A randomised controlled trial to examine the effects of cinacalcet on bone, cardiovascular parameters and mortality in haemodialysis patients with secondary hyperparathyroidism

Background Secondary hyperparathyroidism may lead to increased cardiovascular risk. The use of cinacalcet may improve bone and cardiovascular health with improved parathormone (PTH) and phosphate control. Methods This is an open-label prospective randomised controlled trial to compare progression of cardiovascular and chronic kidney disease mineral and bone disorder (CKD-MBD) parameters. Patients were randomised to receive cinacalcet alongside standard therapy or standard therapy alone. 36 haemodialysis patients who had >90 days on dialysis, iPTH >300pg/mL, calcium >2.1mmol/L and age 18-75 years were included. Following randomization, all 36 patients underwent an intensive 12-week period of bone disease management aiming for iPTH 150-300pg/mL. The primary outcome was change in vascular calcication using CT agatston score. Secondary outcomes included pulse wave velocity (PWV), left ventricular mass index (LVMI), carotid intima-media thickness (CIMT), augmentation index (Aix) and bone measurements. The above measurements were obtained at baseline and 12 months. The difference in mortality between the groups was compared during long-term follow-up. Results There was no evidence of a group difference in the progression of calcication (median change (IQR) cinacalcet: 488 (0 to1539); standard therapy: 563 (50 to 1214)). In a post hoc analysis combining groups there was a mean (SD) phosphate reduction of 0.3mmol/L (0.7) and median (IQR) iPTH reduction of 380pg/mL (-754, 120). Regression of LVMI and CIMT was seen (P=0.03 and P=0.001) and was signicantly associated with change of phosphate on multi-factorial analyses. Over a median follow-up of 32 months, there was no signicant difference in the mortality between the two groups. Conclusions No signicant benet in bone and cardiovascular markers was seen with the addition of cinacalcet to standard therapy over one year. Tight control of hyperphosphataemia and secondary hyperparathyroidism may lead to a reduction in LVMI and CIMT but this needs further investigation. calcication in our study was comparable to that seen in other dialysis patient studies The unrestricted use of vitamin D in our study, the tight control achieved for PTH and phosphate, the small sample size and low baseline calcication scores in some of the patients may account for the reduced progression of calcication between this and some other studies. The tight control, which was also achieved in the standard therapy arm, may have reduced the difference in effect between the two arms of the study. In the aggregated analysis of all patients change of coronary calcication was associated with higher baseline serum phosphate and FGF23 although this association was not shown with aortic calcication. High levels of phosphate and iPTH have been shown to be associated with increased mortality and vascular calcication (20,21). In this study we reduced both phosphate and iPTH in both treatment arms by equal degrees. There is probably a benecial role for both cinacalcet and vitamin D analogues, however cinacalcet allows control of secondary hyperparathyroidism to be achieved more easily in patients The PARADIGM study was a randomised open label study to assess effect of cinacalcet versus vitamin D on biochemical parameters. This showed that both classes of therapy can reduce iPTH to a similar target but that their effects on calcium and phosphate concentrations differ(22). There is data to suggest that Cinacalcet may lower blood pressure, improve cardiac morphology and lower FGF23 and our study has also shown trends consistent with these studies St.Peter et al found no relationship between short term change in iPTH due to cinacalcet use and cardiovascular outcomes although this was after only 1 year and so any benecial changes may not have had time to take effect The relationship between cinacalcet and patient survival has been investigated in the largest study of CKD-MBD, the EVOLVE study The primary end-point of the trial was negative with intention-to-treat analysis but when baseline demographic variations were in the analysis an improvement in survival suggested In pre-specied secondary analyses older patients have been shown to have survival and reduced cardiovascular events if cinacalcet Although no difference in during chronic kidney disease mineral and bone disorder; CMR: cardiac magnetic resonance; EE: estimated effect; IQR: interquartile range; LVH: left ventricular hypertrophy; LVMI: left ventricular mass index; MHRA: Medicines & Healthcare products Regulatory Agency; PTH: parathormone; PWV: pulse wave velocity; SD: standard deviation; RCT: randomised control trial; SHPT: secondary hyperparathyroidism.


