Lucerastat, an oral therapy for Fabry disease: results from a pivotal randomized phase 3 study and its open-label extension

Domain: biology
Published: 2026-01-10
Editorial outcome: major_revision

Abstract
In Fabry disease (FD, OMIM #301500), a rare lysosomal storage disorder, the glucosylceramide synthase inhibitor lucerastat acts as substrate reduction therapy. The Phase 3, prospective, double-blind, placebo-controlled, 6-month, randomized clinical trial, MODIFY (NCT03425539), aimed to evaluate the efficacy, safety, and tolerability of lucerastat in adults with FD with moderate-to-severe neuropathic pain. The single-arm, open-label extension (OLE) (NCT03737214) evaluated the longer-term safety and tolerability of lucerastat over 72 months. Lucerastat 1000 mg twice daily (n = 80), compared with placebo (n = 37), failed to affect neuropathic pain at Month-6, with no significant difference between treatment groups (LSM difference –0.42 [95% CI –1.23, 0.40], p = 0.32) (primary endpoint). In contrast, a decrease in baseline plasma Gb3 was observed at Month-6 in lucerastat-treated participants but not placebo-treated participants (LSM difference –873.53 ng/mL [95% CI –1097.53, –649.53], p < 0.0001; NS due to hierarchical testing). In an unplanned OLE Month-18 interim analysis, the eGFR slope (mL/min/1.73m2/year) in 93 participants with pre- and post-randomization (23-month median lucerastat exposure) eGFR data was –3.50 (–5.04, –1.969) and –1.48 (–2.64, –0.33), respectively. Lucerastat was safe and well tolerated. Lucerastat’s strong pharmacodynamic effect did not translate into an effect on neuropathic pain. The potential effect of lucerastat on renal function requires further investigation.

Introduction
Fabry disease (FD; OMIM 301500) is a rare, genetic, X-linked lysosomal storage disorder caused by pathogenic variants in the GLA gene, which encodes the α-galactosidase A (α-GalA) enzyme1. Deficient α-GalA activity results in globotriaosylceramide (Gb3) accumulation in lysosomes within various cell types, leading to nervous system, gastrointestinal tract, skin, eye, renal, cerebrovascular, and cardiac manifestations, which can reduce quality of life and life expectancy1,2. Current treatment strategies for FD in adults target the underlying pathology of the disease (with enzyme replacement therapy [ERT] or pharmacological chaperone therapy), combined with adjunctive therapies to manage symptoms3.

In FD, neuropathic pain is among the first clinical symptoms (together with gastrointestinal symptoms) that interfere with well-being and daily activities. Neuropathic pain usually arises in childhood4 and is the most common (62% of male and 41% of female patients) and disabling clinical manifestation of FD5–7. It impacts patients’ QoL and is not adequately managed by conventional pain medications or FD-specific therapies2,8,9. Thus, it is an important target for new treatment options. Subsequently, progressive damage to vital organ systems occurs over several decades, resulting in end-stage renal disease and/or life-threatening cardiovascular or cerebrovascular complications, causing substantial morbidity and premature death10. Despite ERT, neuropathic pain and progressive eGFR loss are reported in adults with FD11. Hence, there is a need for more effective therapeutic approaches.

While neuropathic pain is typically assessed using a validated pain scale completed by the patient, kidney function is commonly assessed by estimated glomerular filtration rate (eGFR). In patients with FD, and, depending on the level of kidney involvement, major therapeutic goals are to stabilize or reduce eGFR decline and avoid or delay the progression of chronic kidney disease to end-stage renal disease3.

Lucerastat is an oral glucosylceramide synthase inhibitor under investigation as a substrate reduction therapy (SRT) for the treatment of FD, the principle of which is to reduce Gb3 synthesis and consequently prevent further Gb3 accumulation12–14. Results of preclinical and clinical studies showed that lucerastat reduced Gb3 levels in kidneys and dorsal root ganglions of Fabry mice15 and in cultured fibroblasts from individuals with FD regardless of the GLA variant13, was effective for reducing plasma Gb3 in patients with FD on top of ERT12, and was well tolerated in healthy individuals14,16, individuals with renal impairment (including participants with eGFR <30 mL/min/1.73m2)17, and individuals with FD12. With its oral administration, GLA variant-independent mechanism of action, and tolerability in renally impaired individuals, lucerastat has the potential to address some of the unmet needs associated with current therapy options.

MODIFY (NCT03425539), conducted from 8 May 2018 to 2 September 2021, was a 6-month, randomized, double-blind, placebo-controlled Phase 3 study to determine the clinical efficacy of lucerastat monotherapy and evaluate its safety and tolerability in adults with FD; its open-label extension (OLE) study (NCT03737214), aims at determining the long-term safety and tolerability of lucerastat for up to 72 months18. The primary objective of MODIFY was to evaluate the effect of lucerastat on neuropathic pain. Secondary objectives included the effects of lucerastat on gastrointestinal symptoms, FD biomarkers, and safety and tolerability. Other prespecified efficacy endpoints included the effect of lucerastat on renal function and cardiac parameters. Here, we report the results from MODIFY and an unplanned interim analysis of the combined MODIFY/OLE when ongoing OLE participants had received lucerastat for ≥12 months.

Results
Patient population and baseline characteristics
Overall, 182 participants were screened, and 118 were randomized at 49 sites in 14 countries covering North America, Europe, and Australia (Fig. 1). The most common reason for pre-randomization exclusion was not meeting the inclusion criterion of moderate-to-severe neuropathic pain (24% of screened participants). Of the 118 randomized participants, 117 received at least one dose of study treatment, and 109 (92%) completed the 6-month MODIFY study treatment; 107 (91%) enrolled in the OLE, and 78 (67%) were ongoing at the 18-month interim analysis (Fig. 1). Participants’ characteristics were similar across treatment groups at randomization (Table 1). Around half of the participants were male (47%), and the mean (standard deviation [SD]) age at screening was 39.3 (14.1) years (Table 1). More than 80% of participants had the classic FD subtype, and 80% of the 98 participants tested had pathogenic variants non-amenable to treatment with migalastat; genotypes are shown in Supplemental Table S1.

At baseline, 45 (38%) participants switched from ERT, while 72 (62%) were ERT-naïve. Forty (34%) participants were treated with an ACE inhibitor or ARB, and 92 (79%) were treated with at least one pain medication (Table 1).

Demographic and baseline characteristics for participants with versus without renal impairment are shown in Supplemental Table S2.

The eDiary was adequately completed by 112/118 participants (95%). Overall, the mean (SD) baseline modified Brief Pain Inventory short form (BPI-SF3) score was 6.21 (1.49) (Table 1). Supplemental Table S3 provides more details on neuropathic pain characteristics at baseline.

Three participants on lucerastat (4%) and two on placebo (5%) initiated ERT during MODIFY (all agalsidase beta).

At the interim analysis, the median (Q1, Q3) duration of lucerastat exposure was 23.0 (17.1, 32.8) months for the 114 participants who received at least one dose of lucerastat (in MODIFY and/or OLE), representing 230.4 patient-years of exposure. For most (n = 93) of these participants, pre-randomization eGFR data were available (Supplemental Table S4).

MODIFY: primary and secondary endpoints
Neuropathic pain (primary endpoint), assessed by the modified BPI-SF3 score, was reduced from baseline to Month-6 in both treatment groups (lucerastat and placebo), with a numerically smaller, albeit not statistically significant, reduction with lucerastat versus placebo (least squares mean [LSM] difference −0.42 [95% confidence interval (CI) −1.23, 0.40]) (Fig. 2A).

On secondary endpoints, there was a reduction in plasma Gb3 in the lucerastat group (LSM of the change from baseline to Month-6 −672.68 ng/mL) compared with an increase in the placebo group (200.84 ng/mL), and a relevant treatment effect was detected (LSM treatment difference in favor of lucerastat versus placebo: −873.53 ng/mL; 95% CI: −1097.53, −649.53; p < 0.0001) (Figs. 2B and 3A). However, this difference cannot be considered statistically significant due to the hierarchical testing strategy. Lucerastat did not reduce abdominal pain (LSM difference 0.31 [95% CI –0.53, 1.16], p = 0.47; Fig. 2C) nor diarrhea (win ratio 1.00 [95% CI 0.57, 1.89], p = 0.90; Fig. 2D) versus placebo.

