ratio (with ritonavir/without ritonavir) of 1.26 (95% confidence interval, 1.13-1.40) for the desipramine area under the concentration versus time curve from time 0 to infinity (P < .001). Coadministration of low-dose ritonavir did not significantly affect the dextromethorphan/dextrorphan urinary metabolic ratio and did not convert any extensive metabolizer to a poor metabolizer. Low-dose ritonavir (100 mg twice daily) exerts a modest inhibitory effect on the activity of CYP2D6 in extensive metabolizers, as assessed with desipramine as the index substrate. This effect was not apparent with the dextromethorphan/dextrorphan metabolic ratio as an indicator for CYP2D6 activity. It is expected that the effect of low-dose ritonavir on CYP2D6 will not require standard dose reductions for CYP2D6 substrates.

Exposure to Therapeutic Concentrations of Ritonavir, but Not Saquinavir, Reduces Secreted Aspartyl Proteinase of Candida parapsilosis

The effect of ritonavir and saquinavir, HIV proteinase inhibitors, on the secreted aspartyl proteinase (Sap) activity of Candida parapsilosis was studied. In a proteinase-inducing medium (yeast carbon base-bovine serum albumin), Sap activity in all clinical isolates of C. parapsilosis (n = 20) was observed at 37°C but not at 22°C. The presence of ritonavir at a concentration of 8 µg/ml produced an inhibition close to 50% albumin consumption and also delayed yeast growth; however, saquinavir did not have any effect on growth or on Sap activity. In Sabouraud broth, which does not induce Sap production, no effect was shown on yeast growth by either of the two HIV proteinase inhibitors studied.

Population pharmacokinetics of lopinavir in combination with ritonavir in HIV‐1‐infected patients

To develop a population pharmacokinetic model for lopinavir in combination with ritonavir, in which the interaction between both drugs was characterized, and in which relationships between patient characteristics and pharmacokinetics were identified. The pharmacokinetics of lopinavir in combination with ritonavir were described using NONMEM (version V, level 1.1). First, ritonavir data were fitted to a previously developed model to obtain individual Bayesian estimates of pharmacokinetic parameters. Hereafter, an integrated model for the description of the pharmacokinetics of lopinavir with ritonavir was designed. From 122 outpatients 748 lopinavir and 748 ritonavir plasma concentrations were available for analysis. The interaction between the drugs was described by a time-independent inverse relationship between the exposure to ritonavir over a dosing-interval and the apparent clearance (CL/F) of lopinavir. The model parameters volume of distribution and absorption rate constant were 61.6 l (95% prediction interval (PI) 22.4, 83.7) and 0.564 h(-1) (95% PI 0.208, 0.947), respectively. The model yielded a theoretical value for the CL/F of lopinavir without ritonavir of 14.8 l h(-1) (95%PI 12.1, 20.1), which translates to a value of 5.73 l h(-1) in the presence of ritonavir. The only factor with significant effect on the pharmacokinetics was concurrent use of non-nucleoside reverse transcriptase inhibitors (NNRTI), which increased the CL/F of lopinavir by 39% (P < 0.001). We have developed a model that has defined a time-independent inverse relationship between the exposure to ritonavir and the CL/F of lopinavir, and provided an adequate description of the pharmacokinetic parameters for the latter. Concomitant use of the NNRTIs efavirenz and nevirapine increased the CL/F of lopinavir.

Indinavir protein-free concentrations when used in indinavir/ritonavir combination therapy

To describe the in vivo protein-binding characteristics of indinavir (IDV) in the presence of ritonavir (RTV) relative to total IDV plasma concentrations. The ACTG protocol 5055 was a multicenter study comparing the safety and pharmacokinetics of IDV/RTV at doses of 800/200 and 400/400 mg twice daily in HIV-infected adults. Forty-four patients underwent a 12-h intensive pharmacokinetic assessment after 2 weeks of therapy. Three plasma samples from 35 patients at Cmax, 6 and 12 h post dose were used to determine the unbound IDV concentrations. Unbound IDV was separated in plasma samples using ultra-filtration and measured using high-performance liquid chromatography with UV detection. Mean IDV protein-bound fraction across all time points in the 800/200 and 400/400 arm were 53.4 and 51.8%, respectively. In the 800/200 arm, percentage binding at Cmax was 50% compared with 56% at 12 h (P = 0.008). In the 400/400 arm, percentage binding at Cmax was 49% compared with 54% at 12 h (P = 0.008). The extent of plasma protein binding of IDV in this study was less than in previously published data with IDV alone. Although IDV concentrations differed across the arms, the percentage of IDV protein binding at all time points was not different between the 800/200 and 400/400 arms. However, the percentage of IDV protein binding at Cmax was significantly lower compared with 12 h in each arm, possibly suggesting that IDV protein binding is concentration-dependent. These data suggest that RTV affects IDV protein-binding characteristics and IDV also exhibits concentration dependent binding when administered with RTV.

