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October 01, 2008 (Vol. 28, No. 17)

Tools for Enhancing Vitamin D Therapeutics

Diseases Like CKD, Cancers, and Psoriasis Can Benefit from the Blocking of CYP24 Activity

  • Click Image To Enlarge +
    Figure 1. Ketoconazole vs. vitamin D compound for the inhibition of CYP24.

    Emerging data from new research suggests that abnormally elevated CYP24 expression contributes to the pathology of certain diseases including chronic kidney disease (CKD), cancers, and psoriasis.

    CYP24, a member of the cytochrome P450 enzyme superfamily, is the key enzyme involved in vitamin D catabolism. Increased levels of CYP24 decrease the effectiveness of vitamin D replacement therapies and potentially contribute to local or systemic vitamin D insufficiency. Specific inhibition of CYP24 provides a new approach to treating diseases responsive to vitamin D therapy.

    Enzymes belonging to the cytochrome P450 superfamily catalyze critical biochemical reactions in the body including hydroxylations, epoxidations, oxygenations, and dehalogenations, which are needed to activate or inactivate endogenous-signaling ligands and detoxify xenobiotic compounds such as drugs. Those P450s exclusively involved in the metabolism of endogenous ligands such as fat-soluble vitamins (vitamins A and D), fatty acids, steroids, eicosanoids, and bile acids have attracted interest as potential drug targets because they play pivotal roles in modulating tissue responses to these substances and their synthetic counterparts.

    A number of P450 inhibitors have been developed that utilize functional azole groups to target the heme moiety at the enzyme’s catalytic core. Because heme resides at the core of all P450s, such inhibitors lack specificity and, consequently, can block activity of drug-metabolizing P450s, thus predisposing patients to possible drug-drug interactions (Figure 1). New vitamin D compounds targeting the CYP24 substrate binding pocket, rather than the catalytic site, allow much more potent inhibition with higher specificity.

  • Signaling

    Calcitriol (1α,25(OH)2D3), the active hormone of vitamin D3, is well known for its role in bone metabolism and calcium homeostasis.

    Calcitriol synthesis involves multiple steps, beginning with the formation of the precursor vitamin D3 in the skin following ultraviolet light exposure. Vitamin D3 undergoes two sequential metabolic steps to form calcitriol. The initial step involves 25-hydroxylation in the liver by CYP27A1 to form calcifediol (25(OH)D3), followed by a second step that involves 1α-hydroxylation in the kidney by CYP27B1 to form the active hormone. CYP24 is the enzyme responsible for catabolism of calcitriol via 24-hydroxylation to generate 1,24,25-trihydroxyvitamin D3. CYP24 also catabolizes calcifediol to yield 24,25-dihydroxyvitamin D3.

    In addition to its well-known effects on bone and mineral homeostasis, calcitriol has antiproliferative effects and promotes in vitro differentiation in a wide variety of cell lines. Calcitriol also induces CYP24 expression, thereby limiting its own access to VDR in target tissues because of the resulting increase in its catabolism. CYP24 inhibitors, therefore, have enormous promise in potentiating therapeutic vitamin D hormone effects.

  • Design of CYP24 Inhibitors

    Click Image To Enlarge +
    Figure 2. Compound docking in the substrate binding pocket of CYP24.

    The most effective way to specifically inhibit an enzyme like CYP24 is through its substrate-binding pocket, a unique functional domain. Vitamin D metabolizing enzymes such as CYP24 use specific chemical groups such as a hydroxyl group positioned on the vitamin D scaffold to appropriately position selected molecules in the substrate-access channel.

    Based on this knowledge, Cytochroma used calcitriol as the template and chemically modified either the side chain and/or the A ring to appropriately achieve highly specific and effective inhibition of CYP24 (Figure 2). The resulting compounds were screened using two different assays: an inhibition assay and a cell-based transcription activation assay.

    The first assay used the Chinese Hamster V79 cells stably transfected with CYP24. In this assay, calcitriol was incubated for 40 minutes in the presence of different concentrations of compound after which time the percentage of calcitriol catabolism was measured by HPLC. Ketoconazole, known to inhibit CYP24 (among other P450s), was used as a benchmark. Potent compounds were also tested for their ability to inhibit other P450 enzymes in order to determine their specificity.

    The second assay, a transcriptional activation assay, was used to determine whether the compounds had retained their ability to activate vitamin D signaling through the vitamin D receptor (VDR).

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