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Prednisolone Pharmacokinetics in the Presence and Absence of Ritonavir After Oral Prednisone Administration to Healthy Volunteers

Penzak, Scott R, PharmD*; Formentini, Elizabeth, RNC, MSN, FNP-C; Alfaro, Raul M, MS*; Long, Michael, PharmD*; Natarajan, Ven, PhD; Kovacs, Joseph, MD

JAIDS Journal of Acquired Immune Deficiency Syndromes: December 15th, 2005 - Volume 40 - Issue 5 - p 573-580
doi: 10.1097/01.qai.0000187444.38461.70
Clinical Science
Free

Corticosteroid therapy has been associated with bone toxicities (eg, osteonecrosis) and Cushing syndrome in HIV-infected patients; this may be partially attributable to a pharmacokinetic drug interaction between HIV protease inhibitors and corticosteroids. The purpose of this study was to characterize the influence of low-dose ritonavir on prednisolone pharmacokinetics in healthy subjects. Ten HIV-seronegative volunteers were given single oral doses of prednisone, 20 mg, before (baseline) and after receiving ritonavir, 200 mg, twice daily for 4 and 14 days. After each prednisone dose, serial blood samples were collected and prednisolone concentrations were determined; pharmacokinetic parameter values were compared between the groups. Geometric mean ratios (GMRs, 90% confidence interval [CI]) of the prednisolone area under the plasma concentration versus time curve (AUC0-∞) after 4 and 14 days of ritonavir versus baseline were 1.41 (90% CI: 1.08 to 1.74) and 1.30 (90% CI: 1.09 to 1.49), respectively (P = 0.002 and P = 0.004, respectively). GMRs of prednisolone apparent oral clearance (Cl/F) were 0.71 (09% CI: 0.57 to 0.93) and 0.77 (90% CI: 0.67 to 0.92) after 4 and 14 days of ritonavir versus baseline, respectively (P = 0.0004 and P = 0.0003, respectively). Ritonavir significantly increased the systemic exposure of prednisolone in healthy subjects. Results from this investigation suggest that corticosteroid exposure is likely elevated in HIV-infected patients receiving protease inhibitors.

From the *Warren G. Magnuson Clinical Center, Pharmacy Department, National Institutes of Health, Bethesda, MD; †Science Applications International Corporation-Frederick, Frederick, MD; and ‡Warren G. Magnuson Clinical Center, Department of Critical Care Medicine, National Institutes of Health, Bethesda, MD.

Received for publication May 18, 2005; accepted August 31, 2005.

Supported by the Clinical Center Pharmacy Department, National Institutes of Health and the National Institute of Allergy and Infectious Diseases.

Reprints: Scott R. Penzak, Clinical Pharmacokinetics Research Laboratory, National Institutes of Health, Clinical Center Pharmacy Department, Building 10, Room 1N 257, Bethesda, MD 20892 (e-mail: Spenzak@mail.cc.nih.gov).

Potent combination antiretroviral therapy has been shown to reduce morbidity and mortality in patients with HIV infection.1 Nonetheless, drug interactions and toxicities continue to complicate the pharmacologic management of HIV infection. Patients with HIV infection typically receive at least 3 to 4 antiretroviral medications in addition to other drugs for the treatment of concurrent medical conditions, supportive care, prophylaxis or treatment of opportunistic infections, and immunomodulation.2 Several of these conditions such as asthma, inflammatory arthritis, neoplastic diseases, and renal or liver transplantation are routinely treated with drug regimens that include systemic corticosteroids (eg, prednisone).