Intracellular Accumulation of Nelfinavir and Its Relationship to P-Glycoprotein Expression and Function in HIV-Infected Patients

To compare plasma and intracellular nelfinavir pharmacokinetics, and determine their relationship to P-glycoprotein (P-gp) expression and function in lymphocytes of HIV-infected patients. A pharmacokinetic study of 12 patients receiving nelfinavir plus dual nucleoside analogue therapy. Blood samples were taken at intervals to 12 h. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation, and nelfinavir extracted from cells in the presence of 60% methanol and evaporated to dryness. Both plasma and intracellular nelfinavir samples were assayed by high performance liquid chromatography linked to mass spectrometry. P-gp expression and function were measured by flow cytometric analysis. Data were analysed by non-compartmental analysis using WinNonLin pharmacokinetic software. The mean intracellular nelfinavir AUC(0-12) (mean +/-SE) was about ninefold higher than that of plasma (264,200 +/- 63,420 vs 29,250 +/- 6629 ng/ml/h; P<0.001, and intracellular Cmin and C0 values for nelfinavir were five- to sixfold higher than that of plasma (Cmin: 5712 +/- 2156 vs 1062 +/- 357 ng/ml; C0: 15,860 +/- 3662 vs 2553 +/- 539 ng/ml; P<0.0005). The intracellular nelfinavir Cmax was 15-fold higher than plasma (59,420 +/- 13,940 vs 3986 +/- 822 ng/ml; P<0.0005). There were no differences between plasma and intracellular values for Tmax, elimination half-life or mean residence time. In patients chronically treated with nelfinavir mean P-gp expression was 8.85 +/- 1.3 MFI, there was no correlation between P-gp expression and either intracellular AUC(0-12) (r=-0.35; P=0.29) or intracellular C0 values. There was a correlation between intracellular nelfinavir concentrations and P-gp function at baseline (r=0.59; P<0.05). Basal P-gp-mediated rhodamine efflux was 61.0 +/- 4.2%. In the presence of ritonavir, cellular rhodamine efflux decreased to 25.6 +/- 5.5% (P=0.001), representing an additional reversible efflux potential of 56.1 +/- 9.78%. There was a strong correlation between plasma and intracellular AUC(0-12) for nelfinavir (r=0.75; P=0.011). Nelfinavir undergoes significant intracellular accumulation within the PBMCs of HIV-infected patients, which may be in part related to its moderate ability to inhibit P-gp-mediated drug efflux. The addition of ritonavir further reduced P-gp function. Intracellular accumulation of nelfinavir correlated with P-gp function but not P-gp expression, suggesting pump activity is substrate concentration-dependant. There was a significant correlation between plasma and intracellular nelfinavir concentrations, suggesting one is a good surrogate marker of the other.

Tipranavir: a protease inhibitor from a new class with distinct antiviral activity.

