Changes in fibroblast growth factor 23 levels in normophosphatemic patients with chronic kidney disease stage 3 treated with lanthanum carbonate: results of the PREFECT study, a phase 2a, double blind, randomized, placebo-controlled trial

Men and non-pregnant, non-lactating women aged 18 years or over with CKD stage 3 were included in the study. Patients had to have been under the care of a physician for at least 2 months, and not be expected to begin dialysis for at least 6 months. At screening, patients had to have serum phosphate levels within the normal range (0.808–1.55 mmol/l), an estimated glomerular filtration rate (eGFR) (estimated using the Cockcroft–Gault and Modification of Diet in Renal Disease [MDRD] formulae [25]) of 30–59 ml/min/1.73 m², C-terminal FGF23 (cFGF23) levels ≥ 50.0 RU/ml and 25-hydroxyvitamin D levels ≥ 20 ng/ml. Patients also had to have an adequate protein diet that included two or three portions of protein-rich food per day, as determined at screening. Those who required vitamin D supplements or compounds containing calcium, phosphate, aluminum or magnesium were excluded. Individuals were also excluded if they had: acute kidney failure; rapidly progressing glomerulonephritis; cirrhosis or other clinically significant liver disease; human immunodeficiency virus infection; life-threatening malignancy or multiple myeloma; or any clinically significant illness that would, in the opinion of the investigator, impair their ability to give informed consent or to take part in or complete the study. Patients with a history of gastrointestinal disorders, alcohol or substance abuse, or poor compliance (< 60% or > 120% tablets taken) were excluded, as were vegetarians and patients allergic to iodine.

Patients were withdrawn from the study if: their serum phosphate concentration fell below 0.60 mmol/l; their medical condition deteriorated significantly in the opinion of the investigator; they had a major change in diet; they commenced dialysis or required a kidney transplant; they had unmanageable adverse events; or if they became pregnant.

The study was conducted in accordance with all applicable regulations, the International Conference on Harmonisation guideline for Good Clinical Practice [26], and the Declaration of Helsinki and its subsequent revisions [27]. The study protocol and informed consent documents received ethical approval from the institutional ethical review committee: Comité de Protection des Personnes, Ile de France II. Necker Hospital, Paris, France. All patients provided written informed consent before participating in the study.

This was a phase 2a, double-blind, placebo-controlled, proof-of-concept study carried out between November 2010 and May 2012 (ClinicalTrials.gov identifier: NCT01128179). Participants were screened within a 2-week period, then returned for a baseline visit at which their eligibility for inclusion in the study was confirmed before they were randomized, by sequential allocation of a unique 2-digit number by the investigator that matched a 2-digit number on the study medication. Treatment was assigned by a randomization schedule and patients received either lanthanum carbonate (FOSRENOL^®, Shire, Nyon, Switzerland) 1000 mg three times daily, or a placebo equivalent for 12 weeks (Figure 1A), details of which were in the form of code-break envelopes held at the investigational site. The investigational product was to be chewed rather than swallowed whole, and was recommended to be taken with meals. Patients returned to the clinic site for visits 1, 2, 8 and 12 weeks after the baseline visit, and a follow-up call was scheduled for week 16, or 30 days after the end of treatment if the investigational product was discontinued before study completion.

The primary endpoint was iFGF23 level at week 12. Secondary endpoints were: iPTH, 1,25-dihydroxyvitamin D, phosphate, total calcium and calcium × phosphate product; 24-h urinary phosphate and calcium excretion; urinary creatinine and urea nitrogen concentrations. Tertiary endpoints were: 25-hydroxyvitamin D, ionized calcium, creatinine and cFGF23; GFR as measured by plasma iohexol clearance (iGFR) according to previously described methods [28], and eGFR; CrossLaps^® (a measure of bone resorption; IDS Plc, UK); and bone-specific alkaline phosphatase.

Blood samples were collected at screening and all subsequent visits for measurement of iFGF23, cFGF23, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, bone-specific alkaline phosphatase and iPTH levels. Twenty-four-hour urine samples were collected at all visits from baseline to week 12 for assessment of phosphate, calcium, creatinine and urea nitrogen levels. One day prior to the visit, each patient was asked to discard their first morning urination, to note the time on the urinary bottle, and then to collect the urine of all subsequent micturitions for 24 h. The time of the last urination was also noted on the bottle. eGFR was assessed at screening, baseline and week 12. iGFR, ionized calcium and CrossLaps^® were assessed at baseline and week 12. Vital signs and adverse events were monitored at every visit from screening to the final visit. Adverse events were also recorded on the follow-up call. Compliance was calculated based on the number of tablets remaining in medicine bottles returned at the end of the study.

The study was conducted at a single center (Service de Néphrologie-Dialyse, Clinique du Landy, Paris, France), and measurement of GFR and biochemical evaluations were performed at a single department (Service de Physiologie-Explorations Fonctionnelles, Hôpital Necker-Enfants Malades, Paris, France).