Background
Cardiovascular disease is the leading cause of death in patients with chronic kidney disease (CKD) (1). Studies have shown that derangements of intact parathyroid hormone (iPTH), phosphate and calcium are linked to increased mortality (2,3). A higher iPTH has also been associated with increased calci cation and cardiomyopathy (4,5) Cinacalcet enabled increasing numbers of patients to achieve targets for CKD mineral and bone disorder (CKD-MBD), although there is continued restriction on its use (6,7) Since the start of this trial there has been some evidence that calcimimetics may reduce progression of vascular calci cation (8)(9)(10). Calci cation progression has been studied with cinacalcet and other CKD-MBD medications but the few randomised trials have allowed a disparity of parathyroid hormone or phosphate control between arms so that uncertainty has remained regarding whether it is the medication or the control that has been bene cial (11)(12)(13).
The primary aim of this study was to determine if cinacalcet treatment and standard therapy attenuated progression of vascular calci cation compared to standard therapy in haemodialysis patients when equivalent control of secondary hyperparathyroidism was achieved between treatment arms. Our trial is different from the ADVANCE trial in that we aimed for equivalent biochemical targets in the treatment arms and did not specify a maximum vitamin D dose (14).
The KDIGO guidelines published in 2009 suggest aiming for an iPTH level within the range of 2-9x the upper limit of normal (15). A subsequent observational study by Floege et al showed a survival bene t in patients achieving the tighter KDOQI iPTH target of 150-300pg/mL along with the targets for calcium (2.10-2.37mmol/L) and phosphate (1.13-1.78mmol/L) (16,17). In light of this we performed a post-hoc analysis investigating the whole trial population to determine if any differences in endpoints were associated with changes in PTH, phosphate and other biomarkers.

Materials And Methods
This open label randomized control trial compared cinacalcet versus standard therapy in patients with secondary hyperparathyroidism and dialysis dependent end stage kidney disease to examine the effect of cinacalcet on bone and cardiovascular parameters. All patients gave written informed consent to participate in the trial. Ethical  Patients with secondary hyperparathyroidism who had been receiving dialysis for greater than 90 days were screened for enrolment into the study. Inclusion criteria included an iPTH>300pg/mL, corrected calcium >2.1mmol/L and age 18-75years. Exclusion criteria included previous coronary stents, coronary artery bypass grafts or valve replacement, atrial brillation and severe liver disease.
The randomisation was strati ed by diabetic status and baseline pulse wave velocity. Following randomisation all patients underwent a 12-week intensive management phase with both medical intervention and dietary advice. During this calcium and phosphate concentrations were measured fortnightly and the iPTH was measured at weeks 2 and 12. Unrestricted phosphate binders (i.e. calcium and non-calcium containing preparations) and exible doses of vitamin D sterols were available for use in all patients.
Following the intensive management phase, patients were reviewed every 8 weeks until the nal visit at week 52 unless a medication change or biochemical result warranted more frequent review. Biochemical and haematology tests were measured at each time-point. All patients received a minimum of 12 consultations throughout the trial.
The study protocol is outlined in Fig. 1.
The following investigations were performed at baseline and at 12 months; serum and plasma samples were also taken and stored at these time points.
Scans were performed on the same post-dialysis day at baseline and 12 months and were interpreted by clinicians blinded to randomisation and blood results: Cardiac and abdominal aorta vascular calci cation score (Agaston method, GE CT Lightspeed 16-slice scanner).
Bone mineral density measurements included QCT spine (GE scanner using Mindways software), Dual X-Ray Absorptiometry (DXA) of Hip and spine and peripheral QCT (pQCT) of forearm.
The following bedside investigations were performed immediately after dialysis with the patient at their dry weight as determined by their medical team: Carotid-femoral pulse wave velocity (cfPWV) and augmentation index (Aix) at the radial artery (SphygmoCor® (Atcor Medical)).
