Atazanavir-induced urine crystals demonstrated by infrared spectroscopic analysis.

Atazanavir sulfate, an azapeptide inhibitor of HIV protease, has been associated with urolithiasis. A 60-year-old man with atazanavir-induced urinary sediment crystals verified by infrared spectroscopic analysis is described. He had been receiving highly active antiretroviral therapy (HAART) for HIV infection and also had a history of urinary lithiasis and been undergoing urinalysis once every month. Needle-shaped crystals were seen in his urine sediment and infrared spectroscopic analysis revealed that these were atazanavir crystals. Because the presence of the crystals in urine do not always reveal an abnormality in the urinary test strip analysis, the urinary sediment needed to be observed microscopically in order to prevent future urolithiasis and renal failure in this HIV patient receiving atazanavir.

CPY3A4-mediated α-hydroxyaldehyde formation in saquinavir metabolism.

Saquinavir (SQV) is a protease inhibitor widely used for the treatment of human immunodeficiency virus (HIV) infection. We profiled SQV metabolism in mice using a metabolomic approach. Thirty SQV metabolites were identified in mouse feces and urine, of which 20 are novel. Most metabolites observed in mice were recapitulated in human liver microsomes. Among these novel metabolites, one α-hydroxyaldehyde produced from SQV N-dealkylation was noted and verified for the first time. Meanwhile, the corresponding product (3S)-N-tert-butyldecahydro-isoquinoline-3-carboxamide and its further metabolites were identified in mouse urine. The α-hydroxyaldehyde pathway was confirmed by using semicarbazide as a trapping reagent as well. Using recombinant cytochrome P450 (CYP450) isoenzymes and Cyp3a-null mice, CYP3A was identified as the dominant enzyme contributing to the formation of α-hydroxyaldehyde. This study enhances our knowledge of SQV metabolism, which can be used for predicting drug-drug interactions and further understanding the mechanism of adverse effects associated with SQV.

CYP3A-mediated generation of aldehyde and hydrazine in atazanavir metabolism.

Atazanavir (ATV) is an antiretroviral drug of the protease inhibitor class. Multiple adverse effects of ATV have been reported in clinical practice, such as jaundice, nausea, abdominal pain, and headache. The exact mechanisms of ATV-related adverse effects are unknown. It is generally accepted that a predominant pathway of drug-induced toxicity is through the generation of reactive metabolites. Our current study was designed to explore reactive metabolites of ATV. We used a metabolomic approach to profile ATV metabolism in mice and human liver microsomes. We identified 5 known and 13 novel ATV metabolites. Three potential reactive metabolites were detected and characterized for the first time: one aromatic aldehyde, one α-hydroxyaldehyde, and one hydrazine. These potential reactive metabolites were primarily generated by CYP3A. Our results provide a clue for studies on ATV-related adverse effects from the aspect of metabolic activation. Further studies are suggested to illustrate the impact of these potential reactive metabolites on ATV-related adverse effects.

Absorption, metabolism, and excretion of darunavir, a new protease inhibitor, administered alone and with low-dose ritonavir in healthy subjects.

Absorption, metabolism, and excretion of darunavir, an inhibitor of human immunodeficiency virus protease, was studied in eight healthy male subjects after a single oral dose of 400 mg of [(14)C]darunavir given alone (unboosted subjects) or with ritonavir [100 mg b.i.d. 2 days before and 7 days after darunavir administration (boosted subjects)]. Plasma exposure to darunavir was 11-fold higher in boosted subjects. Total recoveries of radioactivity in urine and feces were 93.9 and 93.5% of administered radioactivity in unboosted and boosted subjects, respectively. The most radioactivity was recovered in feces (81.7% in unboosted subjects and 79.5% in boosted subjects, compared with 12.2 and 13.9% recovered in urine, respectively). Darunavir was extensively metabolized in unboosted subjects, mainly by carbamate hydrolysis, isobutyl aliphatic hydroxylation, and aniline aromatic hydroxylation and to a lesser extent by benzylic aromatic hydroxylation and glucuronidation. Total excretion of unchanged darunavir accounted for 8.0% of the dose in unboosted subjects. Boosting with ritonavir resulted in significant inhibition of carbamate hydrolysis, isobutyl aliphatic hydroxylation, and aniline aromatic hydroxylation but had no effect on aromatic hydroxylation at the benzylic moiety, whereas excretion of glucuronide metabolites was markedly increased but still represented a minor pathway. Total excretion of unchanged darunavir accounted for 48.8% of the administered dose in boosted subjects as a result of the inhibition of darunavir metabolism by ritonavir. Unchanged darunavir in urine accounted for 1.2% of the administered dose in unboosted subjects and 7.7% in boosted subjects, indicating a low renal clearance. Darunavir administered alone or with ritonavir was well tolerated.