Previous work by one of the authors (DP) showed fasting FGF23 levels in patients with CKD to be log-normally distributed with a coefficient of variation of the logged data of 1.13 (author’s unpublished data). A sample size of 33 participants randomized in a 2:1 ratio (22 receiving lanthanum carbonate and 11 receiving placebo) was estimated to be sufficient to detect a ratio of 1.15 in the logged means, or a 40% reduction in mean iFGF23 levels in the lanthanum carbonate group compared with the placebo group at 12 weeks. This assumed 80% power and a two-sided significance level of 5%.

Patients who were randomized and received at least one dose of the investigational product were included in the safety analysis set. Patients in the safety analysis set who had at least one post-dose assessment were included in the full analysis set. The per protocol set consisted of patients who had at least one dose of investigational product in addition to primary data assessment available from week 2 or later, and who did not have any pre-defined protocol deviations that might affect the primary variable. The per protocol set was the primary analysis population for this proof-of-concept study.

Data were presented as descriptive statistics, which are shown as mean ± standard deviation (SD) or percentage change from baseline in the least-squares (LS) mean ± standard error. Statistical analyses were performed using SAS^® version 9.1 (SAS Institute, Cary, NC, USA). Differences in the primary endpoint, iFGF23 levels, between the placebo and lanthanum carbonate groups at week 12 were evaluated using analysis of covariance (ANCOVA) and log-transformed values, with baseline iFGF23 as a covariate. ANCOVA models were also used to evaluate the statistical significance of differences in secondary and tertiary variables using change from baseline to week 12, with treatment group as a factor and the corresponding value at baseline as a covariate. Missing data were accounted for using the last observation carried forward method. In post hoc analyses, ANCOVA was used to evaluate the statistical difference in the percentage change from baseline in the LS means between the lanthanum carbonate and placebo groups by week.

In total, 51 patients were screened, of whom 35 met the inclusion criteria and entered the study. These individuals made up the safety analysis set and were randomized such that 23 patients received lanthanum carbonate and 12 received placebo (Figure 1B). All patients included in the safety analysis set also made up the full analysis set. In the lanthanum carbonate group, two participants did not complete the study: one died (not considered to be treatment related); the other was withdrawn because of a protocol violation (eGFR < 30 ml/min/1.73 m²). Also in the lanthanum carbonate group, four patients completed the study but met exclusion criteria and were not included in the per protocol analysis set.

Baseline characteristics of the per protocol analysis set were similar to the safety analysis set (Table 1); however, there were some numerical differences between the lanthanum carbonate and placebo groups. The median age was 65 years (range: 38–84 years) in the lanthanum carbonate group and 73 years (range: 43–87 years) in the placebo group. In the lanthanum carbonate group, 58.8% of patients were men, compared with 41.7% of those in the placebo group. Most participants were white (lanthanum carbonate group: 70.6%; placebo group: 75.0%), and there was a higher proportion of black patients in the lanthanum carbonate group (29.4%) than in the placebo group (16.7%). Individuals in the lanthanum carbonate group had a lower iGFR (mean: 42.2 ml/min/1.73 m², standard deviation [SD]: 10.1 ml/min/1.73 m²) than the placebo group (mean: 48.0 ml/min/1.73 m², SD: 13.1 ml/min/1.73 m²) at baseline. Mean treatment compliance, measured by the proportion of tablets left in the medicine bottle at the treatment visit, was 87.5% (SD: 20.1%) in the lanthanum carbonate group and 91.0% (SD: 13.0%) in the placebo group.

Numerical changes in mean iFGF23 levels were seen between the lanthanum carbonate and placebo groups throughout the treatment period, although there was no statistically significant difference in the per protocol set between the groups at week 12, the primary endpoint of the study (p = 0.3186 from F-test of logged LS mean iFGF23 values) (Figure 2A). This was also true of the safety/full analysis set, in which there was no statistically significant difference in LS mean iFGF23 at week 12 (p = 0.6330). Across all the analyses, the results were similar for the per protocol set and the safety analysis set, and these groups had similar baseline characteristics; therefore, only efficacy results for the per protocol analysis set are presented.

In the lanthanum carbonate group, mean iFGF23 levels decreased from 70.5 pg/ml (SD: 32.2 pg/ml) at baseline to 51.9 pg/ml (SD: 18.2 pg/ml) at week 1; however, they subsequently increased to 58.8 pg/ml (SD: 20.6 pg/ml) by week 12. For patients given placebo, there was negligible change in mean iFGF23 levels throughout the study (baseline: 63.7 pg/ml (SD: 14.8 pg/ml); week 12: 63.7 pg/ml (SD: 21.7 pg/ml).

The percentage change from baseline in LS mean iFGF23 levels was also retrospectively assessed for each treatment visit (Figure 2B). In this post hoc analysis, the reduction in iFGF23 in the lanthanum carbonate group compared with the placebo group was statistically significant at week 1 (p = 0.0102); however, a statistically significant difference was not seen at subsequent time points.

