Saquinavir Loaded Acetalated Dextran Microconfetti - a Long Acting Protease Inhibitor Injectable.

PURPOSE: Since the adoption of highly active antiretroviral therapy, HIV disease progression has slowed across the world; however, patients are often required to take multiple medications daily of poorly bioavailable drugs via the oral route, leading to gastrointestinal irritation. Recently, long acting antiretroviral injectables that deliver drug for months at a time have moved into late phase clinical trials. Unfortunately, these solid phase crystal formulations have inherent drawbacks in potential dose dumping and a greater likelihood for burst release of drug compared to polymeric formulations. METHODS: Using electrospinning, acetalated dextran scaffolds containing the protease inhibitor saquinavir were created. Grinding techniques were then used to process these scaffolds into injectables which are termed saquinavir microconfetti. Microconfetti was analyzed for in vitro and in vivo release kinetics. RESULTS: Highly saquinavir loaded acetalated dextran electrospun fibers were able to be formed and processed into saquinavir microconfetti while other polymers such as poly lactic-co-glycolic acid and polycaprolactone were unable to do so. Saquinavir microconfetti release kinetics were able to be tuned via drug loading and polymer degradation rates. In vivo, a single subcutaneous injection of saquinavir microconfetti released drug for greater than a week with large tissue retention. CONCLUSIONS: Microconfetti is a uniquely tunable long acting injectable that would reduce the formation of adherence related HIV resistance. Our findings suggest that the injectable microconfetti delivery system could be used for long acting controlled release of saquinavir and other hydrophobic small molecule drugs.

Enhancement of cellular uptake, transport and oral absorption of protease inhibitor saquinavir by nanocrystal formulation.

AIM: Saquinavir (SQV) is the first protease inhibitor for the treatment of HIV infection, but with poor solubility. The aim of this study was to prepare a colloidal nanocrystal suspension for improving the oral absorption of SQV. METHODS: SQV nanocrystals were prepared using anti-solvent precipitation-high pressure homogenization method. The nanocrystals were characterized by a Zetasizer and transmission electron microscopy (TEM). Their dissolution, cellular uptake and transport across the human colorectal adenocarcinoma cell line (Caco-2) monolayer were investigated. Bioimaging of ex vivo intestinal sections of rats was conducted with confocal laser scanning microscopy. Pharmacokinetic analysis was performed in rats administered nanocrystal SQV suspension (50 mg/kg, ig), and the plasma SQV concentrations were measured with HPLC. RESULTS: The SQV nanocrystals were approximately 200 nm in diameter, with a uniform size distribution. The nanocrystals had a rod-like shape under TEM. The dissolution, cellular uptake, and transport across a Caco-2 monolayer of the nanocrystal formulation were significantly improved compared to those of the coarse crystals. The ex vivo intestinal section study revealed that the fluorescently labeled nanocrystals were located in the lamina propria and the epithelium of the duodenum and jejunum. Pharmacokinetic study showed that the maximal plasma concentration (Cmax) was 2.16-fold of that for coarse crystalline SQV suspension, whereas the area under the curve (AUC) of nanocrystal SQV suspension was 1.95-fold of that for coarse crystalline SQV suspension. CONCLUSION: The nanocrystal drug delivery system significantly improves the oral absorption of saquinavir.

Ionic liquid-based dispersive liquid-liquid microextraction followed by RP-HPLC determination of saquinavir in rat serum: application to pharmacokinetics.

An ionic liquid-based dispersive liquid-liquid microextraction followed by RP-HPLC determination of the most commonly prescribed protease inhibitor, saquinavir, in rat plasma was developed and validated. The effects of different ionic liquids, dispersive solvents, extractant/disperser ratio and salt concentration on sample recovery and enrichment were studied. Among the ionic liquids investigated, 1-butyl-3-methylimidazolium hexafluorophosphate was found to be most effective for extraction of saquinavir from rat serum. The recovery was found to be 95% at an extractant/disperser ratio of 0.43 using 1-butyl-3-methylimidazolium hexafluorophosphate and methanol as extraction and dispersive solvents. The recovery was further enhanced to 99.5% by addition of 5.0% NaCl. A threefold enhancement in detection and quantification limits was achieved, at 0.01 and 0.03 µg/mL, compared with the conventional protein precipitation method. A linear relationship was observed in the range of 0.035-10.0 µg/mL with a correlation coefficient (r(2) ) of 0.9996. The method was validated and applied to study pharmacokinetics of saquinavir in rat serum.