Corticosteroid administration has been associated with a variety of toxicities in patients with HIV infection.3-8 There are multiple reports of iatrogenic Cushing syndrome in patients with HIV infection who were receiving fluticasone (oral and nasal inhalation dosage forms) in combination with the HIV protease inhibitor ritonavir.3-5 These reports are consistent with results from a recent investigation in volunteers in which fluticasone propionate aqueous nasal spray (200 μg administered once daily) in combination with ritonavir (100 mg administered twice daily for 7 days) resulted in significant increases in plasma fluticasone concentrations and a marked decrease (86%) in the plasma cortisol area under the plasma concentration versus time curve (AUC).9

Several studies have also identified corticosteroid use as a potential risk factor for the development of osteonecrosis of the femoral head in patients with HIV infection.6-8 Of note, many of the patients in these investigations were on concurrent protease inhibitor-containing antiretroviral regimens, leading to speculation that protease inhibitors may impair the cytochrome P450 (CYP) 3A4-mediated metabolism of corticosteroids, thereby increasing their systemic exposure and toxicity potential. Indeed, other CYP3A4 inhibitors such as certain azole antifungals and diltiazem have been shown to elevate prednisolone concentrations in healthy volunteers administered single doses of prednisone (which is rapidly converted to prednisolone in vivo).10-13 These data suggest that medications that inhibit CYP3A4 (eg, all available HIV protease inhibitors)2 may increase the systemic exposure and toxicity potential of prednisolone.

The purpose of this study was to characterize the influence of the HIV protease inhibitor ritonavir on prednisolone pharmacokinetics in healthy volunteers receiving single oral doses of prednisone before and on days 4 and 14 of a 2-week course of ritonavir administration.

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METHODS

Study Subjects

We conducted a 2-treatment, 2-period, single-sequence drug interaction study in healthy human subjects. Eleven healthy volunteers (6 male) who were 31 ± 7.6 years old (age range: 23-48 years) and weighed 73 ± 14 kg were evaluated in this protocol. Sample size was calculated with regard to reported variability in the prednisolone AUC in healthy volunteers (mean AUC0-24: 1297 ng · h/mL ± 157).11 Based on these data and α = 0.05, a sample size of 10 was calculated to give 90% power to determine a clinically relevant change of 25% in prednisolone AUC with concomitant ritonavir. To be considered for study inclusion, candidates had to be 18 to 50 years old and in good general health, as determined by medical history, a complete blood cell count, and serum chemistries (electrolytes, liver function tests, creatinine, and blood urea nitrogen). Subjects were also required to be HIV-negative (by enzyme-linked immunosorbent assay [ELISA]). Because of ritonavir's predilection for raising serum lipids, individuals with nonfasting cholesterol or triglyceride values >270 mg/dL were excluded from study participation. Additional exclusion criteria included smoking within the previous 6 weeks, regular abuse of drugs and/or alcohol, pregnancy or breast-feeding, receipt of over-the-counter or herbal medications within 30 days of study participation, a history of adverse reaction(s) to corticosteroids or ritonavir, and persistent diarrhea or malabsorption history. Chronic use of prescription medications, including contraceptive steroids, also precluded study participation; exemptions included intermittent use of acetaminophen, nonsteroidal anti-inflammatory medications, loperamide for ritonavir-associated diarrhea, and a daily multivitamin with minerals. Subjects refrained from taking these medications on prednisolone pharmacokinetic sampling days. Subjects also refrained from ingesting grapefruit or grapefruit juice during the study period. Female participants of child-bearing potential were required to use a nonhormonal method of contraception throughout the study. Subjects who received any type of vaccination or were exposed to a viral illness (eg, chicken pox) within 30 days of scheduled study participation were excluded. Informed consent was obtained from all participants, and clinical research was conducted in accordance with guidelines for human experimentation as specified by the US Department of Health and Human Services. The study was approved by the National Institute of Allergy and Infectious Diseases Institutional Review Board.