Tipranavir (TPV) is the first of a new class of non-peptidic protease inhibitors (NPPIs). It is a sulphonamide-containing dihydropyrone, which is highly selective for the HIV protease enzyme and demonstrates potent in vitro activity against wild-type HIV-1 and HIV-2. The IC90 for TPV was 0.1 microM against clinical HIV isolates. Since CYP3A is the major cytochrome P450 isoform for the phase I metabolism of TPV, its exposure is markedly enhanced in the presence of ritonavir (RTV). In one clinical study, using the new self emulsifying drug delivery system (SEDDS) formulation of TPV, plasma concentrations in excess of 20 microM were maintained for 12 hours, allowing for twice-a-day dosing following administration of TPV 300 mg/RTV(r) 200 mg twice a day. The 20 microM target represents 10-fold the IC90 for multiple protease inhibitor (PI)-resistant strains. Both in vitro data and pharmacokinetic results indicate that TPV will be active in vivo against PI-resistant viruses, when given twice a day in combination with low dose RTV. Of 105 HIV viral isolates taken from patients who had been heavily pretreated with PI-based regimens: 90% were fully susceptible to TPV; 8% exhibited intermediate resistance; and 2% were more than 10-fold resistant. In patients who had failed at least two PI-based regimens, only 12.2% of the HIV isolates exhibited four to 10-fold reduced susceptibility to TPV after one year of treatment with a regimen containing the NPPI (Study BI1182.2). A reduction of approximately 1.5 log10 copies/mL in the plasma viral load (pVL) was observed in treatment-naive patients after 15 days of monotherapy with TPV (300 or 1200 mg twice a day) co-administered with RTV (200 mg twice a day) (TPV/r) in a dose-ranging study (Study BI1182.3). The safety and efficacy of TPV (500 or 1250 mg) plus ritonavir (100 mg twice a day) plus two new nucleoside reverse transcriptase inhibitors (NRTIs) was studied in patients failing their first PI-containing regimen (Study BI1182.4). Similar decreases in pVLs (1.44-1.79 log10 copies/mL) were observed after 16 weeks of treatment with either dose of TPV/r. Two doses of TPV/r plus efavirenz (EFV) and a new NRTI have been studied in non-nucleoside reverse transcriptase inhibitor (NNRTI)-naive patients who had failed two or more PI-containing regimens (BI1182.2). Between 50% and 78.9% of patients maintained a pVL < 50 copies/mL for 48 weeks. Clinical studies have shown that TPV/r-associated adverse events are generally gastrointestinal-associated, transient and mild. A phase II study will define the optimal dose of TPV/r for highly treatment-experienced patients. The safety and efficacy of this dose of TPV/r will be evaluated in two phase III studies that will enroll more than 1300 patients worldwide. Tipranavir's robust activity against PI-resistant strains results from its molecular flexibility, which allows it to fit into the active pocket of the protease enzyme in viruses that have become resistant to other PIs.

In-vitro crystallization of indinavir in the presence of ritonavir and as a function of pH.

The aim of this study was to investigate the in-vitro crystallization of indinavir as a function of pH alteration and in the presence of another protease inhibitor, ritonavir. Crystallization processes were studied for indinavir sulfate, indinavir free base and a commercial indinavir capsule dosage form, respectively. Crystallization induction times were determined with varying initial concentration of supersaturated solution, and in the presence or absence of seed material. In-vitro induction times were found to be significantly shorter for the indinavir capsule dosage form compared with that of indinavir sulfate and indinavir free base. Induction times were inversely proportional to the final concentration in pH 7 buffer for all materials, and were significantly shortened in the presence of seeds. The crystal morphology of indinavir varied under different crystallization conditions. This study demonstrated the potential for precipitation of indinavir upon pH elevation, while also suggesting that the presence of impurities or seeding material significantly shortens the induction time for indinavir crystal formation. This induction time period falls well within the gastric emptying time following the intake of a high-caloric meal, and within small intestinal transit time. The results of this study are in agreement with the clinical observation that a high-calorie protein meal significantly reduces the oral bioavailability of indinavir in man, accompanying a pH elevation in the stomach.

An evaluation of the potential for pharmacokinetic interaction between escitalopram and the cytochrome P450 3A4 inhibitor ritonavir