Pharmacokinetic study of saquinavir 500 mg plus ritonavir (1000/100 mg twice a day) in HIV-positive pregnant women.

Antiretroviral therapy during pregnancy is critical to preventing human immunodeficiency virus vertical transmission. Physiological changes during pregnancy can alter drug kinetics. The aim of this study was to assess the pharmacokinetics (PK) of saquinavir (SQV) boosted with ritonavir during pregnancy and postpartum. Fourteen human immunodeficiency virus-positive pregnant women started SQV 500 mg new tablet formulation plus ritonavir at a dose of 1000/100 mg twice a day + 2 nucleoside retrotranscriptase inhibitors during pregnancy. At weeks 24 and 34 of pregnancy and 6 weeks postpartum, a 12-hour PK study was conducted. PK parameters were calculated using Win Nolin software version 4.1. At week 24, the geometric mean values for SQV area under the plasma concentration-time curve from 0-12 hours (AUC0₋12), the maximum observed plasma concentration (C(max)), trough plasma concentration (C(min)), and the elimination half-life (t(1/2)) were 24.80 mg·h⁻¹·mL⁻¹, 4.66 mg/mL, 0.93 mg/mL, and 4.31 hours, respectively. At week 34, AUC0₋12, C(max), C(min), and t(1/2) were 12.71 mg·h⁻¹·mL⁻¹, 3.23 mg/mL, 0.26 mg/mL, and 4.06 hours, respectively. Finally, at 6 weeks postpartum, mean values for SQV AUC0₋12, C(max), C(min), and t(1/2) were 28.94 mg·h⁻¹·mL⁻¹, 3.92 mg/mL, 0.86 mg/mL, and 3.60 hours, respectively. Although PK parameters in week 24 and postpartum were very similar, those for week 34 showed an important reduction: -71.20%, -30.61%, -48.73%, and -5.81% in C(min), C(max), AUC0₋12, and t(1/2), respectively, compared with week 24, but no statistically significant differences were shown between patients. No vertical transmissions were reported. Therapeutic drug monitoring of SQV during pregnancy should be considered, mainly during the third trimester, to ensure adequate drug exposure throughout the entire pregnancy.

Impact of hyperlipidemia on plasma protein binding and hepatic drug transporter and metabolic enzyme regulation in a rat model of gestational diabetes.

It is currently unknown whether gestational diabetes mellitus (GDM), a prevalent obstetrical complication, compounds the changes in drug disposition that occur naturally in pregnancy. Hyperlipidemia occurs in GDM. Using a rat model of GDM, we determined whether excess lipids compete with drugs for plasma protein binding. Because lipids activate nuclear receptors that regulate drug transporters and metabolic enzymes, we used proteome analysis to determine whether hyperlipidemia indirectly leads to the dysregulation of these proteins in the liver. GDM was induced on gestational day 6 (GD6) via streptozotocin injection. Controls received either vehicle alone or streptozotocin with subsequent insulin treatment. Liver and plasma were collected on GD20. Glyburide and saquinavir protein binding was determined by ultrafiltration, and an established solvent method was used for plasma delipidation. Proteomics analysis was performed by using isobaric tags for relative and absolute quantitation methodology with membrane-enriched hepatic protein samples. Relative to controls, GDM rat plasma contained more cholesterol and triglycerides. Plasma protein binding of glyburide and saquinavir was decreased in GDM. Delipidation normalized protein binding in GDM plasma. Proteins linked to lipid metabolism were strongly affected in the GDM proteomics data set, with prohyperlipidemic and antihyperlipidemic changes observed, and formed networks that implicated several nuclear receptors. Up-regulation of drug transporters and metabolic enzymes was observed (e.g., multidrug resistance 1/2, CYP2A1, CYP2B9, and CYP2D3). In this study, GDM-induced hyperlipidemia decreased protein binding and was associated with drug transporter and metabolic enzyme up-regulation in the liver. Both of these findings could change drug disposition in affected pregnancies, compounding changes associated with pregnancy itself.