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Study Design and Treatments

The study schema is displayed in Figure 1. Briefly, HIV-seronegative volunteers received a single oral dose of prednisone, 20 mg, before (baseline) and on days 4 and 14 of a 14.5-day course of ritonavir administration. On the morning of study day 1 (baseline), after an overnight fast, subjects arrived at the clinic and had an intravenous catheter inserted into a forearm vein, from which a baseline (0 hours) blood sample was obtained. Each subject then received a standard breakfast, along with a single 20-mg prednisone tablet (Roxanne Laboratories, Columbus, OH) and 240 mL of water. The breakfast consisted of a bagel with cream cheese, 4 oz of apple sauce, 4 oz of orange juice, and 8 oz of 2% milk. The total number of kilocalories in this meal was 285; the percentages of kilocalories from fat, carbohydrate, and protein were 23%, 66%, and 11%, respectively. Venous blood samples for the determination of prednisolone were obtained 0.5, 1, 2, 3, 4, 6, 8, 12, and 24 hours after the prednisone dose. Of note, prednisone is rapidly converted to prednisolone in vivo through a non-cytochrome P450-mediated pathway. Except for water, subjects refrained from eating and drinking for 4 hours after taking prednisone; participants also engaged in limited ambulatory activity during this period. After collection of the 12-hour sample, the venous catheter was removed, and the remaining blood sample was collected the next morning by venipuncture. All samples were collected into sodium-heparin (green top) tubes and centrifuged at 3200 rpm for 10 minutes, after which plasma was harvested and stored at −80°C until it was analyzed for prednisolone.

FIGURE 1

FIGURE 1

Next, after a 7- to 28-day washout period (to accommodate the schedules of individual participants), subjects began taking ritonavir (Norvir; Abbott Laboratories, North Chicago, IL), 200 mg (2 100-mg capsules), by mouth twice daily with food for 14.5 days. The ritonavir dose (200 mg twice daily) was chosen for this investigation because it provides the highest daily ritonavir dose among clinically relevant boosting regimens recommended by the Department of Health and Human Services (100-200 mg once or twice daily).14 On days 4 and 14 of ritonavir administration, subjects returned to the clinic; at that time, study procedures from day 1 were repeated, except that subjects took ritonavir, 200 mg, along with their prednisone dose. In addition, on days 4 and 14 of ritonavir dosing, a single blood sample was obtained before ritonavir administration (C12) to determine ritonavir plasma concentrations as a means of assessing medication adherence; ritonavir capsule counts were also conducted on these days for the same purpose. Plasma samples for ritonavir determination were processed and stored as described for prednisolone. On the morning of their last ritonavir dose (ritonavir day 15), subjects arrived at the clinic for their 24-hour prednisolone sample; blood was also collected at this time for safety laboratory tests, which included a hepatic panel, electrolytes, a complete blood cell count with differential, and total cholesterol and triglyceride levels.

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Prednisolone Analysis

Prednisolone concentrations in human plasma were determined using a high-performance liquid chromatography (HPLC) liquid-liquid extraction method developed and validated in our laboratory. The HPLC system consisted of a Waters 2795 Alliance HT separations module and a 2996-photodiode array detector set at λ = 250 nm (Waters Corporation, Milford, MA). The HPLC system and the assay parameters were controlled using the Empower (version 5.0) chromatography manager software. Prednisolone and methylprednisolone internal standards were isolated from human plasma via precipitation with perchloric acid (60%), followed by liquid-liquid extraction with 5.0 mL of ethyl acetate/hexane/isoamyl alcohol (80:19:1 vol/vol). Briefly, a plasma sample of 500 μL was mixed with 30 μL of methyl prednisolone internal standard solution (5.0 μg/mL) and 15 μL of concentrated perchloric acid (60%). The samples were vortexed for 30 seconds and centrifuged at 10,000 rpm for 10 minutes at room temperature. The supernate was subsequently transferred to a 16-mm × 150-mm Kimax test tube and mixed with 5.0 mL of ethyl acetate/hexane/isoamyl alcohol (80:19:1 vol/vol). After shaking samples for 30 to 35 seconds and centrifuging at 3200 rpm for 10 minutes, the organic layer was transferred to a clean 13-mm × 100-mm test tube and evaporated to dryness using a Zymark TurboVap for 45 minutes at 40°C. The samples were reconstituted with 100 μL of mobile phase and transferred into HPLC vials. Subsequently, 80.0 μL was injected onto an YMC-Pack Pro RS C18, 5-μm, 4.6-mm × 150-mm reverse-phase analytic column (Waters Corporation) and eluted isocratically at 1.0 mL/min. (30°C) for 25 minutes using a mobile phase consisting of (21:79 vol/vol) tetrahydrofuran and HPLC grade water.

