Effects of oral iron and calcium supplement on the pharmacokinetics and pharmacodynamics of molidustat: an oral HIF–PH inhibitor for the treatment of renal anaemia

The present studies assessed the drug–drug interaction of molidustat, a hypoxia-inducible factor prolyl hydroxylase inhibitor, with iron and calcium supplements, which are common medications in patients with anaemia due to chronic kidney disease (CKD). Forty-two healthy men received molidustat alone (fasted or fed) or combined with oral iron(II) or calcium(II), given immediately before or between 4 h before and 1 h after molidustat in three randomized, open-label, crossover studies (12–15 participants per study). Molidustat AUC and Cmax were assessed as the main pharmacokinetic parameters, and endogenous erythropoietin (EPO) was measured to evaluate pharmacodynamics. Depending on prandial state, concomitant intake of iron(II) reduced molidustat AUC and Cmax by 50–75% and 46–84%, respectively, and EPO AUC(0-24) and Cmax by 31–44% and 36–48%, respectively. The influence of iron(II) declined with increasing the time interval to the intake of molidustat, with reductions in molidustat AUC and Cmax of 9% and 10%, respectively, when iron(II) intake occurred 4 h before molidustat. Accordingly, effects on endogenous EPO were less pronounced with increased time separation between oral iron(II) and molidustat intake. Calcium(II) reduced molidustat AUC and Cmax by 15% and 47%, respectively, without influence on EPO response. All treatments were well tolerated. In contrast to concomitant oral intake of calcium, the effect of oral iron supplements on molidustat pharmacokinetics and pharmacodynamics should be considered, and the two agents should be administered with an appropriate time separation.


Introduction
Anaemia is a common complication of chronic kidney disease (CKD); its prevalence increases as kidney disease progresses, and it affects nearly all patients with stage 5 CKD [1]. It is associated with poor quality of life and increased risk of cardiovascular events, hospitalization, cognitive impairment, and mortality [2,3].
Reduced erythropoietin (EPO) production in the kidney and iron deficiency are both important factors in the pathogenesis of anaemia associated with CKD [4]. Current management of anaemia in patients with CKD consists of a combination of erythropoiesis-stimulating agents (ESAs) and iron supplementation. ESAs mimic the action of endogenous EPO and are effective in elevating haemoglobin (Hb) levels in patients with anaemia due to CKD [5]. However, ESAs are associated with several potential safety concerns, including increased risk Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00228-019-02813-y) contains supplementary material, which is available to authorized users. of cardiovascular events and stroke [6], so alternative therapy options are being developed.
Hypoxia-inducible factor prolyl hydroxylase (HIF-PH) inhibitors offer a potential alternative to the standard of care. EPO transcription is activated by hypoxia-inducible factors (HIF) in response to hypoxia. However, in the presence of oxygen, HIF-PH hydroxylates the HIF-α subunit, which is subsequently targeted for proteasomal degradation, preventing EPO synthesis. Inhibition of HIF-PH results in stabilization of HIF, which leads to endogenous production of EPO and, ultimately, stimulation of erythropoiesis [7]. Molidustat is an orally bioavailable HIF-PH inhibitor being investigated in phase 3 clinical trials in patients with renal anaemia, including patients receiving dialysis treatment [8,9]. The phase 2 DIALOGUE (DaIly orAL treatment increasing endOGenoUs Erythropoietin) programme, which included three studies with a 16week treatment duration and two extension studies, assessed the safety and efficacy of molidustat in different populations of patients with renal anaemia [10,11]. DIALOGUE 1 was a placebo-controlled, fixed-dose trial (25, 50, and 75 mg once daily; 25 and 50 mg twice daily). In DIALOGUEs 2 and 4, molidustat was given as individual dose titration based on Hb values (starting doses ranged between 25-75 mg and 25-150 mg once daily in DIAGLOGUE 2 and 4, respectively). Molidustat treatment was generally well tolerated and corrected and maintained Hb levels within a pre-specified range in patients previously treated with ESAs and in treatment-naïve patients [10]. In healthy volunteers, molidustat was shown to be rapidly absorbed with a maximum plasma concentration of 1 h after drug intake and to elicit dosedependent increases in endogenous EPO after single increasing doses [12]. Molidustat is metabolized via glucuronidation and eliminated in urine as pharmacologically inactive glucuronide [13].
Oral iron supplementation is a common concomitant treatment in patients with anaemia. In vitro experiments indicated that the solubility of molidustat is reduced by 95% in the presence of multivalent cations such as iron(II) and calcium(II) (unpublished data on file, Bayer AG, Wuppertal, Germany) compared to the clinically administered sodium salt. Here, we report the results of three studies that evaluated the effect of co-administration of oral iron(II) on the plasma pharmacokinetics (PK) and pharmacodynamics (PD) of molidustat after intake of a single oral dose under fasted or fed conditions in healthy volunteers. The effect of time separation between oral iron(II) and molidustat administrations on molidustat PK and PD was also investigated to select an optimized time interval between administration of both drugs to avoid clinically relevant interactions. The drug-drug interaction (DDI) between molidustat and calcium acetate was also evaluated in one of the studies.

