1,25-Dihydroxyvitamin D3 but not cinacalcet HCl (Sensipar®/Mimpara®) treatment mediates aortic calcification in a rat model of secondary hyperparathyroidism

Charles Henley1, Matt Colloton1, Russell C. Cattley2, Edward Shatzen1, Dwight A. Towler3, David Lacey1 and David Martin1

1 Department of Metabolic Disorders and 2 Department of Pathology, Amgen Inc, Thousand Oaks, CA 91320, USA and 3 Department of Internal Medicine, Washington University School of Medicine, St Louis, MO 63110, USA

Correspondence and offprint requests to: Dr Charles Henley, Department of Metabolic Disorders, Amgen, Inc., One Amgen Center Drive, MS 15-2-A, Thousand Oaks, CA 91320, USA. Email: chenley{at}amgen.com



   Abstract
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 Abstract
 Introduction
 Subjects and methods
 Statistical analyses
 Results
 Discussion
 References
 
Background. Calcitriol treatment of secondary hyperparathyroidism (HPT) in chronic kidney disease (CKD) patients can lead to increased serum calcium and phosphorus, which have been associated as risk factors for vascular calcification. Cinacalcet HCl (Sensipar®/Mimpara®) {({alpha}R)-(–)-{alpha}-methyl-N-[3-[3-(trifluoromethylphenyl)propyl]-1-napthalenemethanamine hydrochloride} lowers serum parathyroid hormone (PTH), calcium, phosphorus and calcium–phosphorous (CaxP) product in stage 5 CKD dialysis patients; however, its effects on vascular calcification are unknown.

Methods. Cinacalcet HCl (10 or 1 mg/kg, p.o. gavage), 1,25-dihydroxyvitamin D3 (0.1 µg, s.c, calcitriol) or the combination was administered daily for 26 days in a rat model of secondary HPT [5/6 nephrectomy]. After dosing, aortic calcification was determined using the von Kossa staining method. Serum PTH and blood chemistries were determined on days 0, 26 and 0, 14, 26, respectively, prior to and after dosing.

Results. Calcitriol-treated rats had moderate to marked aortic calcification, whereas no significant calcification was observed in vehicle- or cinacalcet HCl-only treated groups. Co-administration of cinacalcet HCl with calcitriol did not attenuate the calcitriol-mediated increase in CaxP product or calcitriol-mediated aortic calcification. Both calcitriol and cinacalcet HCl therapy significantly reduced serum PTH levels. Calcitriol significantly elevated serum calcium, serum phosphorous and CaxP product above pretreatment levels, or those seen with vehicle or cinacalcet HCl. Cinacalcet HCl (10 or 1 mg/kg) decreased serum ionized calcium and decreased calcitriol-induced hypercalcaemia.

Conclusion. Cinacalcet HCl and calcitriol both effectively reduce PTH, albeit via different mechanisms, but unlike calcitriol, cinacalcet HCl did not produce hypercalcaemia, an increased CaxP product or vascular calcification.

Keywords: calcimimetics; calcitriol; cinacalcet HCl; hypercalcaemia; secondary hyperparathyroidism; vascular calcification



   Introduction
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 Abstract
 Introduction
 Subjects and methods
 Statistical analyses
 Results
 Discussion
 References
 
Soft tissue calcification involving primarily the medial portion of the arterial vasculature is a well recognized and common complication of chronic kidney disease (CKD) [1]. Vascular calcification causes increased arterial stiffness and contributes to the high cardiovascular mortality and morbidity in dialysis patients [2,3]. While the causes of calcification in CKD remain to be to elucidated, associated risk factors include age, hypertension, time on dialysis and abnormalities in calcium and phosphate metabolism, such as hyperphosphataemia and a raised serum calcium-phosphorus (CaxP) product [3,4]. Secondary hyperparathyroidism (HPT), defined by excessively elevated parathyroid hormone (PTH) levels, is often observed in patients with advanced CKD and is associated with elevated serum calcium and phosphorus, and CaxP product. Notably, common therapeutic interventions in secondary HPT have come under scrutiny for associations with the development of vascular calcification [5,6].