MODIFY: other prespecified efficacy endpoints and post hoc analyses in subgroups
Renal function endpoints. The LSM of the eGFR slope (mL/min/1.73m2 per month) was +0.26 in the lucerastat group versus +0.08 in the placebo group, corresponding to a change of approximately +3.12 versus +0.96 mL/min/1.73m2 per year, respectively. The LSM treatment difference was not statistically significant (LSM = 0.19; 95% CI: –0.40, 0.78; p = 0.5313).

The UACR change from baseline was not normally distributed. This yielded a LSM percentage change difference of +16.2% (95% CI: −23.9, 77.3; p = 0.4875), indicating no difference between treatment groups.

In a post-hoc analysis by sex, no difference in mean eGFR change from baseline between treatment groups was observed, with a slight increase in males and no significant change from baseline in female participants (Supplemental Fig. S1).

In a post-hoc subgroup analysis of participants with impaired renal function at baseline (i.e., screening eGFR <90 mL/min/1.73m2; n = 40), change from baseline to Month-6 in mean eGFR was +3.8 mL/min/1.73m2 in the lucerastat group (n = 27), whereas in the placebo group (n = 13) the change was –1.6 mL/min/1.73m2 (Supplemental Fig. S2A). There was no observation of note in participants without renal impairment at baseline (n = 77) (Supplemental Fig. S2B).

Cardiac endpoints. Values of the echocardiography-based endpoints remained stable from baseline to Month-6.

Rescue pain therapy. The median number of days on significant rescue pain therapy was 0 in both lucerastat and placebo treatment groups, while the mean number of days on significant rescue pain therapy from baseline to Month-6 was numerically smaller with lucerastat than placebo (4.9 days versus 9.5 days, respectively) (Supplemental Table S5).

Fabry disease biomarkers. Plasma lysoGb3 remained stable in the lucerastat group, with an LSM percentage change from baseline to Month-6 of 1.05% (Fig. 3B). In comparison, the LSM percentage change was 26.82% in the placebo group (LSM difference versus placebo = −20.32%, 95% CI: −34.24, −3.45). This translates into differences between lucerastat and placebo for plasma lysoGb3 (LSM difference at Month-6 −5.88 ng/mL [95% CI −11.62, −0.13]). Urinary Gb3 had an LSM percentage change from baseline to Month-6 of −44.64% in the lucerastat group, while the change for placebo was +47.80% (Fig. 3C). This translates into LSM differences between lucerastat and placebo at 6 months of 8.32 µmol Gb3/mol creatinine [95% CI −11.60, −5.04]. Both differences were nominally statistically significant.

In post-hoc analyses by baseline ERT status, the effect of lucerastat versus placebo on plasma Gb3 observed in the overall population was maintained in both ERT-naïve participants and those switching from ERT; a similar effect was observed on urine Gb3 regardless of baseline ERT status (Supplemental Fig. S3A and S3C). An increase in plasma lysoGb3 was observed in patients switching from ERT at baseline to placebo, but not in patients switching to lucerastat at baseline; no between-group difference was observed in patients who were ERT-naïve at baseline (Supplemental Fig. S3B). The results for plasma Gb3, plasma lysoGb3, and urine Gb3 split by ERT status at baseline and sex are shown in Supplemental Fig. S4A–F.

OPEN-LABEL EXTENSION: interim analysis
The effect of lucerastat observed on plasma Gb3 during MODIFY was maintained over time during the OLE. Patients who switched from placebo to lucerastat at Month-6 responded similarly to those randomized to lucerastat in MODIFY (Fig. 3A–C). The results for plasma Gb3, plasma lysoGb3, and urine Gb3 split by ERT status at baseline and sex are shown in Supplemental Fig. S4A–F.

In the overall population (n = 93), the LSM annualized eGFR slope pre-randomization was −3.50 (95% CI −5.04, −1.96) mL/min/1.73m2 and −1.48 (95% CI −2.64, −0.33) mL/min/1.73m2 post-randomization (Fig. 4). Similar patterns of a lesser decline after randomization were observed by sex, by amenability to migalastat, in ERT-naïve participants, in participants with renal impairment, and in participants who were classified as deteriorating/fast deteriorating (decline of more than 3 mL/min/1.73m2 per year) pre-randomization: in participants with renal impairment, the LSM annualized eGFR slope was −6.18 [95% CI −8.68, −3.69] pre-randomization and −1.88 [95% CI –4.03, 0.26] mL/min/1.73m2) post-randomization, while in participants who were deteriorating/fast deteriorating with an eGFR slope of −9.77 [95% CI −15.42, −4.12] mL/min/1.73m2 pre-randomization, an eGFR slope of −1.16 [95% CI −6.76, 4.44] mL/min/1.73m2 was observed post-randomization on lucerastat (Fig. 4). Of note, pre- and post-randomization eGFR slopes were similar for participants without renal impairment who were on ACE inhibitors/ARB at baseline or who switched from ERT (Fig. 4). Overall, 50 participants (54%) had stable eGFR slope (defined as a decline of less than 3 mL/min/1.73m2/year) prior to randomization, and 70 (75%) while on lucerastat. Twenty-one participants (23%) were fast deteriorating (defined as a decline of at least 5 mL/min/1.73m2/year) pre-randomization, only 10 (11%) while on lucerastat (Table 2 and Fig. 5). Changes from baseline in eGFR over time in MODIFY and OLE overall and split by sex, ERT status at baseline, and eGFR at baseline are shown in Supplemental Fig. S5A–D.

Cardiac endpoints. In participants with normal left ventricular mass at baseline (n = 70), the average LVMi was maintained in a normal range over the duration of the OLE. In participants with left ventricular hypertrophy (n = 26), no further increases in LVMi were noted during the trial (Supplemental Fig. S6).

Safety outcomes. In MODIFY, the prevalence of AEs was similar in the treatment groups (Table 3). The most commonly reported treatment-emergent AEs (TEAEs) with a higher frequency with lucerastat than placebo were nausea and diarrhea. TEAEs reported with a higher frequency with placebo than lucerastat were abdominal distension and decreased GFR. Headache and nasopharyngitis were reported by ≥10% of participants in both groups. AEs leading to the premature discontinuation of study treatment were reported in two participants in each group, including gastroenteritis (n = 1) and vomiting (n = 1) in the lucerastat group. In the placebo group, one participant discontinued due to abdominal distension, fatigue, muscle spasms, flushing, gastrointestinal pain, and headache, while another discontinued due to chest pain, pain, fatigue, palpitations, tachycardia, nausea, and tinnitus. SAEs were reported for five participants (6%) in the lucerastat group and one in the placebo group (3%). No SAE was assessed as related to the study treatment or reported by more than one participant (Supplemental Table S6). No deaths were reported. Analyses of vital signs, physical examination findings, clinical laboratory measurements, and electrocardiographic results did not reveal any clinically relevant effect of lucerastat.

Table 4 shows an overview of AEs occurring or worsening during lucerastat treatment in MODIFY and OLE. The most commonly reported TEAEs were COVID-19, neuralgia, and headache. SAEs reported in more than one participant included atrial fibrillation, transient ischemic attack, and drug withdrawal syndrome (two participants each). There was no sign of inflation of SAEs over time and no pattern in SAEs was observed. There were no deaths and no serious TEAEs related to study treatment during MODIFY or OLE.

Methods
Study design
Protocols and materials for the studies were approved by Independent Ethics Committees or Institutional Review Boards (Supplemental Table S7). The studies were conducted in compliance with the International Council for Harmonization Good Clinical Practice guidelines, the principles of the Declaration of Helsinki, and with the laws and regulations of the countries in which they were performed. Participants provided written informed consent before entry into MODIFY and the OLE, and again at Months 24 and 48 of the OLE.

MODIFY was a multicenter, double-blind, randomized, placebo-controlled, parallel-group Phase 3 study to assess the safety and efficacy of lucerastat oral monotherapy in adults with FD. The study was conducted in 49 hospitals in 14 countries in Europe, North America, and Australia. The study comprised a screening period (6–7 weeks) and a double-blind treatment period (6 months) followed by either a safety follow-up (1–3 months) or a multicenter, single-arm OLE study. In the ongoing OLE study, participants receive lucerastat for up to 72 months. Interim analysis of the OLE study was performed when all ongoing participants completed 12 months of open-label treatment, for a total of at least 12 months for those participants originally randomized to placebo and up to at least 18 months, depending on the treatment arm, for those participants originally randomized to lucerastat.