Calibration curves were linear from 0.020 to 1.0 μg/mL (R2 > 0.998). Percent errors as a measure of accuracy were <15%, and the inter- and intra-assay coefficients of variation were 3.50% to 7.39% and 2.74% to 5.92%, respectively, at 4 different drug concentrations. The limit of quantitation was 0.020 μg/mL, and the limit of detection was 0.010 μg/mL. During the validation process, the short-term stability of the drug in plasma and repeated freezing and thawing of plasma were evaluated. The overall recovery of prednisolone and methylprednisolone using the modified liquid-liquid extraction method was >88%.

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Ritonavir Analysis

Ritonavir was determined using a modified version of a previously described HPLC method.15 Calibration curves consisted of 9 concentration points and were linear from 0.10 to 15.0 μg/mL (R2 > 0.999). Percent errors as a measure of accuracy were <15%, and the inter- and intra-assay coefficients of variation were 3.80% to 6.29% and 2.76% to 6.92%, respectively, at 4 different drug concentrations. The limit of quantitation was 0.100 μg/mL, and the limit of detection was 0.025 μg/mL.

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Pharmacokinetic Analysis

Prednisolone pharmacokinetic parameters were determined using noncompartmental methods with the WinNonlin Professional computer program (version 3.2; Pharsight Corporation, Mountain View, CA). Maximal serum concentrations (Cmax) and time to reach Cmax (Tmax) were determined by visual inspection of the concentration-time profiles. The elimination rate constant (λZ) was estimated as the absolute value of the slope of a linear regression of a natural logarithm of concentration versus time. Half-life (T½) was calculated as ln2/λZ. Values for AUC from 0 hours to the last quantifiable concentration (AUC0-last) were determined by the linear trapezoidal rule. AUC from time 0 to infinity (AUC0-∞) was determined by dividing the last measured concentration by the elimination rate constant (λZ). Apparent oral clearance (Cl/F) was estimated as dose divided by AUC0-∞.

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MDR-1 Genotyping

A secondary aim of this study was to characterize MDR-1 genotypes at positions 3435 and 2677 (which are in linkage disequilibrium with each other) in each of the study subjects. Venous blood samples were obtained from all subjects, and deoxyribonucleic acid (DNA) was isolated from peripheral leukocytes with the Qiamp system (Qiagen, Valencia, CA). MDR1 genotype was determined by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), as previously described.16 Subjects were genotypically classified into groups based on PCR-RFLP analysis: at position 2677, groups were composed of subjects homozygous for guanine (GG), homozygous for thymine (TT), or heterozygous for the mutant allele (GT/A). At position 3435, groups were composed of subjects homozygous for cytosine (CC), homozygous for thymine (TT), or heterozygous for the mutant allele (CT).

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Osteocalcin Serum Concentrations

Osteocalcin (bone Gla-protein [BGP]) serum concentrations were determined as a biochemical indicator of bone turnover. We measured serum osteocalcin concentrations in each subject before he or she began taking any study medications and at the end of the study, after having received 3 doses of prednisone, 20 mg, and a 14.5-day course of ritonavir, 200 mg, twice daily. Blood samples for osteocalcin were collected after a 12-hour fast and immediately sent to the National Institutes of Health (NIH) Department of Laboratory Medicine (DLM) on ice. Osteocalcin serum concentrations were determined by the Clinical Center DLM using a standard immunochemiluminometric assay.