Study design
All three studies had a single-centre, randomized, open-label, crossover design and involved healthy male volunteers. Study 1 investigated the effect of iron(II) sulphate and calcium acetate administered orally immediately before molidustat on the plasma concentration-time profile of molidustat (PK) and endogenous serum EPO (PD) in fasted (≥10 h) individuals. The following three single-dose treatments were administered: molidustat 150 mg (2 × 75 mg immediate-release [IR] tablets); iron(II) sulphate 304 mg (Eryfer 100 mg capsule, CHEPLAPHARM Arzneimittel GmbH, Mesekenhagen, Germany) followed immediately by molidustat 150 mg; and calcium acetate 1900 mg (2 × 950 mg Calcet tablets, Teva GmbH, Ulm, Germany) followed immediately by molidustat 150 mg. In the crossover design, each volunteer received the three treatment regimens with a washout period of at least 96 h between each dose of molidustat.
Study 2 assessed the effect of iron(II) sulphate administered orally from 4 h before to 1 h after molidustat and the effect of enteric-coated iron(II) glycine sulphate complex administered immediately before molidustat on the PK and PD of molidustat in fasted (≥10 h) individuals. The volunteers received the following single-dose treatments: molidustat 150 mg (2 × 75 mg IR tablets); iron(II) sulphate 304 mg (Eryfer 100 mg capsule) 4 h before molidustat 150 mg; iron(II) sulphate 304 mg (Eryfer 100 mg capsule) 2 h before molidustat 150 mg; iron(II) sulphate 304 mg (Eryfer 100 mg capsule) 1 h after molidustat 150 mg; and iron(II) glycine sulphate complex 567.7 mg (Ferro Sanol duodenal 100 mg capsule, enteric-coated, Sanol GmbH, Monheim, Germany) followed immediately by molidustat 150 mg. In the crossover design, each volunteer received all five treatment regimens with a washout period of at least 72 h between each dose of molidustat. Both iron formulations (Eryfer and Ferro Sanol duodenal) contained iron(II) 100 mg per dose. Ferro Sanol duodenal was an enteric-coated formulation, whereas Eryfer consists of an IR tablet. Study 3 assessed the effect of iron(II) sulphate administered from 1 h before to 1 h after molidustat on the PK and PD of molidustat under fed conditions. Molidustat was administered 30 min after the start of a standardized continental breakfast which consisted of 2 slices (40 g) of white bread (toasted), 20 g butter, 25 g jam, 20 g cheese (45% fat), and 200 mL of tea containing 1 cube of sugar. The breakfast had to be finished in 30 min or less. The participants received the following singledose treatments: molidustat 150 mg (2 × 75 mg IR tablets); iron(II) sulphate 304 mg (Eryfer 100 mg capsule) 1 h before molidustat 150 mg; iron(II) sulphate 304 mg (Eryfer 100 mg capsule) followed immediately by molidustat 150 mg; and iron(II) sulphate 304 mg (Eryfer 100 mg capsule) 1 h after molidustat 150 mg. In the crossover design, each participant received each of the four treatment regimens with a washout period of at least 72 h between each molidustat dose.
In all three studies, volunteers were admitted to the study ward at least 12 h before study drug administration, which began in the morning of the next day. Participants were discharged from the ward after a 48-h observation period. The studies were approved by the Ethics Committee of North-Rhine Medical Council, Düsseldorf, Germany, and were conducted in accordance with the International Conference on Harmonization guideline on Good Clinical Practice and with the Declaration of Helsinki. All volunteers gave written informed consent to participate in the study.