A novel therapeutic class for the treatment of secondary HPT, the type II calcimimetics, has recently been approved. Members of this class are small organic compounds that modulate the calcium sensing receptor (CaR), a G-protein coupled receptor that responds to changes in extracellular calcium [7] in the parathyroid glands to diminish PTH secretion [8]. By specifically targeting the molecular mechanism that modulates calcium-regulated PTH release from the parathyroid cell, calcimimetic compounds provide an alternative approach to managing secondary HPT without concomitantly increasing serum calcium and phosphorus, and thus, potentially obviate the development of vascular calcification. In contrast to conventional therapies currently in use, such as vitamin D sterols and phosphate binders, cinacalcet HCl (Sensipar®/Mimpara®), the first type II calcimimetic approved for commercial use, simultaneously reduces the four key biochemical parameters associated with secondary HPT: PTH secretion, CaxP product, and serum calcium and phosphorus [9].

While calcitriol has been associated with vascular calcification in animal models [10], there are currently no studies examining the ability of calcimimetics, particularly cinacalcet HCl, to mediate calcification. In this study, we used an in vivo rodent model of CKD accompanied by secondary HPT to compare the effects of calcitriol with cinacalcet HCl on the potential for production of vascular calcification as well as to examine any potential influence of cinacalcet HCl on calcitriol-mediated calcification.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Statistical analyses
 Results
 Discussion
 References
 
Animals
Experiments were performed under protocols approved by Amgen's Internal Animal Care and Use Committee. Male Sprague-Dawley rats (350–390 g) were purchased from Harlan Sprague-Dawley (Indianapolis, IN). Rats were pair housed with a 12 h/12 h light/dark cycle and given ad libitum access to standard rat chow (1.17% calcium, 1.0% phosphorus) and water.

Subtotal nephrectomy
A rodent model of CKD was induced by 5/6 nephrectomy (Nx), a two-step procedure that reduces the original functional renal mass by five-sixths (5/6). In the first step, animals were anaesthetized using 2% isoflourane (IsoFlo; Abbott Laboratories, Chicago, IL) in a 2 L/min oxygen flow. Once an acceptable anaesthetic plane was achieved, a 5–8 mm incision was made on the right medio-lateral surface of the abdomen, the right kidney exposed and unencapsulated, the renal pedicle clamped and ligated, and the kidney was removed. The ligated pedicle was returned to a neutral anatomical position and the abdomen and skin incisions closed with absorbable suture materials. The animal was moved to a clean recovery chamber prior to return to the colony room.

Following a 7 day recovery, each animal underwent a second surgical procedure on the contralateral kidney. The animal was anaesthetized as previously described and a 5–8 mm incision was made on the left medio-lateral surface of the abdomen and the left kidney was exposed. The left renal artery was visualized and two of the three branches tightly ligated. The kidney was inspected for infarct and returned to an anatomically neutral position within the peritoneal cavity. The abdominal wall and skin incisions were closed, and the rat placed in a clean recovery cage. Sham-operated animals underwent the same procedures without renal manipulation.

Drug administration
Animals were randomized into treatment groups based on the normal distribution of body weights at baseline. Cinacalcet HCl was formulated in 20% Captisol:water (vehicle) on site at Amgen Inc. (Thousand Oaks, CA) to deliver 10 or 1 mg/kg oral dose (gavage) in a 1.5 ml/kg dose volume. Based on exposure levels, the equivalent human dose to 10 mg/kg is 60 mg, which is used clinically (Amgen, data on file). Calcitriol, supplied as 1{alpha}, 25-dihydroxycholecalciferol from Sigma-Aldrich, Inc (St. Louis, MO), was dissolved in 90% ethanol to create a 1 mM stock solution that was stored at –20°C until final dilution in phosphate buffered saline (PBS). Calcitriol (0.1 µg/rat, equivalent to 0.25–0.28 µg/kg, in a dose volume of 0.2 ml PBS) was administered by subcutaneous injection once daily for 26 consecutive days. Since previous work has shown that calcitriol exhibits differing pharmacokinetic and metabolism parameters in rats in relation to humans [11–13], we employed 100 ng of calcitriol per rat (0.25–0.28 ug/kg), to act as a positive control for the induction of calcification. Drug, vehicle, or saline administration (oral gavage) was started 1 week after 5/6 nephrectomy Nx or sham surgery and was continued once a day for 26 consecutive days (n = 8–10 rats/group). It was believed that the use of two different routes of administration would decrease the potential for absorption interactions between calcitriol and cinacalcet HCl. The aortas were removed 24 h after the 26th dose and calcification of aortic sections was determined using von Kossa stain and scoring technique described below.