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Statistical Analysis

Prednisolone pharmacokinetic parameter values (except for Tmax) were compared between baseline and days 4 and 14 of ritonavir administration using repeated measures analysis of variance (ANOVA) and Tukey post-hoc comparisons. The data are presented as geometric mean ratios (GMRs) with 90% confidence intervals (CIs) according to US Food and Drug Administration (FDA) guidelines.17 Tmax was analyzed using the Kruskal-Wallis test. Statistical significance was accepted as a P value <0.05. Ritonavir trough concentrations were determined solely to assess adherence and did not undergo statistical analysis. Statistical computations were performed using Statistica (2001; StatSoft, Tulsa, OK). Descriptive statistics were generated using Microsoft Excel 2002 (Microsoft Corporation, Redmond, WA).

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RESULTS

Pharmacokinetic Parameters

Twelve subjects were enrolled in the study, 2 of whom dropped out because of unrelated health problems before beginning ritonavir. Another subject failed to complete the final pharmacokinetic sampling period (day 14 of ritonavir) because of loss of venous access. To this end, 10 subjects completed the first 2 pharmacokinetic sampling periods and 9 subjects completed the third sampling period (day 14 of ritonavir dosing). Mean prednisolone concentration-time profiles are shown for all 3 sampling periods in Figure 2. Prednisolone pharmacokinetic parameter values before and on days 4 and 14 of ritonavir administration are shown in Table 1. Compared with baseline, on days 4 and 14 of ritonavir administration, the geometric mean prednisolone AUC0-∞ increased from 2261 to 3098 ng · h/mL (GMR = 1.37, 90 CI: 1.27 to 1.47; P = 0.0002) and from 2261 to 2906 ng · h/mL (GMR = 1.28, 90% CI: 1.19 to 1.37; P = 0.001), respectively. All subjects experienced an increase in prednisolone AUC0-∞ on day 4 of ritonavir administration (range: 7%-74%), and 8 of 9 subjects experienced an increase on day 14 of ritonavir administration compared with baseline (range: −6% to 49%). Prednisolone Cl/F was reduced from 8.84 L/h (baseline) to 6.45 L/h (GMR = 0.73, 90% CI: 0.68 to 0.78; P = 0.0002) on day 4 of ritonavir and from 8.84 to 6.88 L/h (GMR = 0.78, 90% CI: 0.64 to 0.92; P = 0.0002) on day 14 of ritonavir. The prednisolone half-life increased from 2.96 hours at baseline to 3.92 hours on day 4 of ritonavir administration (GMR = 1.33, 90% CI: 1.20 to 1.46; P = 0.003); there was no significant difference in the prednisolone half-life between baseline and ritonavir day 14. No significant differences in Cmax and Tmax were noted between the groups (P > 0.05 for all comparisons).

TABLE 1

TABLE 1

FIGURE 2

FIGURE 2

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MDR-1 Genotypes

At position 3435, 6 of the 10 participating study subjects carried the CT genotype, 3 carried the CC genotype, and 1 carried the TT genotype. In most subjects, the GG genotype at position 2677 was linked with the CC genotype at position 3435; this was also true of GT and CT at positions 2677 and 3435, respectively. Exceptions were 2 subjects who possessed the CT allele at position 3435 but expressed the GG allele at position 2677. In addition, there was 1 individual with the TT allele at position 3435 who expressed the GT allele at position 2677. Because of the small number of subjects included in the study, the data set lacked sufficient power to undergo inferential statistical testing. As such, descriptive statistics are used when presenting the results. The mean changes (±SD) in prednisolone AUC0-∞ after 4 and 14 days of ritonavir administration are shown for the different MDR1 genotypes in Table 2; the individual GMRs, depicting the changes in prednisolone AUC0-∞ after 4 and 14 days of ritonavir exposure, are also presented for each of the genotype groups. Similar increases in prednisolone exposure were noted for those genotypes that were in linkage disequilibrium (GG and CC, and GT and CT, respectively). The prednisolone AUC0-∞ increased by 47% and 45% on day 4 of ritonavir in CT and GT individuals, respectively; the differences on ritonavir day 14 were 37% and 40% in the respective CT and GT groups. Mean increases in prednisolone AUC0-∞ for CC and GG individuals were approximately 10% less compared with CT and GT individuals. Only 1 individual expressed the TT allele at position 3435.