Study populations
For all three studies, healthy, white, male volunteers aged 18-45 years with a body mass index (BMI) between 18.0 kg/m 2 and 29.9 kg/m 2 were eligible for enrolment. Key inclusion and exclusion criteria are summarized in Supplementary

Materials
Ferro Sanol duodenal was an enteric-coated formulation, whereas Eryfer and molidustat were administered as IR tablets. Both iron formulations (Eryfer and Ferro Sanol duodenal) contained iron(II) 100 mg per dose. Calcet was administered as tablets containing 950 mg of calcium acetate each.

Pharmacodynamic parameters
In all three studies, blood samples were collected pre-dose and at 4, 6, 8, 12, and 24 h post-dose to assess the PD effects of molidustat on serum endogenous EPO levels as previously described [12]. Absolute values for serum EPO C max , t max , and area under the concentration-time curve from 0 to 24 h (AUC (0-24) ) were determined. Serum EPO C max ratios to baseline were also calculated.

Clinical safety and tolerability
Clinical safety and tolerability were assessed by physical examination, monitoring of vital signs (blood pressure and heart rate), 12-lead electrocardiogram, laboratory safety tests (blood and urine analyses), and occurrence of adverse events.

Statistical methods
In all three studies, geometric means and percentage geometric coefficients of variation were calculated for molidustat AUC, C max , t ½ , and CL/F; t max was described using median and range. Mean molidustat plasma concentrations for each time point were only calculated if values were obtained and were above the LLOQ for at least two-thirds of the participants. For the calculation of the mean value, a data point below the LLOQ was substituted by half this threshold. Logarithms of AUC and C max were analysed using an analysis of variance (ANOVA) including fixed effects for sequence, period, participant (sequence), and treatment. Point estimates and exploratory 90% confidence intervals (CIs) for the ratios of relative bioavailability between molidustat alone and in combination with oral iron(II) or oral calcium acetate in each study were calculated by retransforming the corresponding least squares (LS) mean difference and 90% CIs of the ANOVA.
In all three studies, absolute geometric means and geometric coefficients of variation were calculated for EPO AUC (0-24) (using the trapezoidal rule) and C max ; furthermore, geometric mean ratios to baseline were determined for C max . EPO t max was described using median and range. The logarithms of AUC (0-24) and C max of EPO were analysed using an ANOVA including fixed effects for sequence, period, participant (sequence), and treatment. Point estimates and exploratory 90% CIs for the ratios between molidustat alone and in combination with oral iron(II) or oral calcium acetate were calculated by retransforming the corresponding LS mean difference and 90% CIs of the ANOVA.
Sample size determination was done by evaluating the expected width of the 90% CIs for estimating the drugdrug interaction potential, i.e. ratio of geometric means of AUC between treatments. Assuming an intra-participants' CV of 20%, 90% confidence limits were expected to be 1.16 times the point estimate, which was considered sufficient for these exploratory studies. No further power calculations were performed because the intention of the statistical analysis was to estimate the DDI and not to prove bioequivalence.

Study populations
In study 1, 27 healthy volunteers were screened and 16 were randomized. One participant was withdrawn before receiving any dose of molidustat owing to protocol violation. All 15 remaining participants were included in the safety set (defined as all participants who received at least one dose of molidustat), the PK set (all participants with at least one valid PK profile), and the PD set (all participants with at least one complete EPO profile). In study 2, 33 healthy volunteers were screened and 15 were randomized. The safety analysis set (defined as in study 1) included 15 participants. One subject withdrew his consent in the first treatment period. The PK and PD sets, which both included 14 participants, were defined as all healthy volunteers who received at least two doses of molidustat and who had at least two valid PK profiles or two valid PD profiles (molidustat alone and any with co-treatment), respectively. In study 3, 21 healthy volunteers were screened and 12 were randomized. The safety analysis set (defined as in study 1) comprised 12 participants. One participant was withdrawn during the second treatment period owing to an adverse event (upper respiratory tract infection). The PK and PD sets were defined as in study 2, and both included 11 participants.
For each study, demographic and baseline characteristics are summarized in Table 1. All participants were white men, and the mean ages (29.6-31.2 years), mean weights (74.6-82.1 kg), and mean BMIs (23.6-25.2 kg/m 2 ) were similar across all three studies.