Pharmacokinetics of BILR 355 after multiple oral doses coadministered with a low dose of ritonavir.

The pharmacokinetics and safety of BILR 355 following oral repeated dosing coadministered with low doses of ritonavir (RTV) were investigated in 12 cohorts of healthy male volunteers with a ratio of 6 to 2 for BILR 355 versus the placebo. BILR 355 was given once a day (QD) coadministered with 100 mg RTV (BILR 355/r) at 5 to 50 mg in a polyethylene glycol solution or at 50 to 250 mg as tablets. BILR 355 tablets were also dosed at 150 mg twice a day (BID) coadministered with 100 mg RTV QD or BID. Following oral dosing, BILR 355 was rapidly absorbed, with the mean time to maximum concentration of drug in serum reached within 1.3 to 5 h and a mean half-life of 16 to 20 h. BILR 355 exhibited an approximately linear pharmacokinetics for doses of 5 to 50 mg when given as a solution; in contrast, when given as tablets, BILR 355 displayed a dose-proportional pharmacokinetics, with a dose range of 50 to 100 mg; from 100 to 150 mg, a slightly downward nonlinear pharmacokinetics occurred. The exposure to BILR 355 was maximized at 150 mg and higher due to a saturated dissolution/absorption process. After oral dosing of BILR 355/r, 150/100 mg BID, the values for the maximum concentration of drug in plasma at steady state, the area under the concentration-time curve from 0 to the dose interval at steady state, and the minimum concentration of drug in serum at steady state were 1,500 ng/ml, 12,500 h.ng/ml, and 570 ng/ml, respectively, providing sufficient suppressive concentration toward human immunodeficiency virus type 1. Based on pharmacokinetic modeling along with the in vitro virologic data, several BILR 355 doses were selected for phase II trials using Monte Carlo simulations. Throughout the study, BILR 355 was safe and well tolerated.

[Indinavir-associated tubulointerstitial renal disease]. / Néphropathie tubulo-interstitielle associée à l'indinavir.

Indinavir, used for the treatment of HIV disease, forms distinctive crystals in the urine. The crystalluria has been associated principally with several urinary tract abnormalities which may require discontinuation of the drug. We present a case of progressive leucocyturia and renal impairment occurring during indinavir treatment which illustrates vividly the impact of the crystalluria on the tubulointerstitial renal compartment.

Steady-state disposition of the nonpeptidic protease inhibitor tipranavir when coadministered with ritonavir.