Quantification of 8 HIV-protease inhibitors and 2 nonnucleoside reverse transcriptase inhibitors by ultra-performance liquid chromatography with diode array detection.

BACKGROUND: Most HPLC-UV methods for therapeutic drug monitoring of anti-HIV drugs have long run times, which reduce their applicability for high-throughput analysis. We developed an ultra-performance liquid chromatography (UPLC)-diode array detection method for the simultaneous quantification of the HIV-protease inhibitors (PIs) amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir, and tipranavir (TPV), and the nonnucleoside reverse transcriptase inhibitors (NNRTIs) efavirenz and nevirapine. METHODS: Solid-phase extraction of 1 mL plasma was performed with Waters HLB cartridges. After 3 wash steps, we eluted the drugs with methanol, evaporated the alcohol, and reconstituted the residue with 50 microL methanol. We injected a 4-microL volume into the UPLC system (Waters ACQUITY UPLC BEH C8 column maintained at 60 degrees C) and used a linear gradient of 50 mmol/L ammonium acetate and 50 mmol/L formic acid in water versus acetonitrile to achieve chromatographic separation of the drugs and internal standard (A-86093). Three wavelengths (215, 240, and 260 nm) were monitored. RESULTS: All drugs were eluted within 15 min. Calibration curves with concentrations of 0.025-10 mg/L (1.875-75 mg/L for TPV) showed coefficients of determination (r(2)) between 0.993 and 0.999. The lower limits of quantification were well below the trough concentrations reported in the literature. Inter- and intraassay CVs and the deviations between the nominal and measured concentrations were <15%. The method was validated by successful participation in an international interlaboratory QC program. CONCLUSIONS: This method allows fast and simultaneous quantification of all commercially available PIs and NNRTIs for therapeutic drug monitoring.

Development Of Saquinavir Mesylate Nanoemulsion-Loaded Transdermal Films: Two-Step Optimization Of Permeation Parameters, Characterization, And Ex Vivo And In Vivo Evaluation.

BACKGROUND: Saquinavir mesylate (SQR) tablets are widely used against human immunodeficiency virus. SQR has bioavailability issues owing to its poor aqueous solubility, extensive first-pass metabolism, and even low gastrointestinal tract permeability and absorption. OBJECTIVE: An in-depth optimization process was carried out using factorial design to improve the permeation parameters and thereby the bioavailability of SQR by formulating self-nanoemulsifying drug delivery system (SNEDDS)-loaded polymeric transdermal films. METHODS: The solubility of SQR in different nanoemulsion components was examined. Various combinations of selected components were prepared in an extreme vertices mixture design to identify the useful nanoemulsion zone and to develop SNEDDS with minimum globule size. The optimized SQR-SNEDDS was loaded in polyvinyl alcohol (PVA)-based transdermal films. The Box-Behnken design was used to optimize and evaluate SQR permeability. The prepared films were characterized for thickness, tensile strength, elongation, folding endurance, and accelerated stability studies. The optimized film was examined for ex vivo skin permeation and in vivo pharmacokinetic parameters. RESULTS: The optimized SQR-SNEDDS was prepared in proportions of 0.1, 0.55, and 0.35 of clove oil, labrasol, and Transcutol, respectively. The implemented Box-Behnken design indicated the optimized film consisted of 1.0% PVA, 0.25% propylene glycol, and clove oil as the oil phase. The tensile strength, thickness, percent elongation, and folding endurance of the optimized SQR-SNEDDS film were 0.93 ± 0.013 kg/cm2, 0.22 ± 0.006 mm, 43.1 ± 0.022%, and >200 times, respectively. A higher Cmax and double the AUC were observed for SQR-SNEDDS-loaded film in comparison to pure SQR-loaded films. CONCLUSION: Implementation of a two-step design to optimize and control experimental factors in the preparation of SQR-SNEDDS and its loading onto PVA-based transdermal films was achieved. The films indicated improved ex vivo skin permeation, enhanced bioavailability, and overcame the limitations of the oral dosage form.