TABLE 2

TABLE 2

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Adverse Effects

The study medications were generally well tolerated, and no subject discontinued participation because of an adverse effect. No adverse effects were attributed to single doses of prednisone or to ritonavir on prednisolone pharmacokinetic sampling days. During ritonavir administration, nausea, diarrhea, fatigue, and fullness and/or bloating were the most commonly reported side effects (2 episodes, each reported by different subjects). Single episodes of headache, anorexia, increased appetite, and increased skin sensitivity were also reported. All these adverse effects were considered grade 1 toxicities or less, and none required intervention. In accordance with previously published data, serum lipid elevations occurred in nearly all subjects after ritonavir exposure.18 Nonfasting total cholesterol levels increased from 181 to 198 mg/dL (9% mean increase vs. baseline, range: −8% to 27%) after ritonavir administration. In total, 8 of 10 subjects experienced increases in total cholesterol during the study. Nonfasting triglyceride levels increased by a mean of 56 mg/dL (84% mean increase vs. baseline, range: 6%-211%) after ritonavir administration. All 10 subjects experienced increases in triglyceride levels during the study. One subject, who remained asymptomatic, experienced a grade 2 elevation in aspartate aminotransferase (AST; serum glutamic-oxaloacetic transaminase [SGOT]), which returned to normal when repeated several weeks after the study ended; this same individual had grade 1 elevations in total bilirubin and serum creatinine and a grade 1 reduction in hemoglobin concentration, all of which resolved with repeat testing. No other laboratory abnormalities were observed.

There were no discernible differences in osteocalcin measurements before and after receiving the study medications. The mean serum osteocalcin concentrations before starting study medications versus afterward were 5.8 ± 2.5 ng/mL and 5.5 ± 3.0 ng/mL (P = 0.74). All osteocalcin measurements were within the normal range (1.1-7.2 ng/mL for male subjects and 0.5-7.0 ng/mL for premenopausal female subjects).

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Adherence

Capsule counts were conducted, and ritonavir trough concentrations were determined as separate means of assessing subject adherence. In addition, volunteers were asked to self-report any missed ritonavir doses (prednisone doses were directly observed in the clinic). No volunteers reported missing any ritonavir doses, and this was supported by ritonavir trough concentrations, which were consistent with previously reported values in subjects taking the same regimen (mean 14-day ritonavir trough in this study: 1.58 ± 1.32 μg/mL compared with 1.99 ± 1.98 μg/mL in a previously reported cohort).18

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DISCUSSION

Because of increased longevity associated with highly active antiretroviral therapy (HAART), the concomitant use of corticosteroids and HIV protease inhibitors is ever increasing among patients with HIV infection. In this population, corticosteroids are routinely prescribed for various indications, including renal and liver transplantation, asthma, inflammatory arthritis, and cancer.19-21 Not surprisingly, most patients taking corticosteroids opt to continue their antiretroviral regimen, thereby setting the stage for pharmacokinetic drug interactions.

In the current investigation, low-dose ritonavir (200 mg twice daily) was associated with statistically significant increases in prednisolone exposure (AUC0-∞) and decreases in Cl/F after 4 and 14 days of ritonavir administration. Cmax and Tmax were unaffected by concurrent ritonavir, suggesting that ritonavir did not appreciably alter prednisolone absorption but, instead, inhibited its metabolism through hepatic CYP3A4.