Serum analyses
Blood for chemistry analyses [total serum calcium and phosphorous, blood urea nitrogen (BUN), creatinine, PTH and ionized calcium] was collected before 5/6 Nx (baseline) and again on drug treatment days 0 (pretreatment), 14 and 26 from the retro-orbital sinus of anaesthetized rats, and the serum was analysed. On day 14, blood was collected immediately before drug administration (time 0 h) and 4 h later. On day 26, blood was collected at time 0 and 4 h, and 24 h after the last dose. For measurement of blood ionized calcium levels, blood (50 µL) was collected from the retro-orbital sinus of anaesthetized rats with heparinized capillary tubes and immediately analysed using a Ciba-Corning 634 ISE Ca++/pH Analyzer (Ciba-Corning Diagnostics Corp, Medfield, MA). Separately, blood (0.5 ml) was collected for PTH levels into SST (clot activator) brand blood tubes and allowed to clot. Serum was removed and stored at –70°C until assayed. PTH levels were quantified according to the vendor's instructions using rat PTH(1–34) immunoradiometric assay kit (Immutopics, San Clemente, CA). Calcium and phosphorous were measured using a blood chemistry analyzer (AU 400; Olympus, Melville, NY).

Ex vivo assessment of vascular calcification
Animals were sacrificed by CO2 asphyxiation 24 h after the 26th consecutive dose (corresponding to 33 days following the completion of the subtotal nephrectomy), and necropsy was performed. Terminal blood samples were collected and the region of the aorta from the heart to the diaphragm was excised and fixated in aqueous buffered zinc formalin (Zfix; Anatech Ltd, Battle Creek, MI) and subsequently processed for paraffin embedment.

The aortas were sectioned longitudinally and stained for calcification by the von Kossa method. The stained aortas were evaluated and scored by a board-certified pathologist blinded to the treatment groups. All slides were scored within a single session to avoid drift in scoring outcome. One section per animal was scored on a 0–5 scale with 0 = no calcification, 1 = minimal, 2 = mild, 3 = moderate, 4 = marked and 5 = severe calcification.



   Statistical analyses
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 Abstract
 Introduction
 Subjects and methods
 Statistical analyses
 Results
 Discussion
 References
 
Analysis of variance (ANOVA; {alpha} = 0.05) was used to test for overall treatment effects. When significant treatment effects were detected, post-hoc analyses (Fisher's Protected Least Squares Difference tests) were used for between group comparisons ({alpha} = 0.05). Mann–Whitney non-parametric statistics were used to test for significant differences between baseline measures and 1 week post-5/6 Nx ({alpha} = 0.05). Paired t-test ({alpha} = 0.05) were used to test for differences between predose and 4 h postdose for a given treatment condition and day.



   Results
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 Abstract
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 Subjects and methods
 Statistical analyses
 Results
 Discussion
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Effects of 5/6 Nx on ionized Ca2+, Ca, P and renal and parathyroid function
As expected, prior to 5/6 Nx, renal (Table 1) and parathyroid function (serum PTH), as well as serum chemistries (e.g. ionized calcium and total Ca and P) (Table 2) were normal with no between group differences. Within 1 week after 5/6 Nx, signs of renal impairment and secondary HPT were evident in all 5/6 Nx groups. BUN increased ~2-fold and creatinine increased 2.7-fold above baseline (Table 1). In addition, the average serum PTH (380±23 pg/ml, n = 56) from all 5/6 Nx rats significantly increased (P<0.0001) by 1 week after 5/6 Nx compared to their own presurgery, baseline levels (143±11 pg/ml, n = 56). Mineral homeostasis was significantly perturbed as a result of 5/6 Nx prior to administration of any treatment regimens: ionized calcium significantly decreased (P<0.0001) in all groups 1 week after 5/6 Nx (treatment day 0) compared to baseline (1.479±0.003, n = 64; Figure 1), whereas total phosphorous significantly increased (P<0.0001) an average of 1.2-fold (Table 2). Sham-operated animals showed no changes in any measured parameters. All animals were subsequently started on a 26-day treatment regimen with cinacalcet HCl, vehicle, calcitriol, or a cinacalcet HCl/calcitriol combination in order to compare the effects of these treatments on ionized calcium, aortic vascular calcification, PTH, total calcium and phosphorous, and the CaxP product. It is important to note that body weights stabilized in all of the calcitriol-treated groups during the treatment period, whereas there was a trend for weights to increase in the vehicle and cinacalcet HCl only (1 and 10 mg/kg) treatment groups. Therefore, any potential effects of dietary intake on calcium and phosphorus levels would have a greater impact on the cinacalcet HCl-treated groups than the calcitriol-treated groups.


