This study was conducted in compliance with the Helsinki Declaration and approved by the Ethics Committee of Nagoya University Graduate School of Medicine. The study was registered with the University Hospital Medical Information Network clinical trials registry (UMIN000012503) before the start of the recruitment period.

This study is a randomized, double-blind, placebo-controlled, three-armed, phase II trial to assess the safety and efficacy of creatine monohydrate in patients with SBMA in accordance with the Consolidated Standards of Reporting Trials (CONSORT) guideline. After obtaining informed consent, a primary assessment (T^-1) of the potential participants was performed, whereby participants were screened for eligibility within 4 weeks prior to the start of the study medication by measuring their blood parameter values and serum testosterone levels. Patients with SBMA eligible for this study were allocated randomly in a 1:1:1 ratio to receive placebo, 10 g daily of creatine monohydrate (SAVAS; Meiji Co, Ltd, Tokyo, Japan), or 15 g daily of creatine in a double-blinded fashion. The duration of the intervention was 8 weeks. The study participants underwent four assessments (T⁰, T¹, T², and T³) composed of neurological assessments, examinations, and questionnaires (Figure 1 and Table 1). The study intervention was initiated after baseline assessment (T⁰). For the purpose of safety assessment and compliance, participants were assessed again after 4±1 treatment weeks (T¹). Efficacy assessments were scheduled after 8±1 treatment weeks (T²). Follow-up assessments were performed after the 8-week washout period (T³). All assessments were performed at Nagoya University Hospital.

Participants took creatine or placebo orally after every meal, 3 times a day, for 8 weeks. In the high-dose creatine group, participants took 5 g of creatine monohydrate powder daily after every meal (total 15 g). In the low-dose creatine group, participants took powder medicine containing 3.33 g of creatine monohydrate powder and 1.67 g of lactose daily after every meal (total 10 g). In the placebo group, powder medicine containing 5 g of lactose was taken daily after each meal (total 15 g). Participants started the oral administration within 3 days after the baseline assessment (T⁰). Participants were instructed to pay attention to the following points when taking the medicine: (i) To take powder medicine suspended in sufficient water or juice; (ii) take the powder medicine always after meals, and not before meals; (iii) avoid warm water to suspend powder medicine; and (iv) take all the medicine including the precipitated powder.

To avoid the influence of other treatments on the evaluation of the effectiveness of creatine, participants were prohibited to use the following medicines until the end of the evaluation period; luteinizing hormone-releasing hormone (LH-RH) agonists, LH-RH antagonists, testosterone drugs, 5-alpha-reductase inhibitors, anti-androgen drugs, protein anabolic hormone, progesterone drugs, estrogen drugs, unapproved drugs, and creatine supplements. Participants were also prohibited to start rehabilitation of extremities or undergo castration until the end of the evaluation.

Patients with SBMA were eligible for participating in the study. All participants were recruited from the Department of Neurology at Nagoya University according to the inclusion and exclusion criteria. The inclusion criteria were as follows: (1) Male patients who present one or more of the following motor symptoms: (i) muscle weakness of extremities, (ii) muscle atrophy of extremities, and (iii) bulbar palsy; (2) Patients whose genetic testing results showed that they bear at least 38 CAG repeats within the AR gene; (3) Patients who were twenty (≥ 20) to eighty (< 80) years of age at the time of informed consent; (4) Patients who can visit the hospital regularly as outpatients; (5) Patients whose renal function meets the following criteria: creatinine at < 1.5 × Upper Limit of Institutional Reference Value; (6) Patients who provided written informed consent by themselves.

Patients who met any of the following criteria were excluded from the study: (1) Patients who have taken LH-RH agonists, LH-RH antagonists, testosterone drugs, anti-androgen drugs, estrogen drugs, or unapproved drugs within 48 weeks and 5-alpha-reductase inhibitors within 24 weeks before agreement acquisition; (2) Patients whose serum testosterone level is below the lower limit of normal; (3) Patients who have taken creatine supplementation within 8 weeks before agreement acquisition; (4) Patients who have severe complications, such as malignancy, heart failure, and renal failure; (5) Patients who were determined ineligible for this study by the investigator or coinvestigators.