Substantial protocol amendments included a change from a dichotomous-responder based analysis of the primary endpoint to a continuous analysis (agreed with the FDA to overcome recruitment challenges due to the COVID-19 pandemic)18, and extension of the OLE from 24 to 72 months in light of the favorable treatment effect on plasma Gb3 and safety profile. Furthermore, the statistical section of the OLE was revised to analyze OLE data in combination with that from MODIFY rather than analyzing OLE data separately.

As the OLE is an open-label, uncontrolled, single-arm extension study to determine the long-term safety and tolerability of oral lucerastat in adult participants with FD, an interim analysis was not prespecified in the OLE protocol. However, standard operating procedures do not preclude interim analysis of open-label uncontrolled long-term trials. Interim reporting is generally accepted and judged necessary by Independent Ethics Committees and Institutional Review Boards to reassess at regular intervals the benefit/risk of an investigational drug given long-term to patients and to publish the results if important signals are observed. Indeed, not performing this analysis might seem unfit in a long-term open-label study, which is positioned to identify the long-term safety, tolerability, and potential signs of prolongation of efficacy signals observed in a placebo-controlled study of a novel molecule. While the interim analysis results of the OLE reported here were added to the Investigator Brochure in February 2023, submitted, and approved by all Independent Ethics Committees and Institutional Review Boards, their dissemination through publication does not need Institutional Review Board review.

Protocols and statistical analysis plans for each study are available (see Supplementary Appendix). Of note, the statistical testing strategy was introduced in Global Protocol Version 4 as a result of FDA requests and advice. The order of the secondary endpoints in the statistical testing strategy (plasma Gb3, abdominal pain, stool consistency) is not identical to the order of the secondary endpoints defined in Global Protocol Version 1 (abdominal pain, stool consistency, plasma Gb3). The reason for testing plasma Gb3 before the GI symptom endpoints is that plasma Gb3 is assessed in the full population (mFAS), whilst the GI symptom endpoints are assessed only on a subset of participants with GI symptoms at baseline (mFAS-GIS).

Key inclusion criteria for MODIFY were previously described18. Eligible participants were ≥18 years old with a genetically confirmed FD diagnosis. Participants had to report moderate-to-severe neuropathic pain (defined as an average score of ≥4 on the 11-point numerical rating scale [NRS] Modified Brief Pain Inventory short form [BPI-SF3] over the 4 weeks prior to randomization) during the screening period. Eligible participants were either “naïve” (had never received ERT or had not received it for at least 6 months) or “ERT switch” (had been treated with ERT for at least 12 months and agreed to stop ERT at screening).

Participants with other diseases or conditions associated with pain components that could have confounded the assessment of neuropathic pain were excluded. Participants with eGFR <30 mL/min/1.73m2 (calculated using the Chronic Kidney Disease Epidemiology Collaboration 4 variable equation)29, with any major cardiovascular, kidney, or cerebrovascular medical conditions, or at risk of developing those conditions during the study, were ineligible for MODIFY; those who developed any major cardiovascular, kidney, or cerebrovascular medical conditions during MODIFY, or were at risk of developing them, were ineligible for the OLE study. After randomization in MODIFY, participants had to discontinue study treatment if eGFR was <15 mL/min/1.73m2. Hence, participants with an eGFR >15 mL/min/1.73m2 and <30 mL/min/1.73m2 at the end of MODIFY could still be eligible to enroll in the OLE. Participants taking angiotensin-converting enzyme (ACE) inhibitors and/or angiotensin II receptor blockers (ARBs) had to be on a stable dose for ≥4 weeks before the screening visit. During the trials, initiation or dose adjustments were permitted based on the investigator’s judgment.

Participants could only be reimbursed for expenses incurred in the context of their participation in the trials (e.g., reasonable travel expenses, hotel stay, and, if accepted under local law, loss of earnings).

Randomization and masking
Participants were randomized (2:1) to lucerastat or placebo. Randomization was stratified by sex and ERT status at screening (naïve or ERT switch), and treatment was allocated using a web-based interactive response technology system. Study treatment was provided as identical capsules of 250 mg lucerastat or placebo, and all treatment kits were packaged in the same way. Participants, investigators, site personnel, and sponsor personnel involved in the study conduct were unaware of study treatment allocation. In the event of a medical emergency, investigators were permitted to initiate the unmasking process; no unmasking events occurred in the MODIFY study.

Procedures
Lucerastat and placebo capsules were administered orally twice daily. The starting dose of 1000 mg was adjusted based on eGFR values as previously described17,18. Participants receiving placebo during MODIFY were switched to lucerastat at the beginning of the OLE. The dose was adjusted if a participant’s eGFR crossed boundary thresholds (Supplemental Table S8).

An Individual Pain Management Plan (IPMP) was provided to each participant by the investigator/delegate with customized instructions regarding the use of pain medications during the study. Use of adjuvant pain medication and non-opioid and opioid analgesics was allowed in both studies if medically required, as well as rescue medication in case of unbearable pain. In MODIFY, significant rescue pain therapy was defined as initiation or dose escalation of anti-epileptics, anti-depressants, or opioid analgesic drugs for neuropathic pain, as recorded in the eDiary. Participants were allowed to (re-)initiate and then continue ERT in MODIFY and/or in the OLE study if medically required due to significant FD progression.

Site visits in MODIFY were scheduled at Months 1, 3, 5, and 6 or end of treatment (EOT), with phone calls at Months 2 and 4. Participants who did not enter the OLE were followed up by phone call approximately 1 month after the last dose of study treatment; fertile male participants had a second call 3 months after the last dose of study treatment. In the OLE, visits were scheduled at Months 1, 2, 3, and then every 3 months until EOT, with alternate visits being phone calls.

Key measurements and assessments in MODIFY have been previously described18 and are listed in Supplemental Table S9. All patient-reported outcome assessments, collected daily (MODIFY only) or during site visits (MODIFY and OLE), were recorded in an eDiary. The modified BPI-SF3, NRS-11 for abdominal pain, and Bristol Stool Scale (BSS) (see Supplemental Table S10) (which were recorded daily) were validated and agreed with the FDA18.

Plasma and urine samples for the analysis of the biomarkers plasma Gb3, plasma globotriaosylsphingosine (lysoGb3), and urine Gb3 were collected pre-dose at all scheduled visits in MODIFY and the OLE (Supplemental Table S9). Laboratory measurements in MODIFY and its OLE were analyzed centrally using validated liquid chromatography with tandem mass spectrometry assays. Urine Gb3 was normalized by dividing by the independently determined creatinine concentration. Additionally, participants’ serum creatinine values measured within 2 years prior to randomization in MODIFY (e.g., 2 values per year, 6 months apart from each other, irrespective of why serum creatinine was measured) were collected. Serum creatinine values were used to calculate the eGFR using the Chronic Kidney Disease Epidemiology Collaboration equation29. Echocardiography measurements in both studies were assessed through blinded, centralized evaluation.

Safety data (including AEs and serious AEs [SAEs]) were collected throughout the trial from screening in MODIFY up to 30 days after EOT (in MODIFY or OLE, as applicable).

All data were stored in a database.

Outcomes
Supplemental Table S11 lists all study endpoints for MODIFY and its OLE. In MODIFY, the primary endpoint was the change from baseline to Month-6 in neuropathic pain (measured using the modified BPI-SF3 score of “neuropathic pain at its worst in the last 24 h”, range 0–10)18. The secondary endpoints were change from baseline to Month-6 in plasma Gb3, abdominal pain (measured using the NRS-11 score of “abdominal pain at its worst in the last 24 h”, range 0–10), and number of days with diarrhea (measured using the BSS)18. Other prespecified efficacy endpoints included change from baseline to Month-6 in plasma lysoGb3, urine Gb3, and eGFR slope, and use of rescue pain therapy from baseline to Month-618.

In the OLE, the main efficacy endpoints were the annualized eGFR slope and changes from MODIFY baseline in plasma Gb3, plasma lysoGb3, urine Gb3, and left ventricular mass index (LVMi).

AEs were coded using the Medical Dictionary for Regulatory Activities (version 24.0).

Statistical analysis
Assuming a within-group standard deviation of 3 points, a two-sided type I error of 5%, and a 2:1 randomization ratio, a sample size of 99 participants would provide 87% power to detect a treatment difference of 2 points between lucerastat and placebo for the primary endpoint of MODIFY. These assumptions are based on data from patients on ERT and placebo in previous studies25,30.

