VS-105: a novel vitamin D receptor modulator with cardiovascular protective effects

Abstract

Background and purpose: Vitamin D receptor (VDR) modulators (VDRMs) such as calcitriol, paricalcitol and doxercalciferol are commonly used to manage hyperparathyroidism secondary to chronic kidney disease (CKD). CKD patients experience extremely high risks of cardiovascular morbidity and mortality. Clinical observations show that VDRM therapy may be associated with cardio-renal protective and survival benefits for CKD patients. However, hypercalcaemia remains a serious side effect for current VDRMs, which leads to the need for frequent dose titration and serum Ca (calcium) monitoring. Significant clinical benefits can be derived from a VDRM with cardiovascular protective effects without the hypercalcaemic liability.

Experimental approach: Male Sprague-Dawley rats were 5/6 nephrectomized and 6 weeks later, after they had established uraemia, elevated parathyroid hormone levels, endothelial dysfunction and left ventricular hypertrophy, the rats were treated with VS-105, a novel VDRM. The effects of VS-105 were also tested in cultured HL-60 cells.

Key results: VS-105 induced HL-60 cell differentiation with an EC₅₀ value at 11.8 nM. Treatment (i.p., 3× a week over a period of 2 weeks) of the 5/6 nephrectomized rats by VS-105 (0.004-0.64 µg·kg⁻¹) effectively suppressed serum parathyroid hormone without raising serum Ca or phosphate levels. Furthermore, 2 weeks of treatment with VS-105 improved endothelium-dependent aortic relaxation and attenuated left ventricular abnormalities in a dose range that did not affect serum Ca levels. Similar results were obtained when VS-105 was administered i.p. or by oral gavage.

Conclusions and implications: VS-105 exhibits an overall therapeutic product profile that supports expanded use in CKD to realize the cardiovascular protective effects of VDR activation.

Figure 1

Structure and absorbance wavelength profile of VS-105. To determine the maximal absorbance wavelength (OD_max) and the extinction coefficients for the compound, VS-105 was dissolved in a 50:50 solution (by volume) of de-ionized water and ethanol at 100 µM and scanned by a spectrophotometer.

Figure 2

Effect of VS-105 on HL-60 differentiation. HL-60 cells were treated with different concentrations of VS-105, calcitriol or paricalcitol for 4 days. Cell differentiation was determined as described in Methods. Mean ± SD are shown. Results shown (with triplicate samples in this experiment) are representative of three independent experiments.

Figure 3

Effects of VS-105 on serum creatinine and BUN after 2 weeks of i.p. dosing in the 5/6 NX rats. Sham and 5/6 NX rats were treated with vehicle or VS-105 (i.p., 3× a week) for 12 days as described in Methods (n = 7–10 per group). On Day 0 (before dosing) and Day 13 (24 h after the last dosing), blood samples were collected for the measurement of serum creatinine (A) and BUN (B). Means ± SEM were calculated for each group. Unpaired t-test with 95% confidence intervals of difference was performed to assess differences between baseline Day 0 and Day 13. *P < 0.05, **P < 0.01 versus before treatment.

Figure 4

Effects of VS-105 on serum Ca, Pi and PTH levels after 2 weeks of i.p. dosing in the 5/6 NX rats. Rats were treated as in Figure 3. Blood samples were collected for the measurement of serum Ca (A), Pi (B) and PTH levels (C). Means ± SEM were calculated for each group. Unpaired t-test with 95% confidence intervals of difference was performed to assess differences between baseline Day 0 (before treatment) and Day 13 (after treatment). *P < 0.05, ***P < 0.001 versus before treatment.

Figure 5

Effects of VS-105 on serum Ca and PTH after 2 weeks of oral dosing in the uraemic rats. Sham and 5/6 NX rats were treated with vehicle or VS-105 (oral gavage, once daily) for 12 days as described in Methods (n = 8–11 per group). On Day 0 (before dosing) and Day 13 (24 h after the last dosing), blood samples were collected for the measurement of serum Ca (A) and PTH (B). Means ± SEM were calculated for each group. Unpaired t-test with 95% confidence intervals of difference was performed to assess differences between baseline Day 0 and Day 13. ***P < 0.0001 versus own control group at Week 6.

Figure 6

Endothelial dysfunction in 5/6 NX rats and the effect of VS-105. Sham and 5/6 NX rats were treated with vehicle or VS-105 at indicated doses as described in Figure 3. Vascular function was determined as described in Methods (n = 7–10 per group). (A) Acetylcholine-evoked relaxation. (B) SNP-evoked relaxation. Group mean ± SEM are presented. Statistical analysis was determined using a two-way anova, followed by a Bonferroni post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle.

Figure 7

Left ventricular hypertrophy in 5/6 NX rats and the effect of VS-105. Sham and 5/6 NX rats were treated with vehicle or VS-105 at the doses indicated, as described in Figure 3. (A) Heart was collected and weighed. Left ventricle (LV) was then dissected and weighed (n = 7–10 per group). Heart LV weight (LVW) was first normalized by body weight (BW) and then expressed as % of control (Sham). (B) The diameter of cardiomyocytes was determined as described in Methods. Data were obtained from 30 cells randomly selected from 10 microscopic fields across different rats in each treatment group and expressed as % of control (Sham). Group means ± SEM are presented. One-way anova Dunnett's test with 95% confidence intervals of difference was performed for statistical comparisons. ##P < 0.01, ###P < 0.001 versus Sham; **P < 0.01, ***P < 0.001 versus NX-vehicle.

Figure 8

Cardiomyocyte morphology in 5/6 NX rats and the effect of VS-105. Sham rats were treated with vehicle and 5/6 NX rats were treated with vehicle or VS-105 at the indicated doses as described in Figure 3. The left ventricular tissue sections were prepared and stained with haematoxylin-eosin as described in Methods. Randomly selected areas under 200 × magnification were examined. Pictures shown are representative of 10 fields per section per rat, four rats per treatment group. (A) Sham; (B) 5/6 NX-vehicle; (C) 5/6 NX treated with VS-105 at 0.01 µg·kg⁻¹; (D) 5/6 NX treated with VS-105 at 0.16 µg·kg⁻¹.

Figure 9

Left ventricle fibrosis in 5/6 NX rats and the effect of VS-105. The Sham rats were treated with vehicle and 5/6 NX rats were treated with vehicle or VS-105 at the indicated doses as described in Figure 5. The left ventricular tissue sections were prepared and stained with Masson trichrome as described in Methods. Randomly selected areas under 200 × magnification were examined. Pictures shown are representative of 10 fields per section per rat, four rats per treatment group. (A) Sham; (B) 5/6 NX-vehicle; (C) 5/6 NX treated with VS-105 at 0.01 µg·kg⁻¹; (D) 5/6 NX treated with VS-105 at 0.5 µg·kg⁻¹. (E) Quantitative determination of tissue collagen abundance; group means ± SEM are presented. One-way anova Dunnett's test with 95% confidence intervals of difference was performed for statistical comparisons. ###P < 0.001 versus Sham; ***P < 0.001 versus NX-vehicle.