Molidustat in combination with oral iron(II)
Plasma molidustat PK parameters in the absence and presence of iron(II) in all three studies are shown in Table 2. Based on the ANOVA and compared with molidustat alone, the largest decreases in plasma geometric mean AUC for molidustat in fasted volunteers were observed when iron(II) sulphate (study 1) and iron(II) glycine sulphate (enteric-coated formulation, study 2) were administered immediately before molidustat ( Table 3, Supplementary Table 3). A similar reduction in molidustat AUC was observed in fed volunteers (study 3) after administration of iron(II) sulphate immediately before molidustat (Table 3, Supplementary Table 3). In fasted individuals, when iron(II) sulphate was administered 4 h before, 2 h before, and 1 h after molidustat (study 2), the geometric mean AUC was 9%, 16%, and 26% lower, respectively, than after molidustat alone (Table 3, Supplementary Table 3). Compared with molidustat alone, in fed individuals, the reduction in geometric mean AUC was greater when iron(II) sulphate was administered 1 h after molidustat than when it was administered 1 h before (study 3).
Similarly, the decrease in plasma geometric mean C max for molidustat was largest when iron(II) sulphate (study 1 and 3) or iron(II) glycine sulphate (study 2) was administered immediately before molidustat (Table 3, Supplementary Table 3). In fasted individuals, when iron(II) sulphate was administered 4 h before, 2 h before, and 1 h after molidustat (study 2), the geometric means C max were 10%, 0.3%, and 12% lower, respectively, than after molidustat alone (Table 3,  Supplementary Table 3). Compared with molidustat alone, in fed individuals, the reduction in plasma molidustat C max was less pronounced when iron(II) sulphate was administered 1 h before molidustat than when it was administered 1 h after (study 3).
In study 1, the terminal t ½ increased with concomitant administration of iron(II) sulphate. Similarly, in studies 2 and 3, terminal t ½ increased when oral iron(II) supplement was administered with molidustat, regardless of the time separation between drug administration ( Table 2).
CL/F increased in the presence of iron(II) up to 500%. Given that an increase of clearance (CL) by oral iron(II) is unlikely and that t ½ was not reduced, this effect may be attributed to a decrease in bioavailability rather than an increase in CL.

Molidustat in combination with oral calcium(II)
Plasma molidustat PK parameters in the absence and presence of calcium(II) are shown in Table 2. In fasted individuals, when calcium acetate was administered immediately before molidustat, the geometric means AUC and C max for molidustat were reduced by 15% and 47%, respectively, compared with molidustat alone; the terminal t ½ was also decreased slightly compared with molidustat alone.

Molidustat alone
After oral administration of molidustat 150 mg (2 × 75 mg IR tablets) alone under fasted or fed conditions, endogenous EPO reached peak concentrations of 77.8 IU/L (study 1), 103.6 IU/ L (study 2), or 85.9 IU/L (study 3) approximately 8 h after dosing (Table 4, Supplementary Figs. 1, 2, and 3). Across all three studies, EPO mean AUC (0-24) was in the range of 1171-1498 IU × h/L. Table 4 shows the geometric means for EPO AUC (0-24) and C max and the median for t max after oral administration of molidustat 150 mg (under fasted or fed conditions) to healthy male participants, in the absence and presence of iron(II) sulphate administered from 4 h before to 1 h after molidustat, for all three studies.