A fixed-sequence statistical testing strategy was pre-specified to test the four null hypotheses of MODIFY, starting with the primary endpoint and continuing with the secondary endpoints (in the order of plasma Gb3, abdominal pain, and days with diarrhea). If a hypothesis in the sequence was not rejected, p-values for the subsequent hypotheses were to be considered exploratory.

In MODIFY, efficacy analyses included all randomized participants who took at least one dose of study treatment. Secondary gastrointestinal endpoints were analyzed on the subset of participants experiencing moderate-to-severe abdominal pain and/or diarrhea during the 4 weeks before randomization. Safety data were analyzed in all participants who received at least one dose of study treatment.

Analysis of covariance (ANCOVA) with a factor for treatment group and adjusting for the baseline value and the two stratification factors was used to analyze the primary endpoint and secondary endpoints related to plasma Gb3 and abdominal pain. Missing data were imputed by applying multiple imputation approaches, assuming data are missing at random in the placebo arm but missing not at random in the lucerastat arm (primary endpoint and abdominal pain) or missing at random in both arms (plasma Gb3). The secondary endpoint related to diarrhea was analyzed using rank ANCOVA and win ratio. Sensitivity analyses were conducted to assess the robustness of inferences from the main analyses to deviations from its underlying modeling assumptions regarding missing data.

Subgroup analyses were prespecified for the primary and secondary efficacy endpoints for, among others, subgroups based on sex, ERT-treatment status (naïve versus switch), and baseline eGFR. The latter subgroup was initially defined as eGFR <60 versus ≥60 mL/min/1.73m2, but due to the low number of participants in the former category (Table 1), this was redefined as eGFR <90 versus ≥90 mL/min/1.73m2. Subgroup analyses were also performed for other efficacy endpoints, but these were not prespecified and should be interpreted as exploratory analyses. The limitations of post-hoc subgroup analyses are acknowledged31,32.

An unplanned Month-18 interim analysis of the combined MODIFY/OLE was conducted. Pre-randomization and lucerastat (excluding placebo data from MODIFY) annualized eGFR slopes were estimated using linear mixed models, including time (in years) as a fixed effect and a random intercept and random slope effect. Participants with at least two eGFR values to derive a pre-randomization and lucerastat annualized eGFR slope were included in these analyses. Participants were classified as stable (annualized eGFR slope decline of less than 3 mL/min/1.73m2), deteriorating (annualized eGFR slope decline between 3 and 5 mL/min/1.73m2), or fast deteriorating (annualized eGFR slope decline of more than 5 mL/min/1.73m2) based on their pre-randomization and lucerastat eGFR slope3. Annualized eGFR slopes within subgroups of interest were derived by adding the respective subgroup as a main fixed effect as well as its interaction with time to the linear mixed model described above. All available lucerastat eGFR data (also beyond Month 18) in MODIFY/OLE, including early post-baseline values, were used for the eGFR slope analyses, which assumes no acute hemodynamic effect of lucerastat. Annualized eGFR slopes were reported as least squares mean (LSM).

Descriptive analyses of changes from baseline up to Month 18 in biomarkers, echocardiography, and pain-related variables were performed overall and within subgroups of interest.

For participants who (re-)started ERT during MODIFY or OLE, a treatment policy strategy was applied, i.e., all data after (re-)starting ERT were included in the analyses.

SAS® software version 9.4 was used for statistical analysis and reporting of clinical data. An Independent Data-Monitoring Committee (Supplemental Table S12) was established in MODIFY to monitor safety and efficacy.

MODIFY and its OLE are listed on clinicaltrials.gov (NCT03425539 Date of registration: 07-Feb-2018, https://www.clinicaltrials.gov/study/NCT03425539; NCT03737214 Date of registration: 09-Nov-2018, https://clinicaltrials.gov/study/NCT03737214) and EudraCT (2017-003369-85 Date of registration [first country, GB]: 16-Apr-2018, https://www.clinicaltrialsregister.eu/ctr-search/trial/2017-003369-85/GB/; 2018-002210-12 Date of registration [first country, GB]: 23-Oct-2018, https://www.clinicaltrialsregister.eu/ctr-search/trial/2018-002210-12/GB/). Of note, the trials are registered in EudraCT by the competent authority, i.e., these dates do not refer to the date of submission, but to the date posted on the website. MODIFY was registered prior to patient enrollment, and patient enrollment is complete.