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Table 1. BUN (mg/dL) and creatinine (mg/dL) from serum

 

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Table 2. Calcium-phosphorous product (Ca x P) (mg/dL)2

 


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Fig. 1. Effect of calcitriol, cinacalcet HCl, or both on blood-ionized calcium levels in 5/6 Nx rats. Baseline ionized calcium levels were not different between treatment groups before surgery (1.479±0.003, N = 64). Animals were 5/6 Nx and following a 1 week recovery, dosed daily for 26 days with vehicle, calcitriol, cinacalcet HCl 10 or 1 mg/kg, or calcitriol + cinacalcet HCl 10 or 1 mg/kg. Blood-ionized calcium levels were determined on days 0, 14 and 26 at 4 h postdosing. Daily dosing with cinacalcet HCl (10 or 1 mg/kg) for 26 days mediated a significant decrease in blood-ionized calcium levels. The addition of cinacalcet HCl (10 mg/kg) to calcitriol normalized the calcitriol mediated increase in blood-ionized calcium. Vehicle had little or no effect on blood-ionized calcium levels. Values are mean±SEM, n = 8–10 group. aCinacalcet HCl 10 mg/kg vs vehicle (P<0.001); bcalcitriol vs vehicle (P<0.01); ccalcitriol vs vehicle (P<0.05); dcinacalcet HCl 1 mg/kg vs vehicle (P<0.05).

 
Treatment effects on renal parameters
While creatinine remained elevated throughout the study in all 5/6 Nx groups, levels were significantly higher (P<0.05) in all of the calcitriol-treated groups compared to vehicle- and cinacalcet HCl-treated 5/6 Nx animals (Table 1). All calcitriol-treated animals had significantly (P<0.05) elevated BUN levels throughout the treatment period. BUN levels gradually decreased over the treatment period for those animals treated with either vehicle or cinacalcet HCl alone. These BUN levels were significantly lower than all of the calcitriol-treated animals. Cinacalcet HCl did not attenuate the calcitriol-induced increase in BUN or creatinine.

Treatment effects on ionized calcium
Calcitriol significantly (P<0.01) increased blood-ionized calcium levels following 14 and 26 consecutive doses compared to vehicle treatment over the same period and compared to its own pretreatment (day 0) control values (Figure 1). In contrast, cinacalcet HCl did not produce hypercalcaemia; in fact, cinacalcet HCl effectively lowered ionized calcium levels at both doses (10 mg/kg, P<0.001 and 1 mg/kg, P<0.05) doses compared to vehicle, calcitriol, and all combination calcitriol±cinacalcet HCl groups (Figure 1). Furthermore, cincalcet HCl (10 mg/kg) in combination with calcitriol had significantly lower (P<0.05) ionized calcium compared to animals treated with just calcitriol (Figure 1).

Treatment effects on aortic vascular calcification
Cinacalcet HCl, administered once daily (10 mg/kg oral dose) for 26 consecutive days, did not produce any signs of aortic vascular calcification (calcification score = 0.0; n = 10). When the dose of cinacalcet HCl was reduced to 1 mg/kg only one rat (1 out of 10) demonstrated mild calcification with a group mean calcification score of 0.1±0.1; n = 10. Calcification also was not observed in vehicle-treated (n = 8) rats (Figures 2 and 3). In contrast, all animals in the calcitriol group (n = 8/8) had demonstrable calcification of aortic tissues. Calcification was detected in von Kossa-stained sections as deposits of brown-black material in the media and subintimal areas of the aorta and continuous primary arterial branches (Figure 2). The mean calcification score in the calcitriol group was 3.1±0.4; n = 8 (moderate to marked severity), which was statistically greater than vehicle-treated animals (P<0.05; Figure 3). Animals treated with calcitriol plus cinacalcet HCl (10 mg/kg) had a mean calcification score of 3.8±0.4; n = 10 (P>0.05 vs calcitriol).