The carotid-intima media thickness (CIMT) was measured 1cm below the bifurcation of the carotid artery bilaterally.
Adequate dialysis was determined as a Kt/v >1.2 and / or a URR >65%.
Biochemical and haematology pre-dialysis samples were processed at 12 time points throughout the trial. All biochemical samples were analysed in a central laboratory (Roche modular analyser except iPTH (DPC immulite 2000 chemiluminescent immunoassay)). Haematology results were analysed using Sysmex The primary outcome measure for the trial was change in calci cation score at 12 months. The secondary outcomes were change in arterial stiffness, bone mineral density, cardiac morphology, survival and biomarkers.
At the time of designing this study there were minimal data available upon which to base a potential effect size for the primary endpoint. The trial was designed to have an 80% power to detect a one standard deviation difference in absolute change in calci cation score over 1 year at the 5% signi cance level. A power calculation determined that 32 patients would be required for the study to have adequate power; the recruitment target was set at 40 and 36 patients were randomized.

Statistical analysis
Categorical data were analysed using chi-square analysis. Continuous data were assessed for normality using graphical means and transformed as necessary. All data were then analysed using linear regression adjusted for diabetes and PWV as per randomisation strati cation. Differences over 12 months were assessed by Wilcoxon signed rank test or paired t-test depending on normality of data. Multi-factorial linear regression was used to determine further associations using normalised data where required. The study end points for the long term follow up included the rst of the following: death, renal transplant, last follow up date or end of analysis period, which was 31 st December 2018. Survival analyses was performed using Kaplan-Meier plots and cox regression analyses to study the difference in mortality between the two groups (cinacalcet and control). A post-hoc analysis to examine the overall effects of achieving tight iPTH control was also undertaken, which aggregated the data of the 36 enrolled patients. All statistical analyses were performed using SPSS There were no substantive differences in most baseline parameters between treatment arms except that the baseline median calci cation score, iPTH and mean cfPWV were higher in the standard therapy arm (cinacalcet arm: 814, 665pg/ml, 7.8(2) compared to 3401, 806pg/ml, 9.2(3) respectively). 15 patients were randomised to cinacalcet with standard therapy compared to 21 with standard therapy alone: the difference due to chance imbalance of strata at randomisation. Two patients in the standard therapy arm did not complete the study; one died and the other withdrew in week 51. Adverse events were similar in both arms during the trial (table 3). There was a higher incidence of hypocalcaemia in patients randomised to cinacalcet compared to standard therapy as would be expected. No patients discontinued cinacalcet in the study.

Primary endpoint
The primary outcome was absolute change of total calci cation score from baseline to 1 year (Fig. 3). Although there was no signi cant difference between the study arms (median change cinacalcet 488; standard 563: Estimated effect (EE)=5.0 (-0.1-10, P=0.053) changes were in a direction that favoured cinacalcet. When areas of calci cation were analysed separately no signi cant change was seen with either coronary (median change cinacalcet 43; standard 207: EE =-0.9(-5.9, 4.1 P=0.7) or aortic calci cation (median change cinacalcet 207; standard 293: EE =4.6(-0.5, 9.7 P=0.08). 4 patients who completed the trial had a total calci cation score <30 at baseline; 3 of which still had a calci cation score <30 at 12 months.

Secondary endpoints
Similar iPTH control was seen in both study arms (median iPTH at 12 months (pg/ml): cinacalcet 225 (152,386); standard 294 (145,445)). The area under the curve for PTH exposure was calculated with no signi cant difference found between the groups (P=0.3). Similarly, no signi cant difference was seen with exposure to phosphate over the 12-month period with mean phosphate at 12 months in cinacalcet group 1.62mmol/L (0.56) and 1.62mmol/L (0.65) with standard therapy (P=0.6). There was no signi cant difference in change or nal vitamin D dose, calcium binder dose or calcium dialysate between groups. 12 patients in each treatment arm were prescribed a non-calcium phosphate binder (P=0.3).
