Screen reader users, click here to load entire articleThis page uses JavaScript to progressively load the article content as a user scrolls. Screen reader users, click the load entire article button to bypass dynamically loaded article content. Volume 148, April 2015, Pages 283–28917th Vitamin D WorkshopEdited By JoEllen Welsh, Daniel Bikle and Paul Lips Modified-release oral calcifediol corrects vitamin D insufficiency with minimal CYP24A1 upregulationa b c Received 29 August 2014, Revised 19 November 2014, Accepted 21 November 2014, Available online 22 November 2014•Abrupt calcifediol dosing triggers vitamin D catabolism, limiting iPTH lowering.•Rapid calcifediol administration can induce excessive FGF23 and CYP24 and lower CYP27B1.•Bolus administration of immediate-release calcifediol in CKD patients minimally impacts elevated iPTH.•Modified-release (MR) calcifediol gradually raises 25(OH)D3 and calcitriol.•MR calcifediol effectively lowers iPTH without raising net vitamin catabolism.
Vitamin D insufficiency is prevalent in chronic kidney disease (CKD) and associated with secondary hyperparathyroidism (SHPT) and increased risk of bone and vascular disease. Unfortunately, supplementation of stage 3 or 4 CKD patients with currently recommended vitamin D2 or D3 regimens does not reliably restore serum total 25-hydroxyvitamin D to adequacy (≥30 ng/mL) or effectively control SHPT. Preclinical and clinical studies were conducted to evaluate whether the effectiveness of vitamin D repletion depends, at least in part, on the rate of repletion. A modified-release (MR) oral formulation of calcifediol (25-hydroxyvitamin D3) was developed which raised serum 25-hydroxyvitamin D3 and calcitriol levels gradually. Single doses of either bolus intravenous (IV) or oral MR calcifediol were administered to vitamin D deficient rats. Bolus IV calcifediol produced rapid increases in serum 25-hydroxyvitamin D3, calcitriol and FGF23, along with significant induction of CYP24A1 in both kidney and parathyroid gland.
In contrast, oral MR calcifediol produced gradual increases in serum 25-hydroxyvitamin D3 and calcitriol and achieved similar hormonal exposure, yet neither CYP24A1 nor FGF23 were induced. A 10-fold greater exposure to bolus IV than oral MR calcifediol was required to similarly lower intact parathyroid hormone (iPTH). Single doses of oral MR (450 or 900 μg) or bolus IV (450 μg) calcifediol were administered to patients with stage 3 or 4 CKD, SHPT and vitamin D insufficiency. Changes in serum 25-hydroxyvitamin D3 and calcitriol and in plasma iPTH were determined at multiple time-points over the following 42 days. IV calcifediol produced abrupt and pronounced increases in serum 25-hydroxyvitamin D3 and calcitriol, but little change in plasma iPTH. As in animals, these surges triggered increased vitamin D catabolism, as evidenced by elevated production of 24,25-dihydroxyvitamin D3. In contrast, MR calcifediol raised serum 25-hydroxyvitamin D3 and calcitriol gradually, and meaningfully lowered plasma iPTH levels.
Taken together, these studies indicate that rapid increases in 25-hydroxyvitamin D3 trigger CYP24A1 and FGF23 induction, limiting effective exposure to calcitriol and iPTH reduction in SHPT. They also support further investigation of gradual vitamin D repletion for improved clinical effectiveness.This article is part of a Special Issue entitled "17th Vitamin D Workshop".Keywords; ; ; ; ; ; ; 1. IntroductionVitamin D insufficiency is associated with chronic kidney disease (CKD) and gives rise to secondary hyperparathyroidism (SHPT) which can lead to loss of bone density and elevated rates of fracture in renal patients [1]. Vitamin D therapies are therefore widely used in the management of chronic kidney disease (CKD). Vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol) supplementation is the standard of care for correcting vitamin D insufficiency in CKD [2], while vitamin D hormones (calcitriol and other synthetic hormones) are used to control SHPT [3]. Both of these therapeutic approaches have significant limitations.