Differential roles of P-glycoprotein, multidrug resistance-associated protein 2, and CYP3A on saquinavir oral absorption in Sprague-Dawley rats.

The objective of this investigation was to differentiate the roles of P-glycoprotein (Pgp), multidrug resistance-associated protein 2 (Mrp2), and CYP3A on saquinavir (SQV) oral absorption. With use of single-pass jejunal perfusion (in situ) and portal vein-cannulated rats (in vivo), SQV absorption was studied under chemical inhibition of Pgp [N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2 isoquinolinyl)-ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918)], Mrp2 [(3-(((3-(2-(7-chloro-2-quinolinyl)-(E)-ethenyl)phenyl) ((3-(dimethylamino-3-oxopropyl)thio)methyl)-thio) propanoic acid (MK571)], and/or CYP3A (midazolam). Plasma concentrations of SQV and related metabolites were analyzed by liquid chromatography-tandem mass spectrometry. When given alone, SQV absorption was extremely low both in situ (F(a) = 0.07%) and in vivo [C(max) = 0.068 microg/ml; area under the curve (AUC) = 6.8 microg x min/ml]. Coadministration of GF120918 boosted SQV absorption by more than 20-fold with decreased variation in AUCs (percent coefficient of variation = 30% versus 100%). In contrast, coadministration of MK571 or midazolam increased SQV absorption only 2- to 3-fold without improving the variation in AUCs. SQV oral absorption was not further improved when it was given with GF120918 and midazolam or with GF120918 and MK571. The current results provide, for the first time, direct and explicit evidence that the low oral absorption of SQV is controlled by a secretory transporter, Pgp, and not by limited passive diffusion owing to its poor physicochemical properties. Pgp-mediated transport is also responsible for the highly variable oral bioavailability of SQV. In contrast, intestinal Mrp2 and intestinal CYP3A appear to play minor roles in SQV oral bioavailability. Given the differential and complex roles of Pgp and CYP3A in SQV oral absorption, the optimization of AIDS boosting regimens requires careful consideration to avoid therapy-limiting drug-drug transporter and enzyme interactions.

Ultra-fast quantitation of saquinavir in human plasma by matrix-assisted laser desorption/ionization and selected reaction monitoring mode detection.

We present herein an ultra-fast quantitative assay for the quantitation of saquinavir in human plasma, without prior chromatographic separation, with matrix-assisted laser desorption/ionization using the selected reaction monitoring quantitation mode (MALDI-SRM/MS). The method was found to be linear from 5 to 10,000 ng/ml using pentadeuterated saquinavir (SQV-d5) as an internal standard, and from 5 to 1000 ng/ml using reserpine as internal standard (IS). Accuracy and precision were in the range of 101-108%, 3.9-11% with SQV-d5 and in the range 93-108%, 3.5-15% with reserpine. Plasma samples (250 microl) were extracted with a mixture of ethyl acetate/hexane. MALDI spotting of the extract was automated using electrodeposition and the dried droplet method using alpha-cyano-4-hydroxycinnamic acid (CHCA) as matrix. A 96 spots MALDI plate was prepared within 20 min in a fully unattended manner. Each sample was spotted four times and quantitation was based on the average of their analyte/IS area ratio. Samples were analyzed on a triple quadrupole linear ion trap (QqQ(LIT)) equipped with a high repetition laser source (1000 Hz). The analysis time of one sample was approximately 6 s, therefore 96 samples could be analyzed in less than 10 min. With liquid-liquid extraction sample preparation no significant matrix effects were observed. Moreover, the assay showed sufficient selectivity for samples to be analyzed at the lower limit of quantification (LLOQ) in the presence of other antiretroviral drugs, without prior chromatographic steps. In parallel, to assess the selectivity of the assay with real samples, a liquid chromatography (LC)-SRM/MS method was developed and a cross validation with clinical samples was successfully performed.