Neurologists, ie, investigator or coinvestigators, identified patients with SBMA and provided them with sufficient information about the explanatory statement and the informed consent form before their participation. All participants gave their written informed consent for trial participation prior to the screening. After the screening assessment for eligibility, neurologists evaluated the eligibility of the patients using a checklist and register patients for this trial.

Randomization was performed centrally with the use of an online system (Waritsuke-kun; Mebix, Inc, Tokyo, Japan). Dynamic random allocation was done with minimization on the basis of the patients’ disease duration (0–9 or ≥ 10 years from onset) and past history of LH-RH agonist treatment to reduce the bias. A double-blind study was conducted to achieve a higher standard of scientific rigor in evaluating the efficacy and safety of creatine. A biostatistician from an external facility (MG) was delegated to the “Allocator” who was responsible for treatment allocation. The Allocator ensured that it was impossible to determine whether the study agent was creatine monohydrate or placebo by its appearance or package and randomly allocated patients to the study agents in a repeatable manner. The Allocator ensured that the allocation code/list has been kept in a sealed envelope before opening it, and that the allocation has been concealed before unblinding. During the study period, serum creatine, serum creatinine, urinary creatine, and urinary creatinine were measured by a clinical laboratory measuring institution (LSI Medience Corporation, Tokyo, Japan) to maintain the blindness in this study. The results of the examination were kept at the clinical laboratory measuring institution without disclosure. After unblinding, the examination results of these items were reported to the investigator.

The primary endpoint of this trial was the change in handgrip strength values from baseline to week 8 (T²). Handgrip strength values were measured using an electronic hand dynamometer. For measurement, the patients were instructed to keep their elbows at an angle of 90°, their forearms in a neutral rotation, and their wrists not flexed or pronated. The grip power was measured twice on each side, and the average of the maximal power of both sides was recorded. Secondary outcome measures included muscle strength measured by tongue pressure [30], maximum voluntary isometric contraction (bilateral shoulder flexors and extensors, elbow flexors and extensors, knee flexors and extensors, and ankle flexors and extensors), five components of the Quantitative Myasthenia Gravis score (excluding the ptosis and diplopia sections) [31], 15-foot timed-walk test [32], and rise-from-bed test, which measures the time in changing the position from the supine position on the bed to the sitting position. We also measured changes in the Revised Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS-R) [7,33,34] and the Spinal and Bulbar Muscular Atrophy Functional Rating Scale, which is a validated, disease-specific scale with a high sensitivity to disease progression in SBMA [35]. We evaluated muscle mass with dual-energy X-ray absorptiometry (DXA) using the fan-beam technology (Discovery A; Hologic Inc, Bedford, MA). The sum of the appendicular lean soft tissue mass measured with DXA has been validated by the measurement of the skeletal muscle mass using magnetic resonance imaging and computed tomography [36-38]. As other outcome measures, we analyzed the subjective assessment of swallowing function (Swallowing Disturbance Questionnaire and Swallowing Quality of Life Questionnaire) [39,40], respiratory function values (vital capacity, forced vital capacity [FVC], forced expiratory volume one second percent, peak expiratory flow, and V50/V25), 5-item Amyotrophic Lateral Sclerosis Assessment Questionnaire [41], the Multidimensional Fatigue Inventory [42], and urinary 8-hydroxydeoxyguanosine (8-OHdG) as a marker of oxidative stress [43].

In this study, we intended to enroll 45 patients with SBMA. This was based on previous studies that examined the efficacy of creatine therapy for muscular dystrophy. In those placebo-controlled trials, intergroup differences of the change from baseline were 6.4 to 16.2% (SD 19.8 to 30.7%) in the maximum voluntary muscle strength [25,28,29,44]. The pharmacological mechanism of increasing intramuscular creatine concentration is common in SBMA and muscular dystrophy. In addition, in our preliminary examination, patients with SBMA displayed a lower intramuscular creatine concentration compared with disease controls including muscular dystrophy, suggesting that a larger creatine efficacy may be expected in SBMA. Based on the above, the number of required participants was calculated assuming that the intergroup difference of the change rates of the muscle strength is 28.0% and the SD is 20.0%.