Data from this study (see Table 1) also suggest that the magnitude of the interaction between ritonavir and prednisolone was somewhat diminished by day 14. In particular, the half-life of prednisolone was 33% longer on day 4 compared with baseline, but this difference was reduced to 15% on day 14 (see Table 1). In addition, the increase in prednisolone AUC (and decrease in Cl/F) was less pronounced on day 14 versus baseline compared with day 4 versus baseline (see Table 1). One possible explanation for these results is that ritonavir exposure may diminish with repeated dosing because of its propensity to induce its own CYP-mediated metabolism (ie, autoinduction).22 As such, lower ritonavir concentrations may reduce the degree to which the drug is capable of inhibiting the CYP3A4-mediated metabolism of prednisolone. Presumably, this is the same mechanism that is responsible for the tempering of the interaction between saquinavir and ritonavir when the drugs are coadministered for an extended period.23 Alprazolam is another drug whose plasma concentrations level off over time after undergoing an initial (significant) increase with concurrent ritonavir.24

In patients with HIV infection, low-dose ritonavir (100-200 mg administered once or twice daily) is commonly used to boost plasma concentrations of coadministered protease inhibitors by inhibiting their metabolism through CYP3A425,26; however, because of its potency as a CYP3A4 inhibitor, ritonavir has been associated with many serious drug interactions in patients receiving concurrent therapy with CYP3A4 substrates, including ergot alkaloids, benzodiazepines, hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, tacrolimus, and carbamazepine.2 Whereas the clinical impact of the prednisolone-ritonavir interaction we report here has yet to be fully appreciated, prednisolone can now be added to the list of medications whose plasma concentrations are significantly increased by ritonavir.

Prednisolone, the active metabolite of prednisone, is formed in vivo through the 11β-hydroxydehydrogenase enzyme, which is not part of the CYP system.27 Once formed, however, prednisolone is metabolized by the CYP3A4-mediated 6β-hydroxylase enzyme. In addition to the ritonavir-prednisolone interaction reported here, a variety of other CYP3A4 inhibitors, including diltiazem, ketoconazole, and voriconazole, have been shown to inhibit prednisolone metabolism.10-13 In separate healthy volunteer studies in which these agents were coadministered with oral prednisone, increases in the prednisolone AUC ranged between 21% and 47%, which is similar to our findings.10-13 It is important to note that even small increases in prednisolone exposure, such as those reported by us, can result in adverse clinical effects. In an investigation of renal transplant patients, improvements in blood pressure, glucose intolerance, and lipid levels were seen when chronic low-dose prednisone therapy was decreased or discontinued.28 Conversely, one might expect a clinically relevant worsening of these toxicities in the face of modest increases in corticosteroid exposure, such as those expected to occur with ritonavir administration. Of note, HIV-infected patients on HAART are already predisposed to lipid elevations, hypertension, and glucose intolerance because of their antiretroviral medications and/or underlying HIV infection29-32; hence, further increases in corticosteroid concentrations as a result of drug-drug interactions may pose clinically significant risks in this population.

In addition to CYP3A4, prednisolone is a substrate and ritonavir is a moderate inhibitor of the MDR-1 gene product P-gp.21,33 Along with the CYP3A4 isoform, MDR1 genotypes may also influence the disposition of P-gp substrates and the extent to which a P-gp inhibitor, such as ritonavir, impairs P-gp function.21,34 Prednisolone AUC0-∞ values stratified by different genotypes are presented in Table 2. Because of the small number of subjects in the different genotype groups, these data did not undergo statistical analysis and are purely observational.

Corticosteroid use, with or without a concurrent CYP3A4 inhibitor, may contribute to bone toxicities in HIV-infected patients. Significant reductions in bone density were observed in HIV-seronegative transplant recipients receiving a prednisone-containing regimen in combination with the CYP3A4 inhibitor ketoconazole.35 In another study in HIV-infected patients, Miller et al6 identified systemic corticosteroid use as a potential risk factor in the development of osteonecrosis in asymptomatic HIV-infected adults (relative risk = 3.8, 95% CI: 1.3 to 11.0). It is noteworthy that 14 of the 15 patients who developed osteonecrosis received short courses of corticosteroids (several days to several weeks) and were on a protease inhibitor-containing HAART regimen. Additional studies and case reports have also identified corticosteroid use as a potential risk factor for osteonecrosis in patients with HIV infection.7,8 Finally, in addition to their pharmacokinetic interaction with corticosteroids, protease inhibitors, and possibly other antiretroviral medications, may independently contribute to the development of osteonecrosis, although this premise has yet to be established.36

