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HIV-protease inhibitors impair vitamin D bioactivation to 1,25-dihydroxyvitamin D

Cozzolino, Mario; Vidal, Marcos; Arcidiacono, Maria Vittoria; Tebas, Pablo; Yarasheski, Kevin E; Dusso, Adriana S

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Background: A high prevalence of bone demineralization occurs in people living with HIV/AIDS. The contribution of HIV itself and its treatment is still unclear. Protease inhibitors (PIs) are potent inhibitors of the cytochrome P450 enzyme system. Three cytochrome P450 mixed function oxygenases control serum levels of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), which is responsible for vitamin D3 actions in target tissues including bone. The 25- and 1α-hydroxylases regulate 1,25(OH)2D3 synthesis and 24-hydroxylase 1,25(OH)2D3 catabolism.

Objective: To assess whether HIV-protease inhibitors (ritonavir, indinavir, nelfinavir) impair the activity of the main enzymes involved in 1,25(OH)2D3 homeostasis.

Design and methods: Studies were conducted in the human hepatocyte (H3B)- and monocyte (THP-1) cell lines, expressing 25-hydroxylase and 1α-hydroxylase, respectively. The 24-hydroxylase expression was induced in macrophages by exposure to 1,25(OH)2D3. Conversion rates of vitamin D3 to 25-hydroxyvitamin D3 [25(OH)D3]; 25(OH)D3 to 1,25(OH)2D3 or 24,25(OH)2D3, and 1,25(OH)2D3 degradation were quantified in untreated and HIV-PI-treated cells after C18-cartridge extraction and high-performance liquid chromatography purification of 25(OH)D3- 24,25(OH)2D3- and 1,25(OH)2D3 fractions.

Results: The PIs impair hepatocyte 25(OH)D3- and macrophage 1,25(OH)2D3 synthesis in a reversible, dose-dependent manner. Furthermore, PIs inhibit 1,25(OH)2D3-degradation in macrophages with lower potency than that elicited on 1α-hydroxylase. Thus, reduced macrophage 1,25(OH)2D3 production is the net effect of PIs action.

Conclusions: In intact cells in vitro, HIV-PIs markedly suppress the activities of 25- and 1α-hydroxylase, which are critical in 1,25(OH)2D3 synthesis, while exerting mild inhibition of 24-hydroxylase, responsible for 1,25(OH)2D3 catabolism. If PIs elicit a similar potency in inhibiting these critical steps for 1,25(OH)2D3 homeostasis in vivo, defective 1,25(OH)2D3 production could contribute to the bone demineralization in HIV patients.

From the Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.

Note: *These authors have contributed equally to this work.

Correspondence to Adriana S. Dusso, PhD, Research Associate Professor, Renal Division Box 8126, 660 S. Euclid Ave, St. Louis, MO 63110, USA. Tel: +1 314 362 8243 (office), +1 314 362 8248 (lab); fax: +1 314 362 8237; e-mail: adusso@im.wustl.edu

Received: 3 April 2002; revised: 20 September 2002; accepted: 30 September 2002.

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Introduction

The combination of three different antiretroviral medications, nucleoside analog reverse transcriptase inhibitors, non-nucleoside analog reverse transcriptase inhibitors and HIV-protease inhibitors (PIs), has become the ‘standard of care’ for the treatment of HIV infection in the developed world. The efficacy of highly active antiretroviral therapy (HAART) in inhibiting HIV replication and improving morbidity and mortality of HIV infection is incontrovertible. This progress in therapy, however, is not without problems. All components of HAART regimens can have major acute and long-term toxicities, through mechanisms that are still not well understood [1–12]. The toxicity includes development of insulin resistance and diabetes, dyslipidemia, body fat redistribution (lipoatrophy and lipodystrophy), and lactic acidosis.

Recently, a high incidence of osteopenia and osteoporosis has been observed in HIV-infected individuals. This problem seems to be more frequent in patients receiving potent antiretroviral therapy, although the specific contribution (if any) of the drugs used in combination regimens has yet to be established [13].

The relative contribution of HIV infection itself to bone loss or the immune reconstitution associated with treatment is unclear. However, prior to the use of HAART, bone mineral metabolism in HIV-infected individuals was unaffected or only minimally altered [14]. Individuals that have never been treated with antiretroviral therapy have a slightly increased prevalence of osteopenia/osteoporosis when compared with the general population [15]. Although the availability of PI-based potent antiretroviral therapy (mainly protease inhibitor based) and osteopenia/osteoporosis appear to be coincident, the cause-and-effect has not been established. Furthermore, it is difficult to distinguish between side effects associated with protease inhibitors and those associated with nucleoside analogs in combination with PIs. Some have suggested that nucleoside analogs could play a significant role in the development of this complication [16].