Molidustat in combination with oral iron(II)
When iron(II) sulphate (study 1) or iron(II) glycine sulphate (study 2) were administered in fasted individuals immediately before molidustat, the geometric mean of EPO AUC (0-24) decreased by almost 50% compared with molidustat alone ( Table 5, Supplementary Table 4). In fed healthy volunteers (study 3), compared with molidustat alone, the geometric mean AUC (0-24) for EPO was reduced by 31% when iron(II) sulphate was administered immediately before molidustat ( Table 5, Supplementary Table 4). These reductions in EPO AUC (0-24) were not observed when iron(II) sulphate was administered 4 h before molidustat and partially observed when iron(II) sulphate was administered 2 h before or 1 h after molidustat (study 2) ( Table 5, Supplementary Table 4). Similarly, in fed individuals, the effects of iron supplementation on the EPO AUC (0-24) were less pronounced when iron(II) sulphate was administered 1 h before or 1 h after molidustat (Table 5, Supplementary Table 4).
The decrease in geometric mean C max for EPO was largest when iron(II) sulphate (studies 1 and 3) and iron(II) glycine sulphate (study 2) were administered immediately before molidustat (Table 5, Supplementary Table 4). In fasted individuals, when iron(II) sulphate was administered 4 h before, 2 h before, and 1 h after molidustat (study 2), the geometric means EPO C max were 2%, 15%, and 25% lower, respectively, than after molidustat alone ( Table 5, Supplementary Table 4). Compared with molidustat alone, in fed individuals, the reduction in EPO C max was less pronounced when iron(II) sulphate was administered 1 h before molidustat than when it was administered 1 h after (study 3) ( Table 5, Supplementary  Table 4).

Molidustat in combination with oral calcium(II)
The ANOVA for the comparison between EPO AUC (0-24) and C max after administration of molidustat alone and molidustat plus calcium acetate did not indicate any clinically relevant difference between the two treatments ( Table 5,  Supplementary Table 4).

Clinical safety and tolerability
In study 1, six participants (40%) experienced a total of 10 treatment-emergent adverse events (TEAEs) (Supplementary    Table 6). Overall, two participants experienced three TEAEs that were considered to be related to molidustat by the investigator: one after receiving iron(II)  Fig. 1 Plasma concentrations of molidustat after administration of a single oral dose of molidustat 150 mg alone and together with iron(II) sulphate 304 mg or calcium acetate 1900 mg immediately before molidustat to healthy fasted volunteers in study 1 (geometric mean [SD], PK set, N = 15) h hours, PK pharmacokinetics, SD standard deviation sulphate 2 h before molidustat (mild headache); one after receiving iron(II) glycine sulphate immediately before molidustat (mild headache); and one after receiving iron(II) sulphate 1 h after molidustat (skin blemishes).
In study 3, three participants (25%) experienced a total of nine TEAEs (Supplementary Table 7). No TEAE was considered by the investigator to be related to molidustat.
In all studies, all TEAEs were of mild or moderate intensity. There were no deaths or serious adverse events during these studies. Furthermore, no clinically relevant changes in clinical laboratory parameters, urinalysis, blood pressure, heart rate, or electrocardiogram parameters were observed in any of the studies (data not shown).