Mean FGF23 levels were also assessed using an assay to detect the tertiary variable, cFGF23 (Figure 2C). Post hoc analysis showed that there was a reduction at all weeks in the percentage change from baseline in LS mean cFGF23 levels in the lanthanum carbonate group, whereas these levels increased or were slightly reduced (week 8) in the placebo group (Figure 2D). The between-group difference was significant 2 and 8 weeks after the start of treatment (p < 0.05, ANCOVA), but not after 1 or 12 weeks.

Secondary variables, serum and urinary phosphate and calcium levels were assessed during the study and between-group differences were analyzed statistically at week 12 (Figure 3; Additional file 1: Table S1). Serum phosphate was lower in the lanthanum carbonate group than in the placebo group throughout the study, including the baseline visit, but there was no significant difference in serum phosphate between groups after 12 weeks (Figure 3A). Twenty-four-hour urinary phosphate excretion was generally higher in the placebo group than in the lanthanum carbonate group, and this difference was significant at week 12 (p = 0.0162) (Figure 3B, Additional file 1: Table S1). In the lanthanum carbonate group, phosphate excretion decreased from 18.2 mmol/24 h (SD: 6.8 mmol/24 h) at baseline to 14.4 mmol/24 h (SD: 7.9 mmol/24 h) at week 2 and remained at this level until week 12 (14.5 mmol/24 h [SD: 6.4 mmol/24 h]). Phosphate excretion in the placebo group increased from a mean of 16.2 mmol/24 h (SD: 9.1 mmol/24 h) at baseline to 22.5 mmol/24 h (SD: 10.9 mmol/24 h) by week 12.

Serum total calcium level and calcium × phosphate product were similar in the placebo and lanthanum carbonate groups, and remained constant throughout the study (Figure 3C and 3D). Urinary calcium levels were generally higher in the lanthanum carbonate group than in the placebo group (Figure 3E); however, by week 12, urinary calcium levels had increased in the placebo group (2.72 mmol/24 h [SD: 1.12 mmol/24 h]) and had fallen to below placebo levels in the lanthanum carbonate group (1.78 mmol/24 h [SD: 0.90 mmol/24 h]). There was a significant difference between the decrease in urinary calcium from baseline in the lanthanum carbonate group and the increase from baseline in the placebo group at week 12 (p = 0.0371; Additional file 1: Table S1).

Daily dietary phosphate intake was not measured directly in this study, but was estimated retrospectively using urinary urea nitrogen measurements. Estimated daily phosphate intake was similar both between the two treatment groups, and within each group throughout the study (Figure 3F).

Mean iPTH and 1,25-dihydroxyvitamin D levels for each treatment group were measured as secondary variables throughout the study (Additional file 2: Figure S1). Mean iPTH levels were similar in the placebo and lanthanum carbonate groups: there was a decrease from baseline (placebo: 75.8 pg/ml [SD: 43.9 pg/ml], lanthanum carbonate: 69.4 pg/ml [SD: 30.4 pg/ml]) to week 1 (placebo: 62.2 pg/ml [SD: 38.1 pg/ml], lanthanum carbonate: 51.6 pg/ml [SD: 22.2 pg/ml]) in both groups, but levels returned to close to baseline values by week 12. In patients who received placebo, there was a small numerical decrease in mean 1,25-dihydroxyvitamin D levels by week 12 compared with baseline (-9.5 pg/ml [SD: 19.6]), whereas there was no change from baseline to week 12 in the lanthanum carbonate group (0.1 pg/ml [SD: 12.2 pg/ml]). No significant difference in the change from baseline was seen between the LS means of the lanthanum carbonate and placebo groups for iPTH (p = 0.2995) or for 1,25-dihydroxyvitamin D (p = 0.3252; Additional file 1: Table S1).

Serum or urinary levels of other relevant variables (urinary fractional phosphate excretion, creatinine, urea nitrogen, 25-hydroxyvitamin D, ionized calcium, CrossLaps^®, iGFR and bone-specific alkaline phosphatase) are shown in Additional file 1: Table S1. No significant between-group differences were seen between weeks 0 and 12 for these factors.

Retrospective subgroup analyses were undertaken to assess whether baseline differences between the lanthanum carbonate and placebo groups affected circulating iFGF23 levels over time (Additional file 3: Figure S2). Patients with baseline eGFR below 45 ml/min (CKD stage 3b) showed greater reductions in iFGF23 in both the lanthanum carbonate and placebo groups, than individuals with a baseline eGFR in the range 45–60 ml/min; however, no significant difference was seen between placebo and lanthanum carbonate treatment in either eGFR group. Covariate analyses found no association of the percentage change from baseline of iFGF23 at any time point with baseline cFGF23, baseline iPTH, estimated daily phosphate intake or patient gender.

A summary of adverse events experienced in the study is given in Table 2. In total, 30.4% of patients experienced adverse events in the lanthanum carbonate group compared with 16.7% in the placebo group. The most frequently observed adverse events were gastrointestinal (17.4% for lanthanum carbonate vs 16.7% for placebo), including constipation, dry mouth and acute pancreatitis. Two patients (8.7%) treated with lanthanum carbonate experienced serious adverse events but neither event was considered to be related to the investigational product: one patient experienced acute pancreatitis; the other died following cardiac failure and myocardial ischemia.