Vitamin D is essential for the maintenance of a normal skeleton [17]. The actions of vitamin D on bone remodeling are exerted by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], also known as calcitriol, a potent calcitropic hormone [18]. Vitamin D bioactivation to calcitriol involves 25-hydroxylation of vitamin D in the liver by the enzyme 25-hydroxylase, followed by 1α-hydroxylation of 25-hydroxyvitamin D3 [25(OH)D3] by 1α-hydroxylase, an enzyme expressed in renal proximal tubular cells as well as in several other tissues. Calcitriol is a potent affector of calcium homeostasis. Serum calcitriol concentrations are tightly self-regulated: high concentrations of 1,25(OH)2D3 suppress synthesis through 1α-hydroxylation of 25(OH)D, and they induce the expression of 24-hydroxylase, the primary enzyme that catalyses calcitriol degradation in target tissues [19]. All vitamin D metabolizing enzymes are cytochrome P450 monoxygenases. As HIV-PIs are potent inhibitors of human hepatic cytochrome P450s in vivo [20,21], we examined whether HIV-PIs impair the activity of the enzymes that regulate calcitriol homeostasis, and might therefore contribute to bone demineralization in HIV/AIDS.

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Methods

Materials

The 25-hydroxy [26(27)-methyl 3H] cholecalciferol, specific activity 10–30 Ci/mmol and 1α, 25-dihydroxy [26(27)-methyl 3H] cholecalciferol, specific activity 168 Ci/mmol, were purchased from Amersham Co-orporation (Arlington Heights, Illinois, USA). [3α-3H]-cholecalciferol, specific activity 15 Ci/mmol, was kindly provided by Dr. Hector DeLuca (University of Wisconsin, Madison, Wisconsin, USA). Both 1,25(OH)2D3 and 25(OH)D3 were gifts from Dr. Milan Uskokovic (Hoffman-La Roche, Nutley, New Jersey, USA). Vitamin D3 (cholecalciferol) was purchased from Sigma-Aldrich (St. Louis, Missouri, USA). The protease inhibitors, ritonavir (Abbott Laboratories, Inc. Abbott Park, Illinois, USA), nelfinavir (Agouron Pharmaceuticals, Inc. La Jolla, California, USA) and indinavir (Merck & Co. Rahway, New Jersey, USA) were provided by their manufacturers. Efavirenz, a non-nucleoside analog reverse transcriptase inhibitor used as a control in the experiments was kindly provided by DuPont (Wilmington, Delaware, USA).

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Cell culture

The human monocytic cell line THP-1 (kindly provided by Dr. Beth Lee, Washington University, St Louis, Missouri, USA), was grown in suspension in RPMI 1640 containing 10% fetal bovine serum (FBS). The cells were induced to acquire a macrophage phenotype by exposure to 160 nM phorbol 12-myristate 13-acetate (Sigma-Aldrich) for 24 h in six-well plates at a concentration of 2 × 106 cells/well.

The human hepatocyte cell line Hep3B, obtained from the American Type Culture Collection, was cultured in Earle's minimal essential medium (MEM) containing 10% FBS.

Measurements of enzymatic activity in both cell lines were conducted in serum-free RPMI 1640 or Earle's MEM containing 1% fatty acid-free bovine serum albumin (BSA) to maintain lipophilic vitamin D3 metabolites in solution in the incubation media. Measurements of cell viability after prolonged exposure to PIs were conducted by assessing trypan blue exclusion after a 5 min incubation of cells with the dye.

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Measurement of 1α-hydroxylase activity

The rate of conversion of 25-hydroxycholecalciferol [25(OH)D3] to 1α,25-dihydroxyvitamin D3 was measured in differentiated THP-1 cells (macrophage phenotype) in the resting state and after macrophage activation by exposure to 100 IU/ml of recombinant human interferon-γ for 18h, as specified in each experimental protocol. The reactions were initiated by the addition of 25-hydroxy [26(27)-methyl3H]-cholecalciferol, as substrate for the cells in the absence or presence of the specified HIV-PI concentration. After a 2 h incubation at 37°C, reactions were stopped by addition of 1 ml of acetonitrile. 100 ng of radio-inert 1,25(OH)2D3 in 20 μl of ethanol were added to monitor recovery. The synthesized 3[H]-1,25(OH)2D3 was purified by C18-cartridge solid phase extraction followed by straight phase high-performance liquid chromatography (HPLC) isolation of the 1,25(OH)2D3 fraction using 5% isopropanol in methylene chloride and quantified as previously described [22,23].