Patient and public involvement
A patient survey was conducted via patient organizations (Fabry International Network, Fabry Support & Information Group, National Fabry Disease Foundation) to better understand symptoms of FD and their impact on individuals’ quality of life, as well as to understand perceptions of existing FD treatments. The results were used to inform the design of the Phase 3 study2. The primary and secondary patient-reported outcomes of the present study (neuropathic pain, abdominal pain, and diarrhea) were developed in collaboration with individuals with FD via a concept elicitation and cognitive debrief study18. A plain-language summary is planned to aid dissemination.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability
In addition to Idorsia’s existing clinical trial disclosure activities, the company is committed to implementing the Principles for Responsible Clinical Trial Data Sharing jointly issued by the European Federation of Pharmaceutical Industries and Associations (EFPIA) and the Pharmaceutical Research and Manufacturers of America (PhRMA). The trial protocol and statistical analysis plan are available with this publication (Supplemental Material Appendix A─D). Source data of results included in this article are provided with this paper. After research completion, data will be shared for replication and verification processes with qualified researchers that request access to trial data by submitting a research proposal to the trial sponsor, Idorsia Pharmaceuticals Ltd. For more information and to submit research proposals, contact clinical-trials-disclosure@idorsia.com.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript evaluates the effects of lucerastat, a substrate reduction therapy, in patients with Fabry disease. The\nprimary objective was to determine the effect of lucerastat on neuropathic pain, with secondary objectives assessing\ngastrointestinal symptoms, FD biomarkers, and safety/tolerability. The results indicate that lucerastat failed to improve\nneuropathic pain, abdominal pain, or diarrhea at six months. However, in an interim analysis of the open-label\nextension, lucerastat showed promising effects in improving eGFR in participants with reduced baseline kidney function.\nThe finding of an improvement in eGFR with lucerastat is particularly interesting and warrants further investigation. Below\nare my comments and suggestions to enhance the quality and impact of the manuscript:\n1. Fabry disease is a progressive disorder. What is the rationale for discontinuing effective and approved treatments, such as\nenzyme replacement therapy (ERT), in this study? Were there measures to ensure disease progression did not compromise\npatient outcomes during the trial? Clarification of the ethical and clinical justifications is necessary.\n2. The manuscript reports that mean eGFR increased from baseline to Month 6 in both the lucerastat and placebo groups.\nHow do the authors explain this improvement, particularly in the placebo group?\n3. A significant proportion of participants had the classic subtype of Fabry disease, yet the number of patients with cardiac\ninvolvement appears limited. This could significantly restrict the evaluation of left ventricular mass index (LVMi). Do the\nauthors believe this limitation influenced their ability to detect changes in cardiac outcomes?\n4. Fabry disease typically manifests renal involvement in approximately 80% of patients with the classic phenotype around\nthe age of 40. However, Table 1 indicates a relatively low median UACR value. Could the authors provide an explanation\nfor this finding? Was it influenced by study inclusion criteria or prior treatments?\n5. Due to mosaicism, disease manifestations can differ significantly between male and female patients. Were there any\nnotable differences in the effect of lucerastat on eGFR between genders?\n6. Fabry disease is a slow-progressing condition, and six months might not be sufficient to demonstrate the full therapeutic\npotential of lucerastat. What are the authors' thoughts on this limitation?\n7. Given the significant renal findings, it may be beneficial to revise the title to better reflect the study’s impact on kidney\noutcomes.", "reviewer_2": "The manuscript by Nordbeck et al. reports on a phase 3 RCT of lucerastat along with an open label extension (OLE) study\nfor the treatment of Fabry Disease. The main study showed no benefit of lucerastat, a substrate reduction therapy, compared\nwith placebo on the primary outcome of neuropathic pain reduction. Although the primary outcome was negative, there were\nsome unexpected secondary findings of improvement or stabilization in kidney function with lucerastat, particularly in those\nwith evidence of more rapid progression prior to study entry. The study appears to have been well conducted, reported and\nadequately powered with regards to the main outcome. The limitations described are appropriate. This is a multicentric and\nlarge study for a rare disease, which provides important insight on the use of substrate reduction therapy for clinically\nrelevant outcomes. It provides valid information on the primary outcome of neuropathic pain. There are some issues that\nshould be addressed by the authors:\n1. Due to the COVID pandemic several amendments were made, including a switch of the primary outcome from a binary to\ncontinuous measure for analysis, and combination of the OLE part of the study with the main trial for some of the analysis.\nThe OLE was also lengthened from 24 to 72 months. When the primary outcome was negative, there was very good\nparticipation in the extension study. Were participants informed of this result and allowed or informed that they could\nresume their enzyme replacement therapy during the OLE? Would this not have been considered standard of care?\n2. The improvement of GFR is quite interesting. It would be helpful for the authors to propose a biologic mechanism to\nunderstand this effect. As the median baseline eGFR was quite preserved, are the authors suggesting that lucerastat had a\nfavourable (disease-modifying) effect on the kidneys as early as 2-3 months. Could this be a hemodynamic effect or\nhyperfiltration? Did any pre-clinical or earlier phase studies show similar effects? Was there a washout period after the main\nstudy or after the OLE where eGFR was measured again to assess if the GFR improvement was sustained?\n3. If lucerastat is proposed to have a renoprotective benefit, was there any impact on albuminuria throughout the study?\n4. The authors use the pre-study eGFR decline to understand better those that had a renal benefit with lucerastat.\nAlthough this is interesting, it is a measure that was not in the original study protocol, and was not collected systematically. The\nauthors do show in the supplement that the majority had at least 3-5 creatinine values over time, but it is unclear if these\nrepresent values when a patient was stable or if they were done for some cause. This raises the concern for assessment or\ndetection bias. I would recommend that the description of eGFR decline as compared to placebo be compared only after\nstudy randomization.\n5. It appears that only a few participants were not included in the OLE. Were any placebo treated patients excluded due to\nthe eGFR <30 criteria? If so, removing these individuals may result in the appearance of more stability of mean eGFR in the\nOLE cohort.\n6. An improvement in eGFR could be related to a reduction in NSAID use. Although there was no reduction in pain, was\nthere any reduction in NSAID use?", "reviewer_3": "It is an important manuscript about the treatment of Fabry Disease with Lucerastate.\nHow, some suggest and revision should be done.\nPage3 line 73.\nIn this part, the authors should not mention about the ERT disadvantage,\nSome bias could be noted,\nIf they want to do this, they should put the important part on the discussion for detailed comparison of different treatment\nstrategies such as ERT and\nLucerastat.\nOn page 4, line 77\nOn the section of introduction, mostly mentioned about the kidney function, but they did not mentioned about the heart\nfunction, brain function, etc.\nHowever, if only focus on the kidney function, should we say the heart function is not important?\nSo the manuscript must be more fair for all the important vital organ function statement.\nOn the page 4, line 101, the authors mention about the “surprising effect of lucerastat on kidney function.”\nThis result should be more neutral in the introduction part.\nSuch as “ in this study, we try to see the kidney function after lucerastat.\nOn page 5, line 127\n“with a numerically smaller reduction with lucerastat versus placebo (LSM\ndifference 0.42 [95% confidence interval (CI) -0.40 to 1.23], p=0.32; Figure 2).”\nThis should add on without statistically significant.\nOn page 5, line 127\n“The median number of days on significant rescue pain therapy was 0 in both treatment groups, “\nOn Table S3, “Days on significant rescue pain therapy” on both treatment groups and placebo all showed 0\nBut in this sentence, the result just showed 0 on both treatment group.\nThe authors should clearly showed the both group is MODY and placebo? MODY and OLE?? MODY/OLE and placebo\nHowever is there statistical significance noted relative shorter mean days on the Lucerastat group should be calculated and\nshowed the results.\nIn addition, the pain characeterisitic change? The number of patients with pain-control medication, or other important\nparameters for pain should be mentioned to see the pain change after Lucerastat group,\nBy the way, the S3 just mentioned about the MODIFY study( for 6 months) , but the OLE (extended study) should also be\npresented.\nOn page 5 Line 1333, “LSM difference”\nSome showed “LSM” , some showed “LS mean” , the manuscript should be corrected carefully.\nOn page 6, line 137, the study showed “ in both treatment groups”,\nThe authors should clearly say the both groups meaning. The MODY vs Placebo”?,\nThe MODY vs OLE( extended study)?. In addition, the result of MODY and OLE ( extended group) should be presented here,\nnot just MODY and placebo.\nOn page 6, line 140, the statement: “mean eGFR increased from baseline to Month 6 by 3.8 (SEM 2.2) mL/min/1.73m2””\nIn the result, the authors showed the increased GFR per year, but totally, the decreasing GFR/year was noted ( page 6, line\n143),\nIs this mean that the normal GFR patient received Lucerastat showed a rapid decreasing GFR???\nIn this short period, how many years be calculaged.\nThe figures of MODY/OLE/Placebo patients with GFR < 90 , and normal GFR should be presented.\nBy the way, some readers might think the GFR effect is due to other drug effects such as ACEI, so the comparison with\nplacebo should be important.\nIn addition, the Fabry disease showed an X-linked disease. So the results of renal function in male and female groups\nshould be presented separately.