Discussion
Molidustat is a HIF-PH inhibitor currently in phase 3 of clinical development for the treatment of anaemia in patients with CKD. Its efficacy and safety profiles were evaluated in the DIALOGUE phase 2 clinical trial programme [10]. Given that common concomitant medication in patients with CKD includes iron and calcium supplementations for anaemia [5], the potential for DDI between molidustat and iron or calcium supplements was investigated. The results of these DDI studies demonstrate a substantial reduction in molidustat exposure and endogenous EPO production when iron(II) sulphate preparations (oral iron supplements) were administered orally immediately before molidustat both under fasted and fed conditions. The effect of iron supplementation on molidustat exposure was less pronounced with increasing temporal separation between the two administrations and was significantly reduced when molidustat and iron supplements were administered at least 1 h apart. Co-administration of calcium acetate reduced the rate of molidustat absorption, but molidustat exposure was only slightly affected (−15% reduction in AUC), and endogenous EPO level profiles were unchanged compared with molidustat alone. The similar EPO response observed with both treatments indicates that no relevant clinical interaction is occurring between molidustat and oral calcium supplementation (e.g. calcium acetate).
The decreased exposure of molidustat in the presence of oral iron supplementation may result from reduced absorption owing to chelation, as suggested by unpublished in vitro data that demonstrated that the solubility of molidustat is reduced in the presence of polyvalent cations. The results of coadministration of calcium(II) and molidustat indicate that oral calcium(II) reduces molidustat solubility and, therefore, its rate of absorption; however, the complex seems to dissolve fast enough not to affect the extent of molidustat absorption. In the presence of oral iron(II), the solubility of molidustat is considerably decreased, resulting in markedly reduced rate and extent of absorption when both drugs are administered at the same time. Furthermore, the molidustat terminal t ½ was prolonged, and the concentration-time profile appeared rather flat in the terminal phase, which was probably the result of flip-flop kinetics; this suggested that solubility and absorption were the rate-limiting steps for molidustat exposure in the presence of iron(II). The effect of intravenous iron supplementation was not investigated; however, no impact of  Fig. 3 Plasma concentrations of molidustat after administration of a single dose of molidustat 150 mg alone or together with a single dose of iron(II) sulphate 304 mg given either 1 h before, immediately before, or 1 h after molidustat to healthy fed volunteers in study 3 (geometric mean [SD]; PK set, N = 11) h hours, PK pharmacokinetics, SD standard deviation Table 3 Effect on molidustat PK parameters, expressed as difference in geometric mean, of co-administration of oral iron(II) supplementation (iron(II) sulphate or iron(II) glycine sulphate) or calcium acetate with molidustat in the fed or fasted state S t u d y #       intravenous iron on molidustat exposure is expected because the primary interaction between the two drugs occurs in the gut. Additionally, intravenous iron supplementation uses iron(III), which has less influence on molidustat solubility than iron(II), and free plasma concentrations are too low for precipitation.
Molidustat is reported to increase endogenous EPO levels dose-dependently (EPO C max was 39.8 IU/L and 14.8 IU/L in the molidustat 50 mg and placebo groups, respectively) [12]. The present data suggest that the impact of iron(II) coadministration on EPO exposure was less extensive than on molidustat pharmacokinetics (Supplementary Fig. 4). Therefore, the clinical relevance of this pharmacokinetic interaction will be fully explored once the phase 3 trials are complete.
The present data suggest possible strategies to limit the impact of oral iron supplements on molidustat exposure. Compared with concurrent dosing, the effect of iron supplementation on molidustat exposure was considerably reduced when iron supplements were administered either 1 h before or 1 h after molidustat. Indeed, there was almost no effect on molidustat exposure when iron supplementation was given 4 h before molidustat. Importantly, the effects on endogenous EPO increase were also less when administration of molidustat and iron supplements was separated in time.
The CIs of the ratios for iron either 1 h before or 1 h after molidustat vs molidustat alone for PK and PD were outside the 80-125% range (i.e. relative percentage changes between -20% and + 25%) recommended by the FDA and the EMA guidelines to claim no interaction [15,16]. However, based on previous data, molidustat was well tolerated over a wide dose range (5-200 mg) and is dosed in phase 2 and 3 based on individual response [10,11]. Consequently, it has been recommended that the intake of oral iron and molidustat should be separated by at least 1 h in the phase 3 clinical trials, which are currently ongoing.
Overall, molidustat alone or in combination with iron supplementation was well tolerated with few drug-related TEAEs. Of the six TEAEs related to molidustat administration, one occurred after molidustat alone, one after coadministration with calcium acetate, and four after coadministration with iron(II). Another two TEAEs were related to iron supplementation alone. No clinically relevant postdose changes from baseline were observed in clinical laboratory parameters, urinalysis, blood pressure, heart rate, or electrocardiogram parameters.
The internal control provided by the crossover design, the range of treatment regimens, and the overall number of participants represents strengths of these studies. Using settings that reflect real-world clinical practice and comparing PK and PD outcomes are also strengths. The presented set of studies provides detailed scenarios on how temporal separation of drug intake can reduce the interaction observed compared with concomitant intake; however, these results should be interpreted in the context of several limitations. The interpretation of clinical relevance on changes in EPO levels is limited because the efficacy on Hb response could not be investigated in this single-dose study and in general because the studies were conducted in a selected healthy male volunteer population. The single-dose study design does not offer insight into the PK/PD profile of molidustat during long-term treatment.

Conclusion
Although PK and PD (effect on EPO) parameters of molidustat in healthy volunteers were affected by oral administration of iron(II) sulphate or iron(II) glycine sulphate immediately before molidustat, these effects were reduced by temporal separation of drug intake. This is currently being investigated in ongoing phase 3 clinical trials [8,9]. Calcium supplement (investigated as calcium acetate) co-administration had no relevant effect on the pharmacokinetics or pharmacodynamics of molidustat.
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