To assess the reversibility of the effects of ritonavir on 1α-hydroxylase activity, the THP-1 cells were exposed to ritonavir at the minimal concentration causing maximal inhibition of enzymatic activity for either 1 or 2 h. Ritonavir was then removed from the incubation media and the cells were allowed to recover for 1, 6 or 18 h in 10% FBS RPMI 1640. The media was removed after the indicated recovery period, replaced by serum-free media containing 1% fatty acid-free BSA, and the 1α-hydroxylase activity was measured as described. To mimic more closely the slow pharmacokinetics of ritonavir in vivo, its inhibitory potency on 1α-hydroxylase was also tested in THP-1 cells exposed for 1, 6 or 18 h to circulating levels of the PI.

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Measurement of 24-hydroxylase activity

The 24-hydroxylase expression was induced in THP-1 cells by exposure to 10 nM 1,25(OH)2D3 for 18 h. At the end of the 24-hydroxylase induction, 1,25(OH)2D3 was removed from the incubation media, and the rate of 1,25(OH)2D3 degradation was quantified using tritiated-1,25(OH)2D3. Briefly, the amount of substrate [tritiated-1,25(OH)2D3] remaining after a 2 h incubation with untreated THP-1 cells or with cells treated with 7 μM HIV-PIs (ritonavir, nelfinavir and indinavir) or ketoconazole, was measured following the protocol described for 1α-hydroxylase activity.

Further examination of the potency of ritonavir in inhibiting 24-hydroxylase activity was conducted after a stronger induction of 24-hydroxylase expression in THP-1 cells by exposure to 25 nM 1,25(OH)2D3 for 18 h. The rate of conversion of 25-hydroxy [26(27)-methyl3H]-cholecalciferol to 24,25-dihydroxyvitamin D3, was measured in THP-1 cells following a protocol similar to that described for 1α-hydroxylase. Radio-inert 24,25(OH)2D3 was added (100 ng) to monitor recovery, and the HPLC mobile phase to purify the tritiated-24,25(OH)2D3 synthesized consisted of 4% isopropanol, 11% methylene chloride and 85% hexane, which provides optimal separation of 24,25(OH)2D3 from 25(OH)D3 and 1,25(OH)2D3. The effects of ritonavir on 24-hydroxylase activity were measured by simultaneous incubation of 1,25(OH)2D3-induced THP-1 cells with tritiated substrate [25(OH)D3] and the concentrations of the PI indicated in dose–response studies.

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Measurement of 25-hydroxylase activity

The rate of conversion of [3α-3H]-vitamin D3 to 25(OH)D3 in 2 h was measured in the human hepatocyte cell line H3B [24]. This protocol was similar to that described for measuring 1α- and 24-hydroxylase activities. One-hundred nanograms of radio-inert 25(OH)D3 was added to monitor recovery, and the HPLC mobile phase for isolating tritiated 25(OH)D3 from vitamin D3 consisted of 1% isopropanol in methylene chloride. The effect of ritonavir on 25-hydroxylase activity was measured after a 2 h incubation of hepatocytes with tritiated substrate [vitamin D3] and the concentrations of PI indicated in dose–response studies.

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Statistical analysis

Triplicate determinations of enzymatic activity were performed for every experimental condition, in at least two independent experiments. Unpaired t-test or analysis of variance was used to identify statistically significant differences (P < 0.05) between enzyme activities measured in the presence or absence of protease inhibitors.

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Results

In vitro effects of protease inhibitors on 1,25(OH)2D3 synthesis

The effects of three different protease inhibitors, indinavir, ritonavir and nelfinavir on the conversion of 25(OH)D3 to 1,25(OH)2D3, the most critical step in vitamin D bioactivation, were studied in the human monocytic cell line THP-1. The human monocyte-macrophage 1α-hydroxylase is identical to renal 1α-hydroxylase [25] and its in vitro kinetic parameters (Km, Vmax) mimic closely those of the renal enzyme [26]. The THP-1 cells were induced to acquire a macrophage phenotype by exposure to phorbol esters. Figure 1a shows that concentrations of HIV-PIs in the incubation media, equivalent to the maximal levels in plasma after drug administration (15 μM ritonavir, 10 μM indinavir or 9 μM nelfinavir) caused an inhibition of the basal conversion of tritiated 25(OH)D3 to 1,25(OH)2D3 of 79.4, 63.4 or 31.7%, respectively. The inhibitory effects of ritonavir, indinavir and nelfinavir on macrophage 1,25(OH)2D3 production were enhanced to 98.6, 69.4 and 45.3% respectively, when THP-1 cells were induced to over-express 1α-hydroxylase activity by exposure to 100 IU/ml of interferon-γ, the most potent macrophage activating cytokine (Fig. 1b). In resting and interferon-γ-activated macrophages, incubation with the non-nucleoside reverse transcriptase inhibitor efavirenz (10 μM) resulted in no change in calcitriol synthesis compared with control cells.