The cfPWV and radial Aix increased during the study despite improved control of secondary hyperparathyroidism. The progression was lower in the cinacalcet arm though this was not signi cant ( Table 2). Despite the increase in calci cation and vascular stiffness over the study period there was a reduction in LVMI in both treatment arms (Fig. 4). The difference between arms was not statistically signi cant. No signi cant change was seen for other measured cardiac parameters, CIMT, blood pressure, biomarkers or haemoglobin levels. Bone mineral density showed minimal changes in both arms and there was no signi cant difference.

Post hoc analysis
When the trial population was considered as a whole, statistically signi cant reductions were seen for LVMI and CIMT (P=0.03 and P=0.001, respectively) between baseline and 12 months. This occurred despite progression of vascular calci cation and stiffness. On multi-factorial analysis change of phosphate was signi cantly associated with change of CIMT (EE =0.007 (0.0, 0.13) P=0.04). Change of LVMI was also associated with change of phosphate on unifactorial (EE =0.95 (0.04, 1.86) P=0.04) and multi-factorial analysis (EE =1.23 (0.01, 2.5) P=0.05). In both cases improved phosphate control was associated with desirable reductions in the imaging parameter. Changes in FGF23 or iPTH showed no signi cant association with change in CIMT or LVMI.
Over the trial 22/36 (61%) patients had a reduction of phosphate (mean reduction of phosphate 0.3mmol/L). There was no signi cant difference in starting dose or change of vitamin D doses administered between those who had a reduction in phosphate and those who did not.
During long term follow up there were 16 deaths from randomisation to censoring (median follow up of 32 months). There was no signi cant difference in the number of deaths between the groups (p =0.74) ( Table 4). The Kaplan-Meir survival plot did not show a signi cant survival difference between the groups (Log-Rank, p =0.82). (Fig. 5). In a univariable cox-regression model, cinacalcet status was not associated with all-cause mortality (HR:1.13; 95% CI: 0.39 to 3.2; p =0.82) ( Table 5).

Discussion
In this study we found no signi cant difference in progression of vascular calci cation when cinacalcet was used to control secondary hyperparathyroidism alongside standard therapy compared to standard therapy alone. Cinacalcet has been shown to attenuate the progression of vascular calci cation in rat models but human studies have shown a weaker effect (18,19). In the ADVANCE study there was a modest bene t with cinacalcet as opposed to standard therapy, the median percentage of coronary calci cation progression being 24% in the cinacalcet arm vs. 31% in the control arm (P=0.07). Although the primary endpoint was negative the difference reached signi cance when corrected for baseline phosphate.
Progression of vascular calci cation in our study was comparable to that seen in other dialysis patient studies (12,13). The unrestricted use of vitamin D in our study, the tight control achieved for PTH and phosphate, the small sample size and low baseline calci cation scores in some of the patients may account for the reduced progression of calci cation between this and some other studies. The tight control, which was also achieved in the standard therapy arm, may have reduced the difference in effect between the two arms of the study. In the aggregated analysis of all patients change of coronary calci cation was associated with higher baseline serum phosphate and FGF23 although this association was not shown with aortic calci cation.
High levels of phosphate and iPTH have been shown to be associated with increased mortality and vascular calci cation (20,21). In this study we reduced both phosphate and iPTH in both treatment arms by equal degrees. There is probably a bene cial role for both cinacalcet and vitamin D analogues, however cinacalcet allows control of secondary hyperparathyroidism to be achieved more easily in patients (6). The PARADIGM study was a randomised open label study to assess effect of cinacalcet versus vitamin D on biochemical parameters. This showed that both classes of therapy can reduce iPTH to a similar target but that their effects on calcium and phosphate concentrations differ (22). There is data to suggest that Cinacalcet may lower blood pressure, improve cardiac morphology and lower FGF23 and our study has also shown trends consistent with these studies (23)(24)(25)(26). St.Peter et al found no relationship between short term change in iPTH due to cinacalcet use and cardiovascular outcomes although this was after only 1 year and so any bene cial changes may not have had time to take effect (27). The relationship between cinacalcet and patient survival has been investigated in the largest study of CKD-MBD, the EVOLVE study (28). The primary end-point of the trial was negative with intention-to-treat analysis but when baseline demographic variations were included in the analysis an improvement in survival was suggested (29). In pre-speci ed secondary analyses older patients have been shown to have improved survival and reduced cardiovascular events if taking cinacalcet (30). Although no difference in mortality was seen during long term follow up in our study, we recognise that the study was markedly underpowered to demonstrate this.