Pharmacokinetic monitoring of HIV-1 protease inhibitors in the antiretroviral therapy.

Combination therapy with protease inhibitors (PIs) is used for the treatment of patients infected with the human immunodeficiency virus (HIV). To achieve optimal drug concentrations for viral suppression and avoidance of drug toxicity, therapeutic drug monitoring of PIs levels has been considered essential. In this study an analytical procedure for simultaneous monitoring the plasma concentrations of a total four protease inhibitors: indinavir, lopinavir, nelfinavir and saquinavir was presented. The plasma samples were liquid/liquid extracted and subjected to high performance liquid chromatography (HPLC) analysis. The lopinavir and saquinavir in the patient plasma samples were monitored by ultraviolet detection at 215 nm. Extraction procedure using methyl tert-butyl ether and the mobile phase consisted from acetonitrile with phosphate buffer on a Symmetry C18 column were used to monitor these two compounds. Linearity of the method was obtained in the concentration range of 0.1 - 15.0 mg/mL for all four protease inhibitors. Under steady state conditions, the plasma concentrations of protein inhibitors in 23 patients were determined and area under the plasma concentration-time curve was estimated to maintain optimal viral suppression. We developed a simple and specific method for the simultaneous determination of lopinavir and saquinavir.

Ketoconazole is inferior to ritonavir as an alternative booster for saquinavir in a once daily regimen in Thai HIV-1 infected patients.

OBJECTIVE: To improve the pharmacokinetics of protease inhibitors, boosting with low-dose ritonavir is performed. However, toxicity, storage conditions and high costs of antiretroviral treatment may necessitate interruption of ritonavir. Ketoconazole was investigated as a potential booster of once-daily (o.d.) saquinavir. METHODS: In a single-group, two-period design, 25 virologically and immunologically stable patients on saquinavir/ritonavir 2000/100 mg o.d. were switched to saquinavir/ketoconazole 2000/400 mg o.d. for 2 weeks. Two steady-state pharmacokinetic curves were recorded at both periods. RESULTS: Fourteen females and 11 male patients were included. Median age was 34 years [interquartile range (IQR), 32-42 years], body weight 54 kg (IQR, 47-59 kg) and body mass index 21 kg/m (19-23 kg/m). The mean saquinavir area under the curve (AUC) during boosting with ritonavir was 57.93 +/- 27.96 mg/h/l, maximum observed concentration (Cmax) was 7.50 +/- 3.45 mg/l and concentration at 24 h (Cmin) was 0.35 +/- 0.30 mg/l. When ketoconazole was used, the saquinavir AUC, Cmax, and Cmin were 12.00 +/- 6.97 mg/h/l, 2.43 +/- 1.35 mg/l and 0.03 +/- 0.04 mg/l, respectively. CONCLUSION: Boosting with ketoconazole resulted in 80% lower exposure to saquinavir. Although saquinavir AUC might still be adequate for treatment, concentrations at 24 h reached levels below the recommended trough concentrations of 0.1 mg/l, which may result in selection of resistant HIV-1 viral strains. Therefore, boosting of saquinavir by ketoconazole is not recommended.

The effect of ABCB1 polymorphism on the pharmacokinetics of saquinavir alone and in combination with ritonavir.

This genotype panel study investigated the effect of ABCB1 polymorphism in exon 26 (C3435T), exon 21 (G2677T/A), and exon 12 (C1236T) on saquinavir pharmacokinetics and on the expression and activity of P-glycoprotein (P-gp) in peripheral blood monocytic cells (PBMCs). One hundred and fifty healthy volunteers were genotyped to identify 15 TT3435 and 15 CC3435 individuals. In these individuals, saquinavir pharmacokinetics were assessed after administration of a single oral dose of saquinavir 1,000 mg and saquinavir/ritonavir 1,000/100 mg. PBMC P-gp expression and activity were assessed in 15 and 19 subjects. The co-administration of ritonavir on study day 2 caused a significant increase in saquinavir exposure, in both TT3435 and CC3435 individuals. No correlation was observed between the ABCB1 C3435T, G2677T/A, and C1236T polymorphisms, separately and in haplotypes, with saquinavir pharmacokinetics, administered with or without ritonavir and with PBMC P-gp expression and activity. In conclusion, ABCB1 polymorphism has no pronounced effect on saquinavir exposure.