Fig. 1.

Fig. 1.

Ritonavir (15 μM) was the most potent inhibitor of macrophage 1α-hydroxylase activity, so studies were conducted in THP-1 cells to assess dose–response, duration and reversibility of the inhibitory effects of ritonavir on 1α-hydroxylase activity. Dose–response studies (Fig. 2a) demonstrated that 0.15 μM ritonavir inhibited 33.6% basal 1,25(OH)2D3 production by 33.6% during a 2 h incubation. This ritonavir concentration (0.15 μM) is 100 times lower than the peak circulating ritonavir concentration achieved after oral dosing. Almost complete inhibition of 1α-hydroxylase activity was achieved at a ritonavir concentration > 6.8 μM.

Fig. 2.

Fig. 2.

Figure 2b shows that exposure of THP-1 cells to 15 μM ritonavir for either 1, 6 or 18 h resulted in similar inhibition of 1α-hydroxylase activity, measured in ritonavir-free media. Therefore, 15 μM ritonavir elicits maximal inhibition of 1α-hydroxylase activity in THP-1 cells after 1 h exposure. The THP-1 cell viability was only mildly compromised after prolonged exposure to ritonavir as demonstrated by the low number of cells staining positive for trypan blue [C, 3 ± 0.9‰; 1 h, 4.2 ± 0.5; 6 h, 9.5 ± 1.2; 18 h, 19.5 ± 5.5].

Reversibility experiments were conducted using either 1 or 2 h exposure of THP-1 cells to the minimum concentration of ritonavir (6.8 μM) that caused maximal inhibition of 1α-hydroxylase activity, according to the results of the above studies. The THP-1 cells exposed to 6.8 μM ritonavir for 1 h recovered basal 1α-hydroxylase activity after 2 h incubation in ritonavir-free media. When the THP-1 cells were exposed to 6.8 μM ritonavir for 2 h, 1α-hydroxylase activity was still 51.3% below basal activity after 6 h in ritonavir-free media [controls, 1634 ± 162 d.p.m. of 1,25(OH)2D3/106 cells (n = 3); ritonavir: 868 ± 240 (n = 3); P < 0.05]. Basal 1α-hydroxylase activity was restored after 18 h in ritonavir-free media. Taken together these results demonstrate that a short exposure to circulating levels of ritonavir result in almost complete inhibition of macrophage 1α-hydroxylase activity. Also, although reversible, the longer the exposure to the PI the slower the recovery of basal enzymatic activity after HIV-PI treatment.

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In vitro effects of HIV-PIs on 1,25(OH)2D3 catabolism

Serum levels of 1,25(OH)2D3 are a function of both its synthesis and degradation rates, and 1,25(OH)2D3 can regulate its rate of degradation. We examined the effects of PIs on 24-hydroxylase activity, the enzyme responsible for calcitriol-metabolic inactivation [22]. Table 1 shows the effects of PIs on the rate of 1,25(OH)2D3 degradation by THP-1 cells (induced to express 24-hydroxylase activity through exposure to 10 nM 1,25(OH)2D3 for 18 h). Whereas untreated THP-1 cells degraded 32% of the tritiated 1,25(OH)2D3 within 2 h, simultaneous exposure of THP-1 cells to tritiated 1,25(OH)2D3 and identical concentrations (7 μM) of ketoconazole, ritonavir, nelfinavir or indinavir completely inhibited the ability of the induced 24-hydroxylase to degrade 1,25(OH)2D3. Importantly, all HIV-PIs elicited similar inhibitory effects on 24-hydroxylase. Taken together these results indicate that, in human macrophages, the magnitude of the inhibitory effects of PIs on cytochrome P450 activities is enzyme specific.

Table 1

Table 1

The higher potency of ritonavir in inhibiting 1,25(OH)2D3 synthesis led us to conduct dose–response studies to further assess the net effects of ritonavir on 1,25(OH)2D3 homeostasis. To measure ritonavir potency in inhibiting 24-hydroxylase activity, two modifications were included in the protocols: (1) to further enhance 24-hydroxylase expression, the dose of 1,25(OH)2D3 was increased to 25 nM; (2) the rate of conversion of 25(OH)D3 to 24,25(OH)2D3 was measured instead of 1,25(OH)2D3 degradation to avoid a potential decrease in the specific activity of the substrate [tritiated 1,25(OH)2D3] with the higher concentration of non-radioactive 1,25(OH)2D3 used to induce 24-hydroxylase.