Background: Depression often coexists with a number of disease states, and patients with a diagnosis of depression often receive multiple medications. Thus, it is desirable to avoid coadministration of agents that have a potential for drug interactions in these patients. Although escitalopram and its metabolites are weak to negligible inhibitors of the cytochrome P450 (CYP) 3A4 isozyme and are therefore unlikely to affect plasma concentrations of ritonavir (a CYP3A4 substrate and prototype CYP3A4 inhibitor), ritonavir may potentially affect plasma concentrations of escitalopram, as CYP3A4 is partially responsible for conversion of escitalopram to its major metabolite, S-demethylcitalopram (S-DCT).Objectives: The aim of this study was to investigate the potential for pharmacokinetic interaction between escitalopram and ritonavir after concomitant administration of a single dose of each in healthy young subjects.Methods: In this single-center, randomized, open-label, 3-way crossover study, subjects received each of the following: a single dose of escitalopram 20 mg, a single dose of ritonavir 600 mg, and single doses of both escitalopram 20 mg and ritonavir 600 mg. Blood was collected and plasma was analyzed for the pharmacokinetic parameters (maximum plasma concentration [Cmax], time to Cmax [Tmax], area under the plasma concentration-time curve, plasma elimination half-life, oral clearance, and apparent volume of distribution) of escitalopram, S-DCT, and ritonavir.Results: Of 21 subjects (11 men, 10 women; mean [SD] age, 28.4 [4.4] years) who were enrolled, 18 completed the study. After concomitant administration of escitalopram and ritonavir, no statistically significant differences were noted in the pharmacokinetics of escitalopram, with the exception of apparent volume of distribution, which was reduced by ∼10% (P < 0.001). The pharmacokinetics of S-DCT were unaffected by coadministration of ritonavir, with the exception of Tmax, which was increased in the presence of ritonavir. The pharmacokinetic parameters of ritonavir were also unaffected by coadministration of escitalopram.Conclusion: In general, no pharmacokinetic interaction was observed between escitalopram and ritonavir in the present study.

By-passing of P-glycoprotein Using Immunoliposomes

The over-expression of MDR1 P-glycoprotein has been associated with the development of multidrug-resistance in cancer cells. Methods used to overcome multidrug-resistance often involve the co-administration of inhibitors of P-glycoprotein. Here, we test the hypothesis that an immunoliposome-based drug delivery system may be used as an alternative approach to overcome multidrug-resistance since immunoliposomes penetrate target cells by receptor-mediated endocytosis which allows to by-pass membrane-associated P-glycoprotein. Targeting of immunoliposomes was achieved by the use of an anti-transferrin receptor monoclonal antibody (OX26 mAb). Incorporation of radiolabelled digoxin within OX26-immunoliposomes enhanced cellular uptake of digoxin by a factor of 25 in immortalised RBE4 rat brain capillary endothelial cells. Uptake of liposomal digoxin was insensitive to ritonavir, a P-glycoprotein inhibitor, and was reduced in presence of increasing free concentrations of OX26 mAb or nocodazole, a reversible inhibitor of endocytosis. In contrast, uptake of free digoxin was enhanced by a factor of 1.8 in presence of ritonavir and was insensitive to OX26 mAb or nocodazole. Cellular uptake and intracellular accumulation of liposomal digoxin (55% internalisation within 30 min) was demonstrated by acid wash of the cells and was confirmed by confocal microscopy studies. Endosomal release to the cytosol of propidium iodide loaded immunoliposomes was shown. These in vitro studies suggest that immunoliposome-based drug delivery systems can be used to by-pass P-glycoprotein and thus deliver drugs to the cytosol of a target cell.

Effects of Retroviral Protease Inhibitors on Proteasome Function and Processing of HIV-Derived MHC Class I-Restricted Cytotoxic T Lymphocyte Epitopes