\nWe also very interested in the different improvements in male and female groups.\nOn page 6, line 148, some showed LSM, and some showed LS mean, please correct it.\nOn page 6 , line 153, the Figure 4, should also showed the results of lucerastat and placebo group.\nON page 6, line 162, the statement “ lucerastat treatment stopped the eGFR decline. In these patients”\nWith the results, the authors should showed the results of all the GFR decline in GFR >90, GFR<90, MODY group, OLE\ngroup, placebo group.\nIn addition, results in male and female groups are also important.\nOn page 6, line 162, “The LS mean” should be corrected.\nOn page 7, line 181, the authors showed the decreased plasma Gb3,…;\nThe results in MODY and OLE should be presented.\nIt is interesting in elevated the plasma GB3 in female. So the male data and female data should be presented.\nOn page 8, line 199. The statement,..”TEAEs reported with a higher frequency with placebo than lucerastat were abdominal\ndistension and decreased GFR”\nIt is interesting that all the patient received Lucerstat did not have any side effects of decreasing GFR? Compared with 11%\nin placebo.\nOn page 9, line 228, the authors mentioned about the neuropathic pain in FD,\nHowever, some other studies, such as CPT, QST, and SSR could be done,\nIn addition, more definitely the IENFD( intraepidermal nerve fiber density ) could be done for these groups of patients.\nThe neuropathic pain score could be very subjective, so the authors should mentioned about this.\nBy the way, several different types of pain in Fabry disease were noted.\nEven some showed no pain. the natural characteristic of pain in these groups of patients should be classified, such as the\ninitial pain age, the severity, the characteristics, etc.\nIn addition, the relationship between pain and plasma Gb3 could be mentioned.\nOn page 9, line 250, the authors showed the “lucerastat improved participants’ eGFR slope compared to the pre-\nrandomization slope, “\nThe important vital organ in Fabry are renal, cardiac, and CNS. So why the renal could be effective and no effect on heart\nshould be mentioned and discussed, especially the mechanism . Also, from this result, did the authors could give readers\nsome suggestion in patients with FD??", "reviewer_4": "Peer-review NCOMMS-24-45423A\nThe manuscript describes findings from a pivotal, placebo-controlled, randomized trial examining the efficacy and safety of\nlucerastat in people with Fabry disease. The paper is well written and generally well presented. The statistical methods are\nreasonably robust and well-aligned with the original statistical analysis plan. Overall, the manuscript presents findings of\ngreat medical and scientific interest. However, the current version presents several areas for improvement. Without adequate\nclarification and justification, the paper risks spin bias and invalid claims of subgroup effects, among other issues.\nMajor remarks\n#1. Non-prespecified subgroup analysis. The major limitation of the manuscript refers to the change of focus from the primary\noutcome of neuropathic pain at month 6 to a non-prespecified subgroup analysis focused only on participants with abnormal\nrenal function at baseline, defined as those with an eGFR <90 mL/min/1.73m2. The trial protocol included a predefined\nsubgroup analysis that compared patients based on their eGFR at the screening visit, categorizing them into two groups:\nthose with eGFR ≥ 60 mL/min/1.73 m² and those with eGFR < 60 mL/min/1.73 m². The same analysis appears in the\nstatistical analysis plan. However, the investigators conducted a different subgroup analysis for eGFR. Besides, the cutoff\nused differs substantially from what was planned.\na. If possible, could the authors justify this new analysis and clearly label it in the manuscript as a non-pre-specified\nanalysis?\nb. Additionally, could the authors explain the rationale behind the change in the cutoff? Was this adjustment based on\nclinical reasoning, or was it influenced by the number of participants?\nWe suggest that the main conclusions of the trial in the abstract and main text focus only on the primary outcome of the\nrandomized trial comparison.\n#2. Risk of an invalid claim of a subgroup effect for eGFR. There is an important limitation in the non-prespecified inferences\n(made in Figure 3). These analyses lack support from formal interaction testing that examines the interaction between\ntreatment and eGFR ≥90 vs < 90 mL/min/1.73 m² at baseline. Without this interaction test, the validity of the subgroup effect\ncannot be properly assessed, potentially leading to misleading or unsupported conclusions.\n#3. Fixed-sequence testing and reported P-values. The trial used fixed-sequence testing (page 51 in the statistical analysis\nplan). If the null hypothesis is not rejected for an endpoint in the sequence, no claims of statistical significance can be made\nfor the subsequent endpoints. Hence, if the null was not rejected for the primary outcomes, can the authors:\na. Justify why P-values have been reported for the remaining secondary outcomes.\nAlso, please clarify in the main text that the fixed-sequence testing was pre-specified.\n#4. Outcomes not reported. All outcomes that were initially planned should be reported. For example, could the authors\nclarify why quality of life, depression, and Patient’s rating of change in disease severity outcomes were not included?\n#5. In general, we suggest that the authors avoid causal terminology when describing results of analyses that are not the\nmain analysis of the primary outcome. These are generally exploratory, not properly powered analyses.\n#6. Please provide further information to allow proper interpretation of your primary outcome and some of the secondary\noutcomes. For instance, what is the possible range of scores and what do higher values mean for your primary outcome?\n#7. Could the authors please discuss how relevant is the between-group difference in the biomarkers? Generally, a\nmoderate to large between-group difference in biomarkers would be needed so that a minimally important between-group\ndifference can be observed downstream in clinically relevant outcomes.\n#8. Can the authors make an explicit statement about blinding of outcome assessors in the trial? Because the primary\noutcome is patient reported, it can be considered that outcome assessor is also blinded. But it would be also important that\nthe authors clarify this for other outcomes, not reported by patients, in this trial.\nMinor remarks\n#9. Use of standard errors to convey uncertainty. It would be more appropriate to use 95% confidence intervals rather than\nstandard error, which has been used inconsistently throughout the manuscript (SE or SEM).\n#10. Use of standard errors combined with inappropriate wording of statistical inferences. In the sentence below (page 6),\nthe phrase \"numerically higher increase\" gives a misleading impression of the statistical and clinical superiority of lucerastat\ncompared to placebo.\n#11. We were able to replicate the sample size calculation assuming that a 2:1 randomization was taken into consideration.\nCan the authors explicitly state this in their sample size considerations?\n#12. ANCOVA was not “adjusted” by the treatment group, because treatment group is the main factor of interest.\nPlease rephrase it accordingly."}, "author_response": "We wish to thank the Reviewers for their interest in our manuscript and the valuable and\nconstructive comments we have received. As you will see, we have revised our manuscript based\non these comments, with changes in the manuscript text file highlighted in yellow. We also provide\na clean copy for your convenience.\nPlease find our point-by-point responses below.\nWe hope these modifications have sufficiently addressed the referees’ comments and look forward\nto your final decision.\nKindest regards,\nProf. Hughes\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThis manuscript evaluates the effects of lucerastat, a substrate reduction therapy, in patients with\nFabry disease. The primary objective was to determine the effect of lucerastat on neuropathic pain,\nwith secondary objectives assessing gastrointestinal symptoms, FD biomarkers, and\nsafety/tolerability. The results indicate that lucerastat failed to improve neuropathic pain,\nabdominal pain, or diarrhea at six months. However, in an interim analysis of the open-label\nextension, lucerastat showed promising effects in improving eGFR in participants with reduced\nbaseline kidney function.\nThe finding of an improvement in eGFR with lucerastat is particularly interesting and warrants\nfurther investigation. Below are my comments and suggestions to enhance the quality and impact\nof the manuscript:\n1. Fabry disease is a progressive disorder. What is the rationale for discontinuing effective and\napproved treatments, such as enzyme replacement therapy (ERT), in this study? Were there\nmeasures to ensure disease progression did not compromise patient outcomes during the trial?\nClarification of the ethical and clinical justifications is necessary.\nResponse:\nWe thank the Reviewer for their comments and for allowing us to clarify our rationale for\ndiscontinuing effective and approved treatments. During the MODIFY study, the temporary\ninterruption of ERT in subjects with persisting neuropathic pain despite ERT was considered\nacceptable for three reasons:\n1\n1. Fabry is a slowly progressive disease, and no significant clinical worsening in subjects who\ndiscontinued ERT was expected within the time frame of the study.\n2. Patients with an advanced stage of disease and those at high risk of developing clinical signs of\norgan involvement within the time frame of the study were excluded.\n3. One of the criteria for discontinuing ERT, as recommended by the European Fabry Working\nGroup, includes the lack of clinical response when the sole “indication” for ERT is neuropathic\npain and the patient requests to stop ERT (Biegstraaten et al. 2015).\nIn addition, both the MODIFY and its open-label extension protocols allowed the re-start of ERT if this\nwas judged medically indicated by a significant disease progression. Of the 118 randomized\nparticipants, only 3 (re-)initiated ERT during the 6-month double-blind placebo-controlled period of\nMODIFY as reported in “Results – Patient population and baseline characteristics”.\nNo modification was made to the manuscript.\n2. The manuscript reports that mean eGFR increased from baseline to Month 6 in both the\nlucerastat and placebo groups. How do the authors explain this improvement, particularly in the\nplacebo group?