Figure 3a shows that 24-hydroxylase activity induced by 1,25(OH)2D3 treatment was inhibited by ritonavir in a dose-dependent manner. Importantly, 2.7 μM ritonavir caused almost complete inhibition (81.5%) of 1α-hydroxylase activity, but had no effect on 24-hydroxylase activity in the same cell type (compare dose–response curves in Figs 2a and 3a). Clearly, the inhibitory effects of ritonavir in macrophages appear to be enzyme and concentration specific. On this basis, we examined the effects of ritonavir on 25-hydroxylase activity in a human hepatocyte model because liver 25-hydroxylase catalyzes the first mandatory step in vitamin D3 bioactivation to calcitriol.

Fig. 3.

Fig. 3.

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In vitro effects of ritonavir on the 25-hydroxylase of human hepatocytes

Liver 25-hydroxylase (CYP25) is responsible for 25-hydroxylation of vitamin D3 to form 25(OH)D3; which is the major vitamin D3 metabolite in the circulation and the substrate for the most critical step in 1,25-dihydroxyvitamin D3 synthesis [18]. Kinetic analysis of 25-hydroxylase activity in the human hepatocyte cell line H3B demonstrated the ability of these cells to convert vitamin D3 to 25-hydroxyvitamin D3in vitro. The synthesis of 25-hydroxyvitamin D3 by 2 × 106 cells was within the linear range for up to 6 h (not shown). The 25-hydroxyvitamin D3 production was measured in hepatocytes exposed to 0, 2.7, 6.8 or 15 μM ritonavir for 2 h. Figure 3b shows the dose–response for ritonavir inhibition of hepatocyte 25-hydroxylase activity. Similar to ritonavir inhibition of 1,25(OH)2D3-induced 24-hydroxylase activity, 2.7 μM ritonavir had no effect on hepatocyte 25-hydroxylase activity. A ritonavir concentration of 6.8 μM caused 60% inhibition of basal 25-hydroxylase activity.

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Discussion

These studies were designed to gain insight into potential mechanisms for the high prevalence of bone demineralization in people living with HIV/AIDS and treated with PIs containing HAART.

Based on the known potency of protease inhibitors to inhibit the activity of human hepatic cytochrome P450 enzymes, and the knowledge that three enzymes critical to vitamin D3 bioactivation are cytochrome P450s, we examined the actions of the HIV-protease inhibitors indinavir, ritonavir, and nelfinavir on the activity of these enzymes in human cell lines. The fact that monocyte-macrophage 1α-hydroxylase is identical to renal 1α-hydroxylase [25] allowed us to use the more accessible and well-characterized monocyte-macrophage model [26] to address the hypothesis that HIV-PIs impair 1,25(OH)2D3 production by inhibiting 1α-hydroxylase activity.

In vitro, HIV-PIs inhibited 1α-hydroxylation of 25(OH)D3 to 1,25(OH)2D3, the most critical step in vitamin D3 bioactivation. In the present studies, HIV-PIs almost completely abolished 1,25(OH)2D3 production by resting macrophages and in macrophages induced to enhance 1α-hydroxylase activity through activation by interferon-γ. The potency of HIV-PIs to inhibit macrophage 1α-hydroxylase activity was comparable with that of ketoconazole, an antifungal agent, known to inhibit cytochrome P450-mediated steroidogenesis in several organs including testis, ovary, adrenal gland, kidney and liver [27]. Ketoconazole administration to normal individuals effectively reduces serum calcitriol [28]. More importantly, ketoconazole has been used therapeutically to diminish the excessive calcitriol synthesis in patients with hypercalcemic disorders [29,30].

Each HIV-PI exhibited a different potency for inhibiting 1α-hydroxylase activity. However, each HIV-PI elicited a similar potency for inhibiting 1,25(OH)2D3-24-hydroxylase activity, another cytochrome P450 critical in 25(OH)D- and 1,25(OH)2D3 inactivation.