1063 THE HIV-1 PROTEASE INHIBITOR RITONAVIR (RTV) potently suppresses viral replication and is an effective antiretroviral agent in the clinical setting.1–3 Although originally designed rationally to limit cross-reactive inhibition of structurally similar human aspartic proteases, concern has been raised regarding the negative effects of RTV in murine systems on both the generation of CD8 cytotoxic T lymphocyte (CTL) responses and the processing of major histocompatibility (MHC) class Irestricted peptide epitopes recognized by these effector cells.4,5 We assessed the impact of RTV, and other antiretroviral protease inhibitors, on human proteasome function and the generation of HIV-derived CTL peptide epitopes with well-characterized processing requirements. The proteasome, a large nonlysosomal multisubunit protease complex that accomplishes many of the essential proteolytic functions of the cell, is critically involved in the generation of antigenic peptides destined for the MHC class I pathway. Structurally, the core 20S proteasome comprises four heptameric rings arranged such that the a and b subunits, which range in size from 20 to 35 kDa, form a 700-kDa complex with the configuration a7b7b7a7. Proteolytic activities are restricted to specific threonine residues on certain b subunits, a catalytic mechanism characteristic of N-terminal nucleophile hydrolases.7 At least five distinct peptidase activities, based on amino acid cleavage preferences, have been ascribed to the proteasome; RTV has been shown to inhibit the chymotryptic activity, characterized by cleavage after hydrophobic residues, of isolated murine 20S proteasomes in vitro.4 We confirmed and extended this result with 20S proteasomes purified from human erythrocytes (Fig. 1a4,8). The b subunits MB1 and LMP7 have been identified as prime target sites for the reversible inhibitory effect of RTV on proteasome activity.5 The LMP7 subunit is interferon g (IFN-g) inducible, and replaces the constitutive MB1 subunit in “immunoproteasomes.” We identified two HLA-A2-restricted epitopes from the HIV-1 reverse transcriptase (RT) protein, ILKEPVHGV (Pol, residues 476–484HIV-LAI) and VIYQYMDDL (Pol, residues 346–354HIV-LAI), which are processed through distinct pathways.9 The C-terminal ILKEPVHGV epitope is generated by the constitutive proteasome containing MB1 and is independent of LMP7-associated activity. In contrast, the Nterminal VIYQYMDDL epitope appears to be generated by an alternative antigen-processing pathway and destroyed by the constitutive proteasome; both the expression of LMP7, either in transfection systems or through induction with IFN-g, and inhibition of proteasomal threonine protease activity with lactacystin, substantially enhanced the production of this epitope.9 In this well-characterized system, we were unable to detect any effect of RTV on antigen presentation at therapeutically relevant concentrations (Fig. 1b9). This observation suggests either that the generation of these epitopes is regulated independently of the chymotryptic activity of the proteasome, or that the effects of RTV at concentrations within the therapeutic window are discrepant according to whether the proteasome is examined in purified or cell-associated form. The former possibility is currently difficult to test since the precise anatomical localization of specific enzymatic activities and the effects of LMP7 incorporation on proteasomal hydrolysis patterns remains controversial. Consistent with the latter possibility, RTV had no net quantitative effect on the assembly of HLA class I molecules in pulse–chase experiments, indicating that this process is not limited by impaired production of optimized peptides capable of stabilizing these proteins in nascent form (Fig. 1c10). However, this does not exclude the possibility that the pattern of epitopes produced differs qualitatively in the presence of RTV, and further work is required to resolve this issue. In addition, we could detect no consistent effect of RTV (concentration range, 0.1–10 mM) on the generation of two commonly immunodominant CTL epitopes derived from the HIV-1 Gag protein, SLYNTVATL (p17, residues 77–85HIV-LAI, HLA-A2

Differentiation of gut and hepatic first-pass effect of drugs: 1. Studies of verapamil in ported dogs.

To investigate the relative contributions of the gut and liver to the first-pass loss of verapamil (VL) using an in vivo intestinal-vascular access port (IVAP) dog model. Basic pharmacokinetics of VL were determined after intravenous (IV: 0.5 mg/kg), portal venous (PV: 2 mg/kg), and duodenal (ID: 2 mg/kg) administration in IVAP dogs. Serial blood samples were collected for 8 h after dosing, and plasma was analyzed for unchanged drug by a high-performance liquid chromatography-fluorescence method. Extraction ratios in the liver and intestinal tract were determined from the area under the concentration-time curves for ID, PV, and IV administration. The functional role of CYP450 or secretory transporters such as P-gp on the gut and liver first-pass loss of VL was further studied using ritonavir, a known substrate or inhibitor of these processes. The liver had a high intrinsic capacity for clearing VL because the absolute bioavailability (BA) of VL was 21.7% after PV administration. The BA of VL after ID administration was 23.5%; therefore, intestinal absorption was complete and intestinal extraction was negligible (ER(GI) approximately 0). The BA of VL increased from 23.5% to 66.2% in the presence of ritonavir primarily due to a reduction in hepatic extraction. Although the liver had a high intrinsic capacity for extracting VL, the contribution of gut to the first-pass loss of VL was negligible. Because of the additive effects of intestinal CYP3A-mediated metabolism and secretory transport, a significant gut first-pass effect was expected, but not observed in dogs. These studies demonstrate the utility of the in vivo IVAP dog model for evaluating the relative contribution of the gut and liver to the first-pass loss of drugs and for characterizing the functional role that CYP450 metabolism and/or secretory transporters play in drug-drug interactions and reduced oral bioavailability.