Pharmacokinetics of saquinavir with atazanavir or low-dose ritonavir administered once daily (ASPIRE I) or twice daily (ASPIRE II) in seronegative volunteers.

ASPIRE I and II were prospective, 3-way sequential crossover studies in healthy volunteers to compare the safety and pharmacokinetics of saquinavir/ritonavir (SQV/RTV) with saquinavir/atazanavir (SQV/ATV) administered either once daily (QD, ASPIRE I) or twice daily (BID, ASPIRE II). Treatments were separated by 10 days, and pharmacokinetic analyses were performed on days 11, 32, and 53. SQV pharmacokinetics were significantly higher when dosed with RTV compared to ATV (P < .05 for all comparisons). ATV pharmacokinetics were similar within treatment arms. ATV Cmin increased approximately 60%, and Cmax decreased approximately 35% with BID dosing compared with QD dosing. Women had higher exposure for all 3 protease inhibitors (PIs) compared with men after adjusting for weight. Adverse effects were primarily gastrointestinal-related with SQV/RTV and hyperbilirubinemia with SQV/ATV. Although SQV plasma concentrations were higher when coadministered with RTV, a combination of SQV/ATV administered BID may be a viable alternative in HIV-infected, PI-naive subjects intolerant to RTV.

Simultaneous determination of 8 HIV protease inhibitors in human plasma by isocratic high-performance liquid chromatography with combined use of UV and fluorescence detection: amprenavir, indinavir, atazanavir, ritonavir, lopinavir, saquinavir, nelfinavir and M8-nelfinavir metabolite.

A simple, accurate and fast method was developed for determination of the commonly used HIV protease inhibitors (PIs) amprenavir, indinavir, atazanavir, ritonavir, lopinavir, nelfinavir, M8-nelfinavir metabolite and saquinavir in human plasma. Liquid-liquid extraction was used with hexane/ethylacetate from buffered plasma samples with a borate buffer pH 9.0. Isocratic chromatographic separation of all components was performed on an Allsphere hexyl HPLC column with combined UV and fluorescence detection. Calibration curves were constructed in the range of 0.025-10 mg/l. Accuracy and precision of the standards were all below 15% and the lowest limit of quantitation was 0.025 mg/l. Stability of quality control samples at different temperature conditions was found to be below 20% of nominal values. The advantages of this method are: (1) inclusion and determination of the newly approved atazanavir, (2) simultaneous isocratic HPLC separation of all compounds and (3) increased specificity and sensitivity for amprenavir by using fluorescence detection. This method can be used for therapeutic drug monitoring of all PIs currently commercialised and is now part of current clinical practice.

An isocratic liquid chromatography method for determining HIV non-nucleoside reverse transcriptase inhibitor and protease inhibitor concentrations in human plasma.

An efficient, isocratic high performance liquid chromatography (HPLC) method for determining human immunodeficiency virus (HIV) non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs) in plasma is advantageous for laboratories participating in clinical trials and therapeutic drug monitoring (TDM) programs, or conducting small animal research. The combination of isocratic reversed phase chromatography using an S-3, 3.0 mm x 150 mm column along with low plasma volume (200 microl), rapid liquid-liquid extraction, and detection at a single wavelength (212 nm) over a short run time makes this method valuable. Within and between assay variability ranges from 0.8 to 3.5% and 1.2-6.2%, respectively. Accuracy ranges from 91.0 to 112.8% for four quality controls (50, 100, 1000, and 10,000 ng/ml) for all drugs measured (efavirenz, nevirapine, amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir).

High-performance liquid chromatography assay for the determination of the HIV-protease inhibitor tipranavir in human plasma in combination with nine other antiretroviral medications.