Serum osteocalcin concentrations were measured as a marker of bone turnover before and after a 14-day course of ritonavir. Osteocalcin is a small protein that is synthesized by mature osteoblasts, odontoblasts, and hypertrophic chondrocytes. The turnover of circulating osteocalcin is rapid (T½ of 4-5 minutes), with the protein being promptly cleared by the liver, bone, and kidney.37,38 Osteocalcin is secreted into extracellular spaces by osteoblasts, where it binds to mineralized bone or enters the systemic circulation.39As such, the measurement of serum osteocalcin concentrations is accepted as a sensitive and specific marker of osteoblastic activity that can detect long-term imbalances in bone metabolism (eg, decreased bone formation and normal or increased bone resorption as reflected by low serum osteocalcin concentrations). Because of a growing number of reports suggesting that protease inhibitors and/or corticosteroids contribute to osteonecrosis of the hip in HIV-infected patients, we characterized the influence of a short-term course of ritonavir (14 days) on serum osteocalcin concentrations6-8; subjects also received 3 20-mg prednisone doses in the interim. On analysis of the data, there were no differences in serum osteocalcin concentrations before and after study completion. This is likely attributable to the short duration of protease inhibitor and corticosteroid administration in this study; it also ignores any influence of underlying HIV disease on bone turnover. From these data, it is not clear whether osteocalcin is an effective predictor of osteonecrosis in HIV-infected individuals.

In addition to osteonecrosis, there have been a series of reports of Cushing syndrome in patients with HIV infection receiving fluticasone (oral inhalation and nasal inhalation dosage forms) in combination with the HIV protease inhibitor ritonavir.3-5 Again, the proposed mechanism behind this interaction is inhibition of corticosteroid metabolism by ritonavir. Because of the intermittent dosing of prednisone in our study, we did not monitor plasma or urinary cortisol concentrations to try and elucidate changes in endocrine function.

A potential limitation of our study was our decision not to measure prednisone or unbound prednisolone serum concentrations. Because prednisone is rapidly (and nearly completely) converted to prednisolone through a non-CYP-mediated pathway, however, we could find no advantage to collecting these data. Similarly, we did not measure unbound prednisolone serum concentrations in this study even though ritonavir is 98% to 99% bound to albumin and α1-acid glycoprotein and may have elevated unbound concentrations of prednisolone, which is approximately 95% bound to albumin.40,41 Nonetheless, because of the general clinical irrelevance of protein binding displacement interactions, we decided to forego measuring free prednisolone concentrations. Finally, we did not measure lymphocyte subsets or markers as some drug interaction studies with corticosteroids have done,11 because this information did not pertain to our hypotheses.

The overall consistency in the magnitude and direction of prednisolone AUC changes when combined with ritonavir in this study provides compelling evidence for the validity of the interaction between the 2 medications. Moreover, it is likely that other protease inhibitors, which are also CYP3A4 inhibitors, may elevate prednisolone concentrations in a manner similar to low-dose ritonavir. Enhanced prednisolone exposure in patients with HIV infection may worsen troublesome antiretroviral side effects such as hyperlipidemia, hypertension, and glucose intolerance, which may contribute to serious long-term complications. The clinical effects of this drug-drug interaction deserve further study in HIV-infected patients; in the meantime, clinicians should exercise caution when coadministering corticosteroids and HIV protease inhibitors.

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Keywords:

protease inhibitor; drug interaction; cytochrome P450; corticosteroid

© 2005 Lippincott Williams & Wilkins, Inc.