The in vitro potency of HIV-PIs to alter calcitriol homeostasis in macrophages, however, may not accurately predict pathogenesis in vivo. There are limitations to the incubation conditions used in the present studies because they may not recapitulate HIV-PIs-transport or pharmacokinetics in vivo. Due to the higher potency of ritonavir to inhibit 1α-hydroxylase activity, protocols were conducted with this HIV-PI to mimic its pharmacokinetics in vivo. Protease inhibitors circulate bound to plasma proteins, predominantly albumin and α1-acid-glycoprotein. The level of PI-binding to these carriers ranges from ∼ 60% for indinavir to as high as ∼ 99% for ritonavir [31,32]. It is believed that the unbound PI is the active molecule and that very little bound PI enters the cells [32]. The current results however, challenge the importance of free/bound HIV-PI in cellular responses. Ritonavir, the PI with the highest affinity for carrier proteins, which consequently had the lowest concentration of the free form in the incubation media (containing one-third of the albumin concentration in normal plasma), was the most potent inhibitor of macrophage 1α-hydroxylase activity. Even at an in vitro concentration that was 100 times below maximum circulating levels (0.15 μM) and associated with negligible amounts of free ritonavir, macrophage calcitriol production was reduced. HIV-PI-pharmacokinetics should also be considered before extrapolating our in vitro results to the in vivo situation. In the case of ritonavir, pharmacokinetic studies demonstrated potent inhibitory effects on its own inactivating enzymes in vivo, which prevent its metabolic inactivation [31]. Importantly, in human macrophages in vitro despite identical free/bound levels, cellular uptake and intracellular kinetics, ritonavir did not inhibit with the same potency 1α- and 24-hydroxylases, two cytochrome P450 enzymes involved in vitamin D3 metabolism. Clearly, in the same cell, ritonavir effects are enzyme and concentration specific. The lowest ritonavir concentration that inhibited 1α-hydroxylase activity had no effect on 24-hydroxylase activity. In vivo, inhibition of 24-hydroxylase, the main enzyme responsible for degradation of vitamin D3 metabolites, increases serum 1,25(OH)2D3 levels and augments the response of target tissues to 1,25(OH)2D3 [18]. On the basis of the differential potency of ritonavir for inducing inhibition of enzymes for calcitriol synthesis and catabolism in human macrophages, we predict that ritonavir administration will reduce 1,25(OH)2D3 production. In addition to reducing calcitriol synthesis and degradation, ritonavir markedly impaired 25-hydroxylase activity, the first mandatory hydroxylation in vitamin D bioactivation, in a human hepatocyte cell line. The simultaneous inhibition by ritonavir of the two main enzymes responsible for vitamin D3 activation in vitro suggests that administration of this PI could antagonize normal calcitriol homeostasis. However, in our prior studies of HIV-infected people treated with PI-based HAART who have low bone mineral density, serum 25(OH)D3 and 1,25(OH)2D3 levels do not appear to be dramatically reduced [33]. Tissue levels of 1,25(OH)2D3 have not been evaluated and might well be lower than normal, reflecting the cell specificity for this inhibition. A specific reduction in bone calcitriol production would be predicted to reduce bone remodeling and bone mineral density. Since calcitriol also modulates aromatase expression and activity [34,35], local or systemic calcitriol deficiency might also indirectly contribute to bone demineralization.

Low calcitriol levels in the intestine or the liver, the main sites for drug inactivation, could also prolong the half-life of HIV-PIs in the circulation, and potentially enhance their efficacy on vitamin D metabolizing enzymes. In fact, calcitriol induces the expression of the CYP3A4 isoenzyme of the cytochrome P450 enzyme system, mediating HIV-PI pharmacokinetics, in the adenocarcinoma cell line CACO-2 and in primary human hepatocytes [36,37].

In summary, whereas ritonavir, and to a lesser extent indinavir and nelfinavir, inhibited 1,25(OH)2D3 production in a monocyte-macrophage cell line, all HIV-PI were equally effective at inhibiting 1,25(OH)2D3 degradation. Ritonavir has different potencies for inhibiting 25-, 24- and 1α- hydroxylase activities in intact human hepatocytes and macrophages. If these in vitro effects reflect what occurs in vivo, HIV-PI-induced inhibition of 1,25(OH)2D3 production could contribute to bone demineralization induced by PI-based therapy. Importantly, similar inhibitory effects of HIV-PIs on other cytochrome P450s critical for the synthesis of bone remodeling hormones such as estrogen-synthesizing aromatase, could further contribute to bone loss in HIV patients treated with PI-HAART therapy.

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Acknowledgements

Dr. Tebas is a recipient of the SmithKline Beecham Development Partners Junior Faculty. Maria Vittoria Arcidiacono is the holder of a scholarship from the Universita degli Studi. Milan. Italy

Sponsorship: This study was supported by P30 DK56341-02 to P.T. and A.S.D., and NIH NIDDK-49393, -54163, -59531, to K.E.Y. This study was supported in part by AI5903 and AI01612.