An accurate, sensitive and simple reverse-phase (RP) high-performance liquid chromatography (HPLC) assay has been developed and validated for the simultaneous quantitative determination of tipranavir with nine other antiretroviral drugs in plasma. A liquid-liquid extraction of the drugs in tert-butylmethylether (TBME) from 200 microL of plasma is followed by a reversed phase gradient HPLC assay with UV detection at 210 nm. The standard curve for the drug was linear in the range of 80-80,000 ng/mL for tipranavir; 10-10,000 ng/mL for nevirapine, indinavir, efavirenz, and saquinavir; and 25-10,000 ng/mL for amprenavir, atazanavir, ritonavir, lopinavir, and nelfinavir. The regression coefficient (r(2)) was greater than 0.998 for all analytes. This method has been fully validated and shown to be specific, accurate and precise. Due to an excellent extraction procedure giving good recovery and a clean baseline, this method is simple, rapid, accurate and provides excellent resolution and peak shape for all analytes. Thus this method is very suitable for therapeutic drug monitoring.

Pharmacokinetics of Saquinavir Mesylate from Oral Self-Emulsifying Lipid-Based Delivery Systems.

BACKGROUND AND OBJECTIVES: Although lipid-based drug delivery systems have gained much importance in recent years due to their ability to improve the solubility and bioavailability of poorly soluble drugs, compartmental pharmacokinetic analyses have not been extensively explored. The oral pharmacokinetics of commercial liquid formulation and a developed semisolid system containing saquinavir mesylate (SQVM) were compared in Beagle dogs. A compartmental analysis after intravenous bolus administration of this drug (1 mg/kg) was also performed. METHOD: Pharmacokinetic profiles were analyzed using both non-compartmental and compartmental approaches. Plasma concentration of the drug was determined by high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS). RESULTS: The disposition curve of SQVM given intravenously was better described by a three-compartment model. In contrast, plasma profiles obtained following the oral administration were fitted to a two-compartment model with lag time due to the fact that the distribution phase was masked by the absorption phase in these formulations. CONCLUSION: The proposed semisolid lipid system was found to be a promising formulation for commercial purposes given the similarity of SQVM absorption rate to that from the commercial liquid formulation.

Effect of omeprazole on the pharmacokinetics of saquinavir-500 mg formulation with ritonavir in healthy male and female volunteers.

INTRODUCTION: Recent studies have described reduced absorption of certain protease inhibitors when administered with agents known to increase gastric pH. No clinically significant interactions between saquinavir absorption and gastric pH have previously been shown. We evaluated the effect of omeprazole, a proton-pump-inhibitor, on the pharmacokinetics of the recently developed saquinavir-500 mg formulation co-administered with ritonavir. METHODS: Eighteen healthy subjects (n = 6 women and 12 men) received 1000/100 mg saquinavir/ritonavir twice daily in an open-label study for 15 days. On days 11-15, subjects were administered omeprazole 40 mg daily with the morning dose. Serial plasma samples were collected for 12-h pharmacokinetic profiles of saquinavir and ritonavir on days 10 and 15 and safety analysis on days 1, 4, 10, 15 and 29. RESULTS: The geometric mean and 95% confidence interval (CI), for the area under time-concentration curve (AUC; ng h/ml), trough plasma concentration (C trough; ng/ml) and maximum observed plasma concentration (Cmax; ng/ml) of saquinavir were 20599 (14396-29360) and 37511 (28733-48970); 737 (482-1127) and 1521 (1039-2227); 3227 (2370-4393) and 5611 (4507-7710) on days 10 and 15, respectively, with geometric mean ratios of 1.82, 2.06 and 1.75. No significant changes were observed in saquinavir elimination half life, ritonavir pharmacokinetic parameters or in safety laboratory tests. No unexpected adverse events attributed to study medication were noted. CONCLUSIONS: In the presence of omeprazole, total saquinavir plasma exposure is significantly increased (82% increase in AUC). The mechanism of this interaction requires elucidation. Despite the significant increase in saquinavir exposure, no short term toxicities were observed.