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Fig. 2. Aortic medial calcification. Animals were 5/6 Nx and then dosed with calcitriol, cinacalcet HCl or vehicle for 26 days, and aortas were harvested 24 h after the final dose. Sections were stained according to the von Kossa method.

 


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Fig. 3. Calcitriol-induced aortic calcification. Administration of calcitriol, but not cinacalcet HCl (10 or 1 mg/kg), daily for 26 days mediates aortic calcification in 5/6 Nx rats. Administration of cinacalcet HCl with calcitriol for 26 days did not attenuate calcitriol-mediated aortic calcification. Values are means±SEM (n = 8–10 group). The calcification score scale ran from 0 (no calcification) to 5 (severe calcification).

 
Treatment effects on serum PTH
Serum was collected for PTH determinations in all rats on the last treatment day (day 26) immediately before dosing and again 4 h postdosing in n = 8–10 rats/group. Cinacalcet HCl significantly (P<0.01) reduced PTH levels measured on treatment day 26 from 468±57 pg/ml, n = 10 (prior to the last dose) to 123±35 pg/ml, n = 10 by 4 h after an oral dose of 10 mg/kg (Figure 4). The lower dose of cinacalcet HCl (1 mg/kg) did not significantly reduce PTH levels at 4 h postdose (447±98 pg/ml, n = 10) compared to predose levels (628±103 pg/ml, n = 10; P = 0.11, Paired t-test, two-tailed). As expected, vehicle treatment had no effect on serum PTH (Figure 4). Calcitriol produced a sustained, significant reduction in serum PTH compared to vehicle-treated rats; levels were low on day 26 before and 4 h after dosing with no difference between pre- versus 4 h postdose (Figure 4). The combination of calcitriol plus cinacalcet HCl (10 mg/kg) also produced a similar sustained PTH lowering pattern; however, with the combination therapy PTH was significantly lower 4 h postdosing compared to predose values (P<0.05) (Figure 4).



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Fig. 4. Serum PTH levels following various treatments in 5/6 Nx rats. Animals were 5/6 Nx and following a 1 week recovery, dosed daily for 26 days with vehicle, cinacalcet HCl 10 mg/kg, calcitriol, or calcitriol + cinacalcet HCl 10 mg/kg. Serum PTH levels were determined on day 26 prior to dosing (black) and at 4 h postdosing (white). Cinacalcet HCl (4 h postdose), calcitriol and the combination treatments significantly (P<0.01) reduced serum PTH levels when compared to vehicle treated animals. aTreatment 4 h (open) vs vehicle 4 h (open) (P<0.01); bpredose (solid) vs vehicle (solid) (P<0.05); ctreatment 4 h (open) vs predose (solid) (P<0.05).

 
Treatment effects on total serum calcium, phosphorous and calciumxphosphorus (CaxP product)
Although total serum calcium did not change appreciably after 5/6 Nx, calcitriol treatment alone significantly (P<0.05) increased total calcium throughout the treatment period (Table 2). In contrast, calcium did not increase in vehicle or cinacalcet HCl-treated animals. In fact, total calcium was reduced in cinacalcet HCl-treated animals in relation to vehicle-treated animals (P<0.05) throughout the study (Table 2). The combination of cinacalcet HCl (10 mg/kg) with calcitriol significantly (P<0.05) decreased total calcium compared to calcitriol + vehicle 4 h after the 14th and 26th dose (Table 2, treatment days 14 and 26).

5/6 Nx mediated a significant increase in serum phosphorous prior to any treatment regimen compared to sham-operated animals (Table 2; P<0.05). The 5/6 Nx-induced increase in serum phosphorous was transient in vehicle-treated animals; serum phosphorous levels returned to within sham-operated levels by treatment day 14 (Table 2). In contrast, serum phosphorous was significantly increased (P<0.005) in animals treated with calcitriol alone or in combination with cinacalcet HCl (calcitriol + cinacalcet HCl 10 mg/kg or +1 mg/kg) compared to vehicle-treated animals throughout the study period. Cinacalcet HCl at 10 mg/kg increased serum phosphorous levels 4 h postdosing on days 14 (P = 0.0005) and 26 (P = 0.0045) and at 1 mg/kg only at day 14 (P = 0.012) compared to vehicle-treated animals. Although cinacalcet HCl increased serum phosphorus above vehicle-treated levels, phosphorous levels, in general, were lower in the cinacalcet HCl-treated animals than in animals treated with the combination of calcitriol and cinacalcet HCl (P<0.05).