\nResponse:\nThis is an interesting point, and we thank the Reviewer for highlighting it. Overall, there was indeed\nno mean decrease in eGFR in the placebo group, and instead, a small increase (+1.3 mL/min/1.73m2)\nwas observed. However, of the 118 participants randomized in MODIFY, 78 had no renal impairment\nat baseline (a small increase in renal function was observed in these participants for both lucerastat\nand placebo), and 40 had renal impairment. Notably, in participants with abnormal kidney function\nat baseline, eGFR decreased on placebo during MODIFY, as shown in Figure 3 and described on page\n6.\nIn Fabry disease, renal deterioration is not a linear process, and patients with a normal range of renal\nfunction can remain stable for a long time, even classic male patients, and then experience\nproteinuria and a rapid deterioration (Germain 2010). No deterioration but a small improvement was\nobserved on placebo in participants without renal impairment at baseline or in the overall\npopulation, with unclear cause.\nWe have included a sentence in the Discussion to explain the small improvement in eGFR in the\noverall placebo group (page 10, lines 266-268).\n3. A significant proportion of participants had the classic subtype of Fabry disease, yet the number\nof patients with cardiac involvement appears limited. This could significantly restrict the evaluation\nof left ventricular mass index (LVMi). Do the authors believe this limitation influenced their ability to\ndetect changes in cardiac outcomes?\nResponse:\nLVMi was limited by the absence of cardiac hypertrophy at baseline. We have added a sentence in\nthe Discussion to clarify this point (page 11, line 288-290).\n4. Fabry disease typically manifests renal involvement in approximately 80% of patients with the\nclassic phenotype around the age of 40. However, Table 1 indicates a relatively low median UACR\nvalue. Could the authors provide an explanation for this finding? Was it influenced by study\ninclusion criteria or prior treatments?\nResponse:\nThe Reviewer raises an interesting point here. The relatively low median UACR in the study\npopulation with a mean age of 40 years can be explained by several factors:\n- Patients with severe renal insufficiency (eGFR <30 mL/min/1.73 m2) or on dialysis for the\ntreatment of chronic kidney disease were excluded.\n- More than 50% of the patients enrolled were female, in which the progression of the disease is\nusually slower than in males.\n- The female participants were, on average, 10 years older than the male participants. Despite\nthis, the median UACR was much lower in females than males (20.0 vs 28.6 mg/g).\n- Approximately 60% of the patients were ERT-naïve or pseudo-naïve. One can assume that\nthese patients were in a less advanced stage of the disease than patients who required ERT.\nNo modification was made to the manuscript.\n5. Due to mosaicism, disease manifestations can differ significantly between male and female\npatients. Were there any notable differences in the effect of lucerastat on eGFR between genders?\nResponse:\nWe thank the Reviewer for allowing us to clarify this point. No notable differences in the effect of\nlucerastat on eGFR between male and female participants were observed. The annual eGFR slope\nLS mean improved on lucerastat in both sex by more than 50% (from -2.84 to -1.04 mL/min/1.73 m2\nin 46 female participants and from -4.07 to -1.94 mL/min/1.73 m2 in 47 male participants).\nNo modification was made to the manuscript.\n6. Fabry disease is a slow-progressing condition, and six months might not be sufficient to\ndemonstrate the full therapeutic potential of lucerastat. What are the authors' thoughts on this\nlimitation?\nResponse:\nWe agree with the Reviewer. This is why we integrated the interim analysis of the open-label extension\nwhere lucerastat patients were continued on lucerastat and where we see this amelioration of eGFR\nslope in patients who were rapidly deteriorating. The assessment of symptoms such as neuropathic\npain and gastrointestinal symptoms based on patient-reported outcomes are subjective by nature\n3\nand require the use of a placebo arm comparator. The placebo-controlled period in MODIFY was\nlimited to 6 months for ethical reasons. In the Discussion, we acknowledge that the inhibition of Gb3\nsynthesis by lucerastat may not have caused sufficient substrate reduction in the dorsal root ganglia\nin the 6-month treatment period (page 9, lines 241-244). The open-label extension was designed to\nassess the long-term effect of lucerastat on objective parameters such as eGFR slope in comparison\nto the slopes observed in those patients prior to their inclusion in the MODIFY study.\nNo modification was made to the manuscript.\n7. Given the significant renal findings, it may be beneficial to revise the title to better reflect the\nstudy’s impact on kidney outcomes.\nResponse:\nWe agree with the reviewer and have modified the title as suggested. The revised title is now:\nEffect of lucerastat on kidney function in Fabry disease: Results from a pivotal phase 3 study\nand its open-label extension\nReviewer #2 (Remarks to the Author):\nThe manuscript by Nordbeck et al. reports on a phase 3 RCT of lucerastat along with an open label\nextension (OLE) study for the treatment of Fabry Disease. The main study showed no benefit of\nlucerastat, a substrate reduction therapy, compared with placebo on the primary outcome of\nneuropathic pain reduction. Although the primary outcome was negative, there were some\nunexpected secondary findings of improvement or stabilization in kidney function with lucerastat,\nparticularly in those with evidence of more rapid progression prior to study entry. The study appears\nto have been well conducted, reported and adequately powered with regards to the main outcome.\nThe limitations described are appropriate. This is a multicentric and large study for a rare disease,\nwhich provides important insight on the use of substrate reduction therapy for clinically relevant\noutcomes. It provides valid information on the primary outcome of neuropathic pain. There are\nsome issues that should be addressed by the authors:\n1. Due to the COVID pandemic several amendments were made, including a switch of the primary\noutcome from a binary to continuous measure for analysis, and combination of the OLE part of the\nstudy with the main trial for some of the analysis. The OLE was also lengthened from 24 to 72\nmonths. When the primary outcome was negative, there was very good participation in the\nextension study. Were participants informed of this result and allowed or informed that they could\nresume their enzyme replacement therapy during the OLE? Would this not have been considered\nstandard of care?\nResponse:\nwas unblinded, the participants were informed of the results, which were included in an updated\nversion of the patient information leaflet. Participants were asked to re-consent to continue\nparticipating in the OLE study.\nFurthermore, irrespective of the MODIFY results, both MODIFY and OLE study protocols included the\noption to re-start ERT if judged clinically needed by the investigator (this is also described in the\nmanuscript in “Methods-Procedures” page 14, lines 385-387). This information was included in the\npatient information leaflet and the consent process since the start of the studies.\nNo modification was made to the manuscript.\n2. The improvement of GFR is quite interesting. It would be helpful for the authors to propose a\nbiologic mechanism to understand this effect. As the median baseline eGFR was quite preserved,\nare the authors suggesting that lucerastat had a favourable (disease-modifying) effect on the\nkidneys as early as 2-3 months. Could this be a hemodynamic effect or hyperfiltration? Did any pre-\nclinical or earlier phase studies show similar effects? Was there a washout period after the main\nstudy or after the OLE where eGFR was measured again to assess if the GFR improvement was\nsustained?\nResponse:\nWe agree with the reviewer and have modified the Discussion to include a proposed mechanism of\naction to explain the effect of lucerastat on eGFR, as follows (page 10, lines 274-279):\n“The mechanism of action of lucerastat on eGFR is assumed to be primarily based on the reduction\nof lipid inclusions in the kidney cells. A pre-clinical study indicated that, compared with untreated\ncontrols, lucerastat treatment significantly reduced the storage of α-GalA substrates, including Gb3,\nin the kidneys of Fabry mouse models after 20 weeks (Welford et al. 2017). In the previous clinical\ntrial in patients with Fabry disease, the duration of lucerastat treatment was too short (12 weeks) to\nmake any meaningful conclusion on the effect of lucerastat on renal function (Guérard et al. 2018)”.\nThe Introduction has also been revised to include the observations on the effect of lucerastat in the\nkidneys of Fabry mice (page 4, line 83).\nAs there was no withdrawal period after the main study or the OLE, we were unable to assess if the\neGFR improvement was sustained.\n3. If lucerastat is proposed to have a renoprotective benefit, was there any impact on albuminuria\nthroughout the study?\nResponse:\nThis is an interesting point, and we thank the Reviewer for highlighting it. As mentioned by Reviewer\n#1, UACR was low at baseline, with a median of 23.0 mg/g (Table 1). There was no difference in the\nchange in UACR from baseline to Month 6 between lucerastat and placebo in the MODIFY study, as\ndetermined by an additional post-hoc analysis of UACR on log-transformed data, which yielded a\ndifference between treatment groups.\nNo modification was made to the manuscript.\n4. The authors use the pre-study eGFR decline to understand better those that had a renal benefit\nwith lucerastat. Although this is interesting, it is a measure that was not in the original study\nprotocol, and was not collected systematically. The authors do show in the supplement that the\nmajority had at least 3-5 creatinine values over time, but it is unclear if these represent values when\na patient was stable or if they were done for some cause. This raises the concern for assessment or\ndetection bias. I would recommend that the description of eGFR decline as compared to placebo\nbe compared only after study randomization.\nResponse:\nRespectfully, we disagree with the Reviewer. The collection of available serum creatinine data from\nthe period preceding randomization was planned in the original protocol of MODIFY. Specifically, in\norder to build a pre-randomization slope, at least 2 values per year, 6 months apart, during the last\ntwo years before randomization were to be collected (if available). We do agree, however, that we\ncannot exclude some bias in the timing of creatinine measurements, and this point is mentioned as\na limitation in the Discussion (page 11, lines 296-299). However, the overall slope should not be\naffected by the selected time points.\nNo modification was made to the manuscript.