Effect of quercetin on the plasma and intracellular concentrations of saquinavir in healthy adults.

STUDY OBJECTIVES: To determine if quercetin, a bioflavonoid that inhibits p-glycoprotein, alters plasma saquinavir concentrations, and to explore the potential influence on intracellular concentrations. DESIGN: Prospective pharmacokinetic analysis. SETTING: University-affiliated general clinical research center. SUBJECTS: Ten healthy adults (four women, six men) with a mean +/- SD age of 30.7 +/- 9.4 years. INTERVENTION: All subjects received saquinavir 1200 mg 3 times/day with food on days 1-11 and quercetin 500 mg 3 times/day with food on days 4-11. MEASUREMENTS AND MAIN RESULTS: On days 4 and 11, nine blood samples and four peripheral blood mononuclear cell samples were drawn during a steady-state dosing interval. Pharmacokinetic parameters were calculated by using standard noncompartmental techniques. Plasma saquinavir concentrations were similar regardless of quercetin administration. Geometric mean ratios for the area under the concentration-time curve during an 8-hour dosing interval (AUC0-8), maximum concentration in the dosing interval, and minimum concentration in the dosing interval were 0.99 (95% confidence interval [CI] 0.65-1.50), 0.99 (95% CI 0.64-1.54), and 1.06 (95% CI 0.68-1.67), respectively. Intracellular saquinavir concentrations displayed substantial intra- and intersubject variability, which limited the ability to determine the influence of quercetin coadministration (geometric mean ratio for AUC0-8 = 0.51 [95% CI 0.14-1.95], six patients). CONCLUSION: Quercetin coadministration did not influence plasma saquinavir concentrations. Because of substantial inter- and intrasubject variability, more study is necessary to determine if saquinavir intracellular concentrations are altered by coadministration of quercetin.

Lack of in vivo correlation between indinavir and saquinavir exposure and cytochrome P450 3A phenotype as assessed with oral midazolam as a phenotype probe.

STUDY OBJECTIVE: To investigate a potential correlation between exposure to oral midazolam, a commonly used cytochrome P450 (CYP) 3A probe, and saquinavir and indinavir exposure. DESIGN: Open-label, prospective, pharmacokinetic study. SETTING: Outpatient research center. SUBJECTS: Thirty-six healthy volunteers aged 22-50 years. INTERVENTION: Subjects received a single oral dose of midazolam 8 mg; 4 hours later, blood was drawn to determine their serum midazolam concentrations. Midazolam phenotyping was followed by successive administration of the protease inhibitors indinavir and saquinavir, with blood sampling and pharmacokinetic analyses performed at steady state. MEASUREMENTS AND MAIN RESULTS: Pharmacokinetic parameters of each protease inhibitor were evaluated to assess for a potential relationship with 4-hour concentrations of midazolam. No correlations between phenotype results for midazolam and any pharmacokinetic parameter for indinavir or saquinavir were identified (r(2)=0.00002-0.073). When the results were analyzed based on race, significant correlations were identified in five African-American subjects, including correlations between 4-hour midazolam levels and apparent oral clearance of saquinavir (r(2)=0.734, p=0.064), area under the plasma concentration-time curve from 0-8 hours (r(2)=0.914, p=0.011), minimum concentration (r(2)=0.857, p=0.024), and maximum concentration (r(2)=0.969, p=0.002). These findings for African-American subjects were not seen with indinavir. No correlation was found between indinavir and saquinavir pharmacokinetic parameters (r(2)=0.017-0.261). CONCLUSION: Oral midazolam was not a useful probe for predicting saquinavir or indinavir exposure at steady state. Reasons for the lack of correlation likely included differences between midazolam and protease inhibitor P-glycoprotein specificity, differences in the relative contribution of CYP3A5-mediated metabolism, and/or variation in intestinal and hepatic CYP3A specificity. The strong correlation between midazolam phenotype and pharmacokinetic parameters for saquinavir in African-American subjects indicated a racial difference in one or more of these confounding variables.