The pharmacokinetic and metabolite profiles of the antiretroviral agent tipranavir (TPV), administered with ritonavir (RTV), in nine healthy male volunteers were characterized. Subjects received 500-mg TPV capsules with 200-mg RTV capsules twice daily for 6 days. They then received a single oral dose of 551 mg of TPV containing 90 microCi of [(14)C]TPV with 200 mg of RTV on day 7, followed by twice-daily doses of unlabeled 500-mg TPV with 200 mg of RTV for up to 20 days. Blood, urine, and feces were collected for mass balance and metabolite profiling. Metabolite profiling and identification was performed using a flow scintillation analyzer in conjunction with liquid chromatography-tandem mass spectrometry. The median recovery of radioactivity was 87.1%, with 82.3% of the total recovered radioactivity excreted in the feces and less than 5% recovered from urine. Most radioactivity was excreted within 24 to 96 h after the dose of [(14)C]TPV. Radioactivity in blood was associated primarily with plasma rather than red blood cells. Unchanged TPV accounted for 98.4 to 99.7% of plasma radioactivity. Similarly, the most common form of radioactivity excreted in feces was unchanged TPV, accounting for a mean of 79.9% of fecal radioactivity. The most abundant metabolite in feces was a hydroxyl metabolite, H-1, which accounted for 4.9% of fecal radioactivity. TPV glucuronide metabolite H-3 was the most abundant of the drug-related components in urine, corresponding to 11% of urine radioactivity. In conclusion, after the coadministration of TPV and RTV, unchanged TPV represented the primary form of circulating and excreted TPV and the primary extraction route was via the feces.

Effects of escin on indinavir crystallization time in the urine of patients with HIV-I infection: a multicenter, randomized, open-label, controlled, four-period crossover trial.

BACKGROUND: The combination of indinavir, a protease inhibitor, and reverse-transcriptase inhibitors is widely used in the treatment of HIV-1 infection. However, precipitation of indinavir crystals in the renal tubular lumen due to the drug's aqueous insolubility may result in characteristic symptoms of flank pain or classic renal colic. An in vitro study has shown that addition of escin to synthetic urine containing indinavir delayed the crystallization time of indinavir. OBJECTIVE: This study examined the efficacy and tolerability of the addition of escin to highly active antiretroviral therapy containing indinavir to delay the crystallization time of indinavir in urine. METHODS: This was a multicenter, randomized, open-label, controlled, 4-period crossover trial in which each period lasted 4 weeks. HIV-1-infected adults receiving treatment with indinavir plus 2 nucleoside analogue reverse-transcriptase inhibitors in whom plasma viral loads had been undetectable (HIV-1 RNA <200 copies/mL) for at least 6 months were randomly assigned to 1 of 2 groups based on the timing of the initiation of escin. Group I received escin during the second and third treatment periods, and group II received escin during the first and fourth treatment periods. The primary end point was the in vitro crystallization time of indinavir in 24-hour urine specimens, determined at the end of each 4-week period. Tolerability was assessed based on the number of patients with a rebound in plasma viral load and on the numbers of clinically and biologically relevant adverse events (including those requiring discontinuation of treatment). Clinical and laboratory evaluations were performed throughout each 4-week period. RESULTS: Fifty HIV-1-infected patients were enrolled, 47 were randomized to treatment (40 [85.1%] men, 7 [14.9%] women; median [interquartile range] age, 36 [34-45] years), and 30 completed the study. Urine pH and plasma and urine indinavir concentrations were unaffected by the addition of escin to antiretroviral treatment. The mean time to the onset of crystallization was 14.7 minutes with escin (95% Cl, 11.8-17.5) and 9.9 minutes without it (95% Cl, 6.7-13.1). Therefore, the addition of escin increased the mean crystallization time by 5.5 minutes (95% Cl, 1.5-9.5; P = 0.008), representing the overall capacity of study treatment to inhibit indinavir crystallization in the urine. Three of 47 patients had mild gastrointestinal symptoms associated with escin treatment. No episodes of nephrolithiasis were recorded during the study or after the completion of study treatment. CONCLUSION: The results of this prospective clinical trial of the effect of escin on indinavir crystallization time support the possibility that indinavir-associated nephrolithiasis may be prevented by means other than overhydration. Further research is needed in greater numbers of patients over longer follow-up times.

Highly sensitive determination of saquinavir in biological samples using liquid chromatography-tandem mass spectrometry.