The therapeutic interventions examined in this trial mediated significant effects on the levels of total serum calcium and phosphorus, which translated into significant effects on the CaxP product. The CaxP product increased (P<0.05) in all groups one week after 5/6 Nx compared to sham-operated (normal) rats (Table 2). Calcitriol alone significantly (P<0.0001) exacerbated the increase in CaxP product following 14 and 26 days of treatment (Table 2) compared to vehicle-treated, cinacalcet HCl-treated (10 and 1 mg/kg) or sham-operated rats. The calcitriol-induced increase in CaxP product after 14 or 26 daily injections was not attenuated by concurrent treatment with cinacalcet HCl 10 mg/kg or 1 mg/kg. The CaxP product decreased (P<0.0001) during the 26 day treatment period in animals treated with cinacalcet HCl (10 or 1 mg/kg) compared to all calcitriol-treated animals (Table 2).



   Discussion
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 Abstract
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 Subjects and methods
 Statistical analyses
 Results
 Discussion
 References
 
The salient finding in this study is that calcitriol treatment produced aortic vascular calcification in uraemic rats that was unaffected by concomitant treatment with cinacalcet HCl. This may be related to the dose of calcitriol used and further studies will be required to determine if calcimimetics are able to prevent or alter the progression of calcitriol-induced calcification if lower doses of calcitriol are used. Calcification was prominent within the media and subintima and was, on average, moderate to marked in severity in all calcitriol-treated groups. In contrast, cinacalcet HCl treatment alone did not produce any significant signs of aortic medial calcification in a rodent model of secondary HPT when administered at 10 or 1 mg/kg per day for 26 days. It is established that vitamin D sterols, administered in high doses to treat secondary HPT, are associated with significant cardiovascular disease (vascular calcification) [14]. In light of this, the present finding that cinacalcet HCl does not produce vascular calcification in an animal model of secondary HPT is a novel finding of significant importance to the pharmacological management of PTH in secondary HPT. Together with the demonstrated clinical efficacy in the reduction of PTH, CaxP, Ca and P [9], these data suggest that cinacalcet may play an important role in the treatment of patients with secondary HPT.

Although the causes of calcification in CKD patients with secondary HPT are unknown, administration of vitamin D sterols to treat secondary HPT significantly increased serum calcium and phosphorous, which have been implicated in the development of vascular calcification in CKD [3,15]. We observed a slight increase in Ca x P product 1 week after subtotal nephrectomy that was exacerbated in secondary HPT animals treated with calcitriol. In contrast, cinacalcet HCl-treated secondary HPT animals did not demonstrate an increase in Ca x P product. This was primarily a result of the effects of cinacalcet HCl on calcium, for cinacalcet HCl-treated rats exhibited increases in phosphorus levels compared to controls. Both calcium and phosphorus levels were elevated in calcitriol-treated secondary HPT rats. In both cases, the increase in phosphorus can be explained by the reduction of PTH, an essential regulator of phosphorus reabsorption. In the normal kidney, an increase in serum phosphorus results in a decrease in ionized calcium and an increase in PTH secretion, which in turn, decreases renal proximal tubule phosphorus reabsorption and normalizes serum phosphorus levels. Therefore, given this and the demonstrated ability of cinacalcet HCl to lower PTH levels, it is not surprising that rats with an intact or partially intact renal system treated with cinacalcet HCl exhibit increased serum phosphorus levels. Our finding of increased phosphorus with cinacalcet HCl is in agreement with previous reports in experimental animals treated with the calcimimetic NPS R-568 or cinacalcet HCl [8,16]. However, unlike the above preclinical animal studies, cinacalcet HCl decreases serum phosphorous in patients who are receiving haemodialysis [9]. This effect, coupled with its ability to reduce PTH, CaxP product and serum Ca, strengthens the case for the use of cinacalcet HCl as a therapeutic agent in the treatment of secondary HPT.