High iPTH levels have also been shown to be associated with left ventricular hypertrophy (LVH) in haemodialysis patients and the relationship between iPTH and LVH is thought to be independent of blood pressure (31,32). A reduction in LVMI was seen in both arms of our study with no signi cant difference between treatment arms. These data are interesting as this occurred despite progression of vascular stiffness and vascular calci cation, and without changes in any other key parameters such as blood pressure or dialysis duration, and suggests that tighter control of phosphate and/or iPTH may be associated with a reduction in LVMI. The trend to greater improvement observed in the cinacalcet arm may be explained by a number of mechanisms including the presence of the calcium sensing receptor (CaSR), identi ed on cardiac myocytes, which has been shown to affect DNA synthesis and may affect cell remodelling and growth (33,34) An overall reduction in CIMT was also seen in this study when data in the two arms were aggregated.
We wished to further explore the regression of LVMI and CIMT and therefore performed post hoc analysis of the trial data, making use of an increased sample size and survival data. In these analyses we found a signi cant association between reduction in phosphate and regression of LVMI, reduction of CIMT and improved survival. These associations were not seen with iPTH reduction or change in FGF23 although this may be linked to the limitations of small patient numbers or short duration of the trial. There are many studies detailing that increased phosphate is associated with an increased mortality risk in CKD and dialysis patients (35,36). Higher serum phosphate is associated with LVH in the general population, predialysis and dialysis patients and it follows, but is not causally proven, that lowering phosphate may be bene cial (37)(38)(39). There are many studies comparing different phosphate binders and morbidity but there is an evidence gap with respect to the reduction of elevated phosphate leading to improved patient outcomes (12,13). Our study has shown that patients having a reduction in phosphate had an improved survival over four years although the patient numbers were very small, and itis important to emphasise that this was a post-hoc analysis Although interesting, a larger clinical trial would be needed to verify our ndings.
Our overall ndings support the view that tight control of both phosphate and iPTH, by whatever means, may slow progression of calci cation and reduce LVMI and CIMT. However, the potential detrimental effects of the medications used to achieve targets need to be considered. There are studies suggesting increased calci cation with high dose vitamin D and calcium containing phosphate binders (40,41). A proportion of patients within our study also developed low iPTH levels, despite close monitoring and dose adjustments, and therefore had increased likelihood of developing adynamic bone disorder. Both of these complications are associated with a worse overall outcome (42).
The main limitation of this study was the small sample size. In light of the ADVANCE study it is clear that a larger sample size would be required to show conclusive changes in vascular calci cation. However, despite only 36 patients entering the study, the changes in calci cation favoured cinacalcet use, re ecting the tight control of phosphate and iPTH achieved in our study, brought about by diligent and frequent monitoring by a clinician, which would be di cult to achieve in the setting of a larger trial. This could be achievable in clinical practice albeit at the expense of signi cant clinician resource. Although the post hoc analysis of our RCT generated interesting results regarding phosphate control, the study was not designed or powered to examine the effects of changes in phosphate level. However, our ndings are hypothesis generating and further studies designed to con rm or refute the validity of these ndings are indicated.
In conclusion, when control of secondary hyperparathyroidism was obtained to equivalent degrees in haemodialysis patients, no signi cant difference in change in calci cation was seen between the cinacalcet and standard therapy arms. The control of SHPT and phosphate appears to be important given the overall reduction in LVMI seen despite progression of other cardiovascular parameters. The trial was funded via an unrestricted educational grant from Amgen, who supplied the cinacalcet for this trial but had no role in the design of this study, data collection, analysis or interpretation of the results. Change in LVMI between the treatment arms at one year