A selective and sensitive method for the determination of the HIV protease inhibitor saquinavir in human plasma, saliva, and urine using liquid-liquid extraction and LC-MS-MS has been developed, validated, and applied to samples of a healthy individual. After extraction with ethyl acetate, sample extracts were chromatographed isocratically within 5 min on Kromasil RP-18. The drug was detected with tandem mass spectrometry in the selected reaction monitoring mode using an electrospray ion source and 2H(5)-saquinavir as internal standard. The limit of quantification was 0.05 ng/mL. The accuracy of the method varied between -1 and +10% (SD within-batch) and the precision ranged from +4 to +10% (SD batch-to-batch). The method is linear at least within 0.05 and 87.6 ng/mL. After a regular oral dose (600 mg) saquinavir concentrations were detectable for 48 h in plasma and were well correlated with saliva concentrations (r(2)=0.9348, mean saliva/plasma ratio 1:15.1). The method is well suited for low saquinavir concentrations in different matrices.

Prospective study of urinalysis abnormalities in HIV-positive individuals treated with indinavir.

Indinavir is a potent protease inhibitor widely used in combination with reverse-transcriptase inhibitors to treat human immunodeficiency virus (HIV) disease. Individuals treated with indinavir are prone to develop urinary complications, including renal colic, renal calculi, lower urinary tract symptoms, and indinavir crystalluria. Although renal stones secondary to indinavir have been described and characterized, little is known about the onset, frequency, and significance of the crystalluria. To document the longitudinal characteristics of indinavir crystalluria and associated urine abnormalities, 54 asymptomatic indinavir-naive HIV-positive individuals had urinalysis testing initially weekly and then monthly during the first year of indinavir treatment. Six hundred eight urinalyses were performed (11 +/- 2 urinalysis/subject), including 579 microscopy examinations performed by a nephrologist (10 +/- 2 examinations/subject). Baseline urinalysis results were essentially normal. After the start of treatment, indinavir crystalluria was frequently observed (67% of subjects). After the first 2 weeks, indinavir crystalluria remained constant at a frequency of approximately 25% of urine sediments examined at each test point. Other urine abnormalities, principally leukocytes (>/=10/high-power field) and casts, were observed in 39% of subjects. These abnormalities were more severe in five subjects, with concomitant increasing serum creatinine levels in three of them. Additional urine findings include the predominance of low pH (/=1.025 in 66% of urinalyses). In conclusion, abnormal urinalysis results were noted frequently during the first year of treatment with indinavir. The main findings were the high proportion of subjects with crystalluria and the relatively high frequency of crystalluria observed consistently throughout. These findings may occasionally be associated with other urine abnormalities, presumably secondary to indinavir crystalluria.

Detection of indinavir crystals in urine: dependence on method of analysis.

OBJECTIVES: To determine the frequency of crystalluria in patients treated with the human immunodeficiency virus protease inhibitor indinavir and to compare methods of detecting crystalluria. METHODS: A total of 308 freshly voided urine specimens from 168 patients treated with indinavir were evaluated by manual microscopy of sediment and microscopy with an automated workstation and by dipstick analysis. RESULTS: Crystals were detected in 22%, 31%, or 32% of specimens using, respectively, an automated workstation, manual microscopy, or both methods. Proteinuria or hemoglobinuria occurred significantly more often in specimens with (28%) than without (18%) crystals. Frequency of crystalluria was unrelated to specific gravity, but it increased at higher pH. Crystals were detected in 21% of specimens with pH less than 6 and 42% of specimens with pH of 6 or higher. CONCLUSIONS: Crystalluria occurs in more than 30% of urine specimens from patients treated with indinavir, but detection rates vary substantially with method of analysis. Manual microscopy detected crystalluria 41% more often than did an automated workstation.

Determination of L-756 423, a novel HIV protease inhibitor, in human plasma and urine using high-performance liquid chromatography with fluorescence detection.