Although cinacalcet HCl significantly reduced the Ca x P product in our animal model of secondary HPT when used alone, this effect was not observed when administered concurrently with calcitriol. This is in contrast to Phase 3 clinical observations [9]. While both cinacalcet HCl and calcitriol effectively reduce PTH, the mechanisms by which these compounds achieve this endpoint are different and may explain the enhanced efficacy of the combination treatment regimes. Calcitriol suppresses PTH by a genomic mechanism on PTH production [17], whereas cinacalcet HCl inhibits the release of PTH [8]. The inhibition of PTH secretion by cinacalcet HCl is cyclic due to the metabolism and clearance of this compound [8]. Peak efficacy for attenuating serum PTH levels by cinacalcet HCl in rats is ~4 h post-administration, which is consistent with the present results and the pharmacokinetic profile for this compound from previous rodent studies [8]. Furthermore, in this model, treatment with cinacalcet HCl does not produce aortic vascular calcification. It is important to note that calcitriol produces a sustained lowering of PTH levels, thereby chronically interfering with renal maintenance of phosphorus homeostasis. The sustained increase in phosphorus coupled with increased calcium yields an elevated Ca x P product over the entire treatment period. In addition to phosphorus and Ca x P product levels, studies have indicated that mineral disposition and arterial calcification are associated with expression of certain bone matrix proteins [1]. This suggests that calcification is likely to be more complex than mineral precipitation and may, in fact, be an actively mediated process [18,19].

Recent data from Jono et al. [20,21] provide additional insights into potential mechanisms of calcitriol vascular toxicity. Through the vitamin D receptor, calcitriol suppresses vascular smooth muscle cell (VSMC) expression of PTH-related protein (PTHrP), an autocrine factor that inhibits alkaline phosphatase (AP) expression via the PTH/PTHrP receptor. By down-regulating autocrine PTHrP signalling, calcitriol induces the VSMC expression of AP. This osteogenic enzyme degrades pyrophosphate, the major inhibitor of tissue calcification [22] and generates inorganic phosphate for calcium phosphate mineral deposition. Thus, pharmacological doses of calcitriol used to suppress PTH production may concomitantly suppress vascular PTHrP expression that defends against calcific vasculopathy. The apparent selective actions of cinacalcet HCl on endocrine tissues that express the CaR may reduce circulating PTH levels without compromising the vascular PTHrP paracrine defence mechanism. Although vascular calcification may not be entirely dependent upon an elevated Ca x P product, it is interesting to note that all animals receiving calcitriol alone or in combination with cinacalcet HCl exhibited a sustained increase in the Ca x P product and vascular calcification; this association was not evident in cinacalcet HCl- or vehicle-treated animals.

Calcitriol significantly elevated ionized calcium above pretreatment, vehicle control, or cinacalcet HCl levels. Cinacalcet HCl was observed to significantly decrease ionized calcium in animals with established calcitriol-induced hypercalcaemia. The lowering effects of cinacalcet HCl on ionized calcium in our animal model of secondary HPT are in agreement with clinical observations [9]. Although the reduction in serum calcium by cinacalcet HCl may mediate symptomatic hypocalcaemia, recent clinical trials have shown that cinacalcet HCl-mediated hypocalcaemia occurs in a low percentage of patients and is often transient and asymptomatic [9]. The doses of cinacalcet HCl used in the present studies in rodents produce similar exposure levels that are observed in the clinic, although the data generated from the present uraemic animal study should be extrapolated to human disease with caution. However, these results do suggest that cinacalcet HCl would be a useful agent for the reduction of PTH and reordering of mineral metabolism while avoiding the vascular sequelae often associated with vitamin D sterol-based treatments.

In summary, although both calcitriol and cinacalcet HCl reduce PTH levels in 5/6 Nx rats by different mechanisms, only calcitriol treatment mediates extensive aortic calcification. Combination therapy with cinacalcet HCl and calcitriol resulted in similar levels of vascular calcification to that of calcitriol alone, and reduced levels of blood-ionized calcium compared to calcitriol alone, thereby potentially allowing for combination therapy with reduced vitamin D sterol doses.



   Acknowledgments
 
We acknowledge the assistance of William W. Stark, Jr, PhD in the preparation of the manuscript and Sheila Scully for reproduction of figures.

Conflict of Interest Statement. None declared.



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Statistical analyses
 Results
 Discussion
 References
 

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Received for publication: 11. 8.04
Accepted in revised form: 23. 3.05