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References

1.Eastone JA, Decker CF. New-onset diabetes mellitus associated with use of protease inhibitor.Ann Intern Med 1997, 127:948.
2.Visnegarwala F, Krause KL, Musher DM. Severe diabetes associated with protease inhibitor therapy [letter].Ann Intern Med 1997, 127:947.
3.Lo JC, Mulligan K, Tai VW, Algren H, Schambelan M. `Buffalo hump’ in men with HIV-1 infection.Lancet 1998, 351:867–870.
4.Deeks SG, Smith M, Holodniy M, Kahn JO. HIV-1 Protease inhibitors. A review for clinicians.JAMA 1997, 277:145–153.
5.Brinkman K, ter Hofstede HJ, Burger DM, Smeitink JA, Koopmans PP. Adverse effects of reverse transcriptase inhibitors: mitochondrial toxicity as common pathway.AIDS 1998, 12:1735–1744.
6.Carr A, Samaras K, Chisholm DJ, Cooper DA. Abnormal fat distribution and use of protease inhibitors.Lancet 1998, 351:1736.
7.Carr A, Samaras K, Burton S, Law M, Freund J, Chisholm DJ, et al. A syndrome of peripheral lipodystrophy, hyperlipidaemia and insulin resistance in patients receiving HIV protease inhibitors.AIDS 1998, 12:F51–58.
8.Carr A, Samaras K, Thorisdottir A, Kaufmann GR, Chisholm DJ, Cooper DA. Diagnosis, prediction, and natural course of HIV-1 protease-inhibitor-associated lipodystrophy, hyperlipidaemia, and diabetes mellitus: a cohort study.Lancet 1999, 353: 2093–2099.
9.Carr A, Miller J, Law M, Cooper DA. A syndrome of lipoatrophy, lactic acidaemia and liver dysfunction associated with HIV nucleoside analogue therapy: contribution to protease inhibitor-related lipodystrophy syndrome.AIDS 2000, 14:F25–F32.
10.Behrens G, Dejam A, Schmidt H, Balks HJ, Brabant G, Korner T, et al. Impaired glucose tolerance, beta cell function and lipid metabolism in HIV patients under treatment with protease inhibitors.AIDS 1999, 13:F63–70.
11.Henry K, Melroe H, Huebsch J, Balks HJ, Brabant G, Korner T, et al. Severe premature coronary artery disease with protease inhibitors.Lancet 1998, 351:1328.
12.Yarasheski KE, Tebas P, Sigmund C, Dagogo-Jack S, Bohrer A, Turk J, et al. Insulin resistance in HIV protease inhibitor-associated diabetes.J Acquir Immune Defic Syndr 1999, 21:209–216.
13.Nolan D, Upton R, McKinnon E, John M, James I, Adler B, et al. Stable or increasing bone mineral density in HIV-infected patients treated with nelfinavir or indinavir.AIDS 2001, 15: 1275–1280.
14.Paton NI, Macallan DC, Griffin GE, Pazianas M. Bone mineral density in patients with human immunodeficiency virus infection.Calcif Tissue Int 1997, 61:30–32.
14.McGowan I, Cheng A, Coleman S, Johnson A, Genant H. Assessment of bone mineral density (BMD) in HIV-infected antiretroviral-therapy-naïve patients.Eighth Conference on Retroviruses and Opportunistic Infections. Chicago, February 2001 [abstract 628].
16.Carr A, Miller J, Eisman JA, Cooper DA. Osteopenia in HIV-infected men: association with asymptomatic lactic acidemia and lower weight pre-antiretroviral therapy.AIDS 2001, 15:703–709.
17.Holick MF. Vitamin D and bone health.J Nutr 1996, 126:1159S–1164S.
18.Brown AJ, Dusso A, Slatopolsky E. Vitamin D.Am J Physiol 1999, 277:F157–175.
19.Whitfield GK, Hsieh JC, Jurutka PW, Selznick SH, Haussler CA, MacDonald PN, et al. Genomic actions of 1,25-dihydroxyvitamin D3.J Nutr 1995, 125:1690S–1694S.
20.von Moltke LL, Greenblatt DJ, Grassi JM, Granda BW, Duan SX, Fogelman SM, et al. Protease inhibitors as inhibitors of human cytochromes P450: high risk associated with ritonavir.J Clin Pharmacol 1998, 38:106–111.
21.Eagling VA, Back DJ, Barry MG. Differential inhibition of cytochrome P450 isoforms by the protease inhibitors, ritonavir, saquinavir and indinavir.Br J Clin Pharmacol 1997, 44:190–194.