A method for the determination of L-756 423, a novel HIV protease inhibitor, in human plasma and urine is described. Plasma and urine samples were extracted using 3M Empore extraction disk cartridges in the C18 and MPC (mixed-phase cation-exchange) formats, respectively. The extract was analyzed using HPLC with fluorescence detection (ex 248 nm, em 300 nm), and included a column switching procedure to reduce run-time. The assay was linear in the concentration range 5 to 1000 ng/ml when 1-ml aliquots of plasma and urine were extracted. Recoveries of L-756 423 were greater than 84% over the calibration curve range using the described sample preparation procedures. Intra-day precision and accuracy for this assay was less than 9% RSD and within 7%, respectively. Inter-day variabilities for the plasma (n=17) and urine (n= 10) were less than 5% and 3% for low (15 ng/ml) and high (750 ng/ml) quality control samples. Bovine serum albumin (0.5%) was used as an additive to urine to prevent precipitation of L-756 423 during the storage of clinical samples. The assay was used in support of human clinical trials.

Indinavir crystallization and urolithiasis.

The crystallization of indinavir in synthetic urine at different pH values and indinavir concentrations was kinetically studied. It was found that precipitation time notably decreases at urinary pH values above 6.0. The effects of some products as potential crystallization inhibitors were studied. Some natural saponins such as escin and glycyrrhizic acid provoked a notable increase in the precipitation time of indinavir, this pointing out their possible use to prevent renal tubular solid deposition.

Indinavir crystalluria: identification of patients at increased risk of developing nephrotoxicity.

OBJECTIVE: To determine whether the protease inhibitor indinavir sulfate, which is extremely insoluble at physiologic pH levels and which is known to be associated with nephrolithiasis, is associated with crystalluria at a usual therapeutic dose. METHODS: Freshly voided urine from 27 male human immunodeficiency virus patients being treated with indinavir at a dose of 800 mg, tid, in an outpatient setting and from 20 healthy subjects undergoing routine physical examination was subjected to dipstick urinalysis and microscopic examination of urinary sediments. RESULTS: Three (11%) of 27 patients treated with indinavir developed highly characteristic crystalluria during the course of therapy. No such crystals were observed in the urine of the 20 healthy subjects. CONCLUSION: Indinavir crystalluria was identified in asymptomatic patients treated with usual therapeutic doses of the drug. Screening urines of patients taking indinavir may be useful in identifying patients at risk for developing nephrotoxicity.

Disposition of indinavir, a potent HIV-1 protease inhibitor, after an oral dose in humans.

Indinavir, N-[2(R)-hydroxy-1(S)-indanyl]-5-[2(S)-tertiary- butylaminocarbonyl-4-(3-pyridylmethyl)piperazino]-4(S)- hydroxy-2(R)-phenylmethylpentanamide (L-735,524,MK-639, ayl-4- Crixivan), is a potent and specific inhibitor of the HIV-1(3 protease for the treatment of AIDS. Disposition of [14C]indinavir was investigated in six healthy subjects after single oral administration of 400 mg. AUC, Cmax, and Tmax values for indinavir were 492 microM x min, 4.7 microM, and 50 min, respectively. The AUC value for the total radioactivity in plasma was 1.9 times higher than that of indinavir, indicating the presence of metabolites. The major excretory route was through feces, and the minor through urine. Mean recovery of radioactivity in the feces was 83.4%. In the urine, mean recoveries of the total radioactivity and unchanged indinavir were 18.7% and 11.0% of the dose, respectively. HPLC radioactivity and LC-MS/MS analyses of urine showed the presence of indinavir and low levels of quaternary pyridine N-glucuronide (M1), 2',3'-trans-dihydroxyindanylpyridine N-oxide (M2), 2',3'-trans-dihydroxyindan (M3) and pyridine N-oxide (M4a) analogs, and despyridylmethyl analogs of M3 (M5) and indinavir (M6). M5 and M6 were the major metabolites in urine. The metabolic profile in plasma was similar to that in urine. Quantitatively, the metabolites in feces accounted for >47% of the dose, which along with the urinary excretion of approximately 19%, suggested that the absorption of the drug was appreciable. In the feces, radioactivity was predominantly due to M3, M5, M6, and the parent compound. Thus, in urine and feces, the prominent metabolic pathways were oxidations and oxidative N-dealkylations. Excretion of the quaternary N-glucuronide metabolite in the urine, which is a minor metabolite in human, was specific to primates.