Vitamins D2 and D3 (collectively “vitamin D”) are absorbed less readily than more polar vitamin D compounds [4], and the degree of absorption can vary considerably between patients [5]. Once absorbed, vitamin D must undergo two sequential hydroxylations to be active: first at carbon 25 by CYP2R1 or CYP27A1 to form 25-hydroxyvitamin D, and then at carbon 1 by CYP27B1 to form 1,25-dihydroxyvitamin D [6]. Hepatic 25-hydroxylation varies widely in efficiency and, together with variable absorption, complicates the determination of optimal dose [7] ; [8]. Significant percentages of CKD patients receiving vitamin D supplements do not attain targeted levels of serum 25-hydroxyvitamin D [9] ; [10]. Recommended repletion [11] comprises intermittent high dose regimens which may trigger accelerated vitamin D catabolism [12]. A comprehensive review of the topic concluded that vitamin D supplementation is generally ineffective in clinical management of CKD patients [13].Vitamin D hormones induce the desired clinical responses in target tissues, such as increased intestinal calcium uptake and suppression of iPTH production, by directly activating the vitamin D receptor [14].
Production of 1,25-dihydroxyvitamin D by renal CYP27B1 is controlled by feedback inhibition, thereby protecting tissues from overexposure. However, vitamin D hormone therapy is not subject to feedback regulation and can readily cause oversuppression of iPTH, hypercalcemia and hyperphosphatemia, leading to adynamic bone disease and vascular calcification [15]. Hormones also accelerate vitamin D catabolism and raise target tissue resistance by inducing CYP24A1 [16] which can mitigate the desired therapeutic responses and exacerbate vitamin D insufficiency.The limitations of current vitamin D supplementation and hormone replacement therapies have led us to re-examine calcifediol (25-hydroxyvitamin D3) as a potentially effective intervention for restoring adequate serum levels of 25-hydroxyvitamin D and safely controlling SHPT. Calcifediol is more readily absorbed than vitamin D [17] ; [18] and requires only 1-hydroxylation for activation, which remains under physiological feedback regulation.
We investigated whether gradual delivery of calcifediol, using a modified-release (MR) formulation for oral administration, would minimize CYP24A1 upregulation, thereby improving its effectiveness. The nonclinical and clinical studies described herein compared MR and bolus intravenous (IV) calcifediol with regard to effects on serum levels of vitamin D metabolites, plasma iPTH, serum FGF23, and tissue expression of the catabolic enzyme CYP24A1.2. Materials, methods and results2.1. AnimalsAdult male Sprague Dawley rats (6–8 weeks of age) from Hilltop Lab Animals Inc., (Scottdale, PA, USA) were maintained on a vitamin D deficient diet for 8 weeks after which detectable serum 25-hydroxyvitamin D was negligible. Two groups of twenty-five rats were administered a single 0.4 mL IV injection of either calcifediol (4.5 μg) or vehicle (30:50:20, v/v/v propylene glycol:saline:ethanol). Two additional groups of 25 rats were administered by gavage hard shell gelatin capsules containing an MR formulation of calcifediol (4.5 μg) or the MR calcifediol formulation alone (comprising a wax matrix).
The MR formulation progressively released calcifediol over a 12-hour period during in vitro dissolution testing. Serum or plasma were collected post-dose at 0, 0.08, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 h.2.1.2. Plasma iPTHDetermined with the rat iPTH ELISA kit (Immutopics, San Clemente, CA, USA).2.1.3. Serum FGF23Measured using an FGF23 ELISA kit (Kainos Laboratories, Tokyo, Japan).2.1.4. CYP24A1, CYP27B1 and PTH mRNAKidney and parathyroid gland tissue samples were excised and frozen in RNAlater® and were processed using an automated hard tissue homogenizer. RNA was isolated using TRIzol® Reagent (Invitrogen). The ThermoScript™ RT-PCR System kit (Invitrogen) was used to create cDNA from 10 μg of RNA. The TaqMan® probes specific for rat Cyp24A1 (Cat. # Rn01423141_g1), Cyp27B1 (Rn00678309_g1), PTH (Rn00566882_m1) and GAPDH (Rn99999916_s1) were designed and manufactured by Applied Biosystems Inc., (Foster City, CA). Quantitative real-time PCR was performed using an ABI Prism 7000 sequence detection system (Applied Biosystems) using Taqman Universal PCR Master Mix (ABI #4304437).