22.Dusso AS, Kamimura S, Gallieni M, Zhong M, Negrea L, Shapiro S, et al. gamma-Interferon-induced resistance to 1,25-(OH)2 D3 in human monocytes and macrophages: a mechanism for the hypercalcemia of various granulomatoses.J Clin Endocrinol Metab 1997, 82:2222–2232.
23.Dusso AS, Finch J, Brown A, Ritter C, Delmez J, Schreiner G, et al. Extrarenal production of calcitriol in normal and uremic humans.J Clin Endocrinol Metab 1991, 72:157–164.
24.Strugnell S, Calverley MJ, Jones G. Metabolism of a cyclopropane-ring-containing analog of 1 alpha-hydroxyvitamin D3 in a hepatocyte cell model. Identification of 24-oxidized metabolites.Biochem Pharmacol 1990, 40:333–341.
25.Monkawa T, Yoshida T, Hayashi M, Saruta T. Identification of 25-hydroxyvitamin D3 1alpha-hydroxylase gene expression in macrophages.Kidney Int 2000, 58:559–568.
26.Dusso A, Brown A, Slatopolsky E. Extrarenal production of calcitriol.Semin Nephrol 1994, 14:144–155.
27.Miossec P, Archambeaud-Mouveroux F, Teissier MP. [Inhibition of steroidogenesis by ketoconazole. Therapeutic uses].Ann Endocrinol (Paris) 1997, 58:494–502.
28.Glass AR, Eil C. Ketoconazole-induced reduction in serum 1,25-dihydroxyvitamin D.J Clin Endocrinol Metab 1986, 63: 766–769.
29.Glass AR, Eil C. Ketoconazole-induced reduction in serum 1,25-dihydroxyvitamin D and total serum calcium in hypercalcemic patients.J Clin Endocrinol Metab 1988, 66:934–938.
30.Glass AR. Ketoconazole-induced stimulation of gonadotropin output in men: basis for a potential test of gonadotropin reserve.J Clin Endocrinol Metab 1986, 63:1121–1125.
31.Koudriakova T, Iatsimirskaia E, Utkin I, Gangl E, Vouros P, Storozhuk E, et al. Metabolism of the human immunodeficiency virus protease inhibitors indinavir and ritonavir by human intestinal microsomes and expressed cytochrome P4503A4/3A5: mechanism-based inactivation of cytochrome P4503A by ritonavir.Drug Metab Dispos 1998, 26:552–561.
32.Koeplinger KA, Raub TJ, Padbury GE, Zhao Z. Equilibrium distribution of HIV antiviral drugs into human peripheral blood mononuclear cells (PBMC) is controlled by free drug concentration in the extracellular medium.J Pharm Biomed Anal 1999, 19:399–411.
32.Tebas PYK, Whyte M, Claxton S, DeMarco D, Powderly W. Serum an d urine markers of bone mineral metabolism in HIV infected patients taking protease inhibitor containing potent antiretroviral therapy.Antiviral Therapy Second International Workshop on Adverse Drug Reactions and Lipodystrophy in HIV. Toronto, September 2000.
34.Tanaka S, Haji M, Takayanagi R, Sugioka Y, Nawata H. 1,25-Dihydroxyvitamin D3 enhances the enzymatic activity and expression of the messenger ribonucleic acid for aromatase cytochrome P450 synergistically with dexamethasone depending on the vitamin D receptor level in cultured human osteoblasts.Endocrinology 1996, 137:1860–1869.
35.Kinuta K, Tanaka H, Moriwake T, Aya K, Kato S, Seino Y. Vitamin D is an important factor in estrogen biosynthesis of both female and male gonads.Endocrinology 2000, 141:1317–1324.
36.Schmiedlin-Ren P, Thummel KE, Fisher JM, Paine MF, Watkins PB. Induction of CYP3A4 by 1 alpha,25-dihydroxyvitamin D3 is human cell line-specific and is unlikely to involve pregnane X receptor.Drug Metab Dispos 2001, 29:1446–1453.
37.Schmiedlin-Ren P, Thummel KE, Fisher JM, Paine MF, Lown KS, Watkins PB. Expression of enzymatically active CYP3A4 by Caco-2 cells grown on extracellular matrix-coated permeable supports in the presence of 1alpha,25-dihydroxyvitamin D3.Mol Pharmacol 1997, 51:741–754.
Keywords:

protease inhibitors; vitamin D; bone mineral metabolism; cytochrome P450

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