\n5. It appears that only a few participants were not included in the OLE. Were any placebo treated\nparticipants excluded due to the eGFR <30 criteria? If so, removing these individuals may result in the\nappearance of more stability of mean eGFR in the OLE cohort.\nResponse:\nWe thank the Reviewer for allowing us to clarify this point. After randomization in MODIFY,\nparticipants had to discontinue study treatment if eGFR was <15 mL/min/1.73m2. Hence,\nparticipants with an eGFR >15 mL/min/1.73m2 and <30 mL/min/1.73m2 at the end of MODIFY could\nstill be eligible to enroll in the OLE. This has been clarified in the “Methods-Participants” section\n(page 13, lines 361-363).\nAs shown in Figure 1, of the 37 placebo-treated participants in MODIFY, 34 completed the 6-month\ntreatment, and all were enrolled in the OLE. Among the 3 participants who did not complete the\ntreatment in MODIFY, 1 was lost to follow-up, and 2 participants prematurely discontinued due to\nadverse events. These participants had eGFR values ≥90 mL/min/1.73m2 throughout their\nparticipation in MODIFY. Of note, an eGFR <30 mL/min/1.73m2 was only an exclusion criterion for the\nMODIFY trial.\n6\n6. An improvement in eGFR could be related to a reduction in NSAID use. Although there was no\nreduction in pain, was there any reduction in NSAID use?\nResponse:\nThe Reviewer raises an interesting point here. The use of NSAIDs, which was recorded daily in the\neDiary, was fluctuating as NSAIDs were used PRN by the patient. We, therefore, did not look at the\nuse of NSAIDs in MODIFY, as daily fluctuations of such treatment over a 6-month period would\nrender any analysis of the potential impact on eGFR difficult to interpret.\nNo modification was made to the manuscript.\nReviewer #3 (Remarks to the Author):\nIt is an important manuscript about the treatment of Fabry Disease with Lucerastate\nHow, some suggest and revision should be done.\nPage3 line 73.\nIn this part, the authors should not mention about the ERT disadvantage,\nSome bias could be noted,\nIf they want to do this, they should put the important part on the discussion for detailed comparison\nof different treatment strategies such as ERT and Lucerastat.\nResponse:\nWe agree with the Reviewer and have deleted the sentence on the pitfalls of ERT and migalastat.\nOn page 4, line 77\nOn the section of introduction, mostly mentioned about the kidney function, but they did not\nmentioned about the heart function, brain function, etc.\nHowever, if only focus on the kidney function, should we say the heart function is not important?\nSo the manuscript must be more fair for all the important vital organ function statement.\nResponse:\nWe agree with the Reviewer and have added a sentence on other organ involvement (page 4, lines\n78-79).\nOn the page 4, line 101, the authors mention about the “surprising effect of lucerastat on kidney\nfunction.”\nThis result should be more neutral in the introduction part.\nSuch as “ in this study, we try to see the kidney function after lucerastat.\nResponse:\nwhich showed a surprising effect of lucerastat on kidney function.”\nOn page 5, line 127\n“with a numerically smaller reduction with lucerastat versus placebo (LSM\ndifference 0.42 [95% confidence interval (CI) -0.40 to 1.23], p=0.32; Figure 2).”\nThis should add on without statistically significant.\nResponse:\nWe agree with the Reviewer and have clarified the sentence to show that it was not statistically\nsignificant. We have also deleted the P value.\nOn page 5, line 127\n“The median number of days on significant rescue pain therapy was 0 in both treatment groups, “\nOn Table S3, “Days on significant rescue pain therapy” on both treatment groups and placebo all\nshowed 0\nBut in this sentence, the result just showed 0 on both treatment group.\nThe authors should clearly showed the both group is MODY and placebo? MODY and OLE??\nMODY/OLE and placebo\nHowever is there statistical significance noted relative shorter mean days on the Lucerastat group\nshould be calculated and showed the results.\nIn addition, the pain characeterisitic change? The number of patients with pain-control medication,\nor other important parameters for pain should be mentioned to see the pain change after\nLucerastat group,\nBy the way, the S3 just mentioned about the MODIFY study( for 6 months) , but the OLE (extended\nstudy) should also be presented.\nResponse:\nWe thank the Reviewer for their comments and for allowing us to clarify this information. Significant\nrescue pain therapy was only collected in the eDiary in MODIFY, as it is a variable that would be\ndifficult to evaluate in the OLE in the absence of a placebo control arm. The “Methods-Procedures”\nsection and the Results section have been updated to make this clearer (page 14, lines 383-385).\nNo statistical difference was noted relative to the numerically smaller mean number of days with\nsignificant rescue pain medication in the lucerastat group compared with the placebo group. This\nhas been updated in the “Results-primary and secondary endpoints” (page 5, lines 130-133).\nThe study focused on assessing the worst intensity of neuropathic pain using the validated tool\nmodified BPI-SF3 rather than evaluating the change in pain characteristics. The pain questionnaire\nand the definition of neuropathic pain have been included in Supplemental Materials Table S10.\nAt the time of screening, each patient was provided with an Individual Pain Management Plan\n(IPMP) by the investigator. The IPMP included customized instructions regarding the use of pain\nmedication during the study including the use of rescue pain medication in case of unbearable\n8\npain. The section “Procedures” has been amended to reflect the use of IPMP during the study (page\n13, lines 380-387). In total, 79% of the patients were using pain medication at the time of\nenrollment in MODIFY, as stated in section “Patient population and baseline characteristics” (page\n5, lines 115-116), with more details provided in Table 1 (page 26).\nOn page 5 Line 1333, “LSM difference”\nSome showed “LSM” , some showed “LS mean” , the manuscript should be corrected carefully.\nResponse:\nWe agree with the Reviewer and have changed “LS mean” to “LSM” throughout the manuscript.\nOn page 6, line 137, the study showed “ in both treatment groups”,\nThe authors should clearly say the both groups meaning. The MODY vs Placebo”?,\nThe MODY vs OLE( extended study)?. In addition, the result of MODY and OLE ( extended group)\nshould be presented here, not just MODY and placebo.\nResponse:\nWe thank the Reviewer for their comments and for allowing us to clarify this information. Indeed, in\nthe section “Renal function endpoints,” we report results for MODIFY (6 months placebo-\ncontrolled) (first and second paragraphs) and the MODIFY+open-label extension (third paragraph\nstarting with “In the 114 participants exposed to lucerastat in MODIFY/OLE,….”.)\nWe have also clarified the definition of each group (in terms of renal impairment at baseline) in the\nfirst sentence of the second paragraph of this section.\nOn page 6, line 140, the statement: “mean eGFR increased from baseline to Month 6 by 3.8 (SEM\n2.2) mL/min/1.73m2””\nIn the result, the authors showed the increased GFR per year, but totally, the decreasing GFR/year\nwas noted ( page 6, line 143),\nIs this mean that the normal GFR patient received Lucerastat showed a rapid decreasing GFR???\nIn this short period, how many years be calculaged.\nResponse:\nWe thank the Reviewer for their comments and for allowing us to clarify this information. The first\nstatement (“mean eGFR increased (..)”) referred to change from baseline to Month 6 in the Modify\ntrial, whereas the second statement (“decreasing eGFR/year”) referred to the eGFR slope in the\nMonth-18 interim analysis of the longer Modify/OLE study, which was indeed negative. This slope\nwas, however, less negative than the historical eGFR slope.\nNo modification was made to the manuscript.\nThe figures of MODY/OLE/Placebo patients with GFR < 90 , and normal GFR should be presented.\nthe comparison with placebo should be important.\nResponse:\nWe thank the Reviewer for their comments and for allowing us to clarify this information. The figure\nshowing the change from baseline over time in eGFR in MODIFY in participants without renal\nimpairment has been included in the Supplemental material (Figure S3). The results in this\nsubgroup have been added to the Renal function endpoints section (page 6, lines 143-148).\nIn addition, the Fabry disease showed an X-linked disease. So the results of renal function in male\nand female groups should be presented separately.\nWe also very interested in the different improvements in male and female groups.\nResponse:\nWe thank the Reviewer for their comments and for allowing us to clarify this information. The\nfigures showing the change from baseline over time in eGFR in MODIFY in males and female have\nbeen included in the Supplemental material (Figure S4). The results are described in section “Renal\nfunction endpoints,” (page 6, lines 149-151).\nOn page 6, line 148, some showed LSM, and some showed LS mean, please correct it.\nResponse:\nWe agree with the Reviewer and have changed “LS mean” to “LSM” throughout the manuscript.\nOn page 6 , line 153, the Figure 4, should also showed the results of lucerastat and placebo group.\nResponse:\nWe respectfully disagree with the Reviewer. Figure 5 (previously Figure 4) displays the long-term\neffect of lucerastat on annualized eGFR slope versus the pre-randomization annualized eGFR slope\nusing data from MODIFY/OLE. In the OLE, there was no placebo arm.\nNo modification was made to the manuscript.\nON page 6, line 162, the statement “ lucerastat treatment stopped the eGFR decline. In these\npatients”\nWith the results, the authors should showed the results of all the GFR decline in GFR >90, GFR<90,\nMODY group, OLE group, placebo group.\nIn addition, results in male and female groups are also important.\nResponse:\nWe thank the Reviewer for allowing us to clarify this comment. The results of eGFR slopes for the\nsubgroups of subjects with baseline eGFR >=90, <90, and classic males and females can be found\nin Supplemental Materials Figure S53. We have also added Supplemental Materials Figure S4\nshowing the mean change from baseline over time in eGFR in male and female participants in\nMODIFY.\nResponse:\nWe agree with the Reviewer and have changed “LS mean” to “LSM” throughout the manuscript.\nOn page 7, line 181, the authors showed the decreased plasma Gb3,…;\nThe results in MODY and OLE should be presented.\nIt is interesting in elevated the plasma GB3 in female. So the male data and female data should be\npresented.\nResponse:\nWe thank the Reviewer for allowing us to clarify this point. In MODIFY, the change in plasma Gb3\nwas one of the secondary endpoints, and results are described in the section “Results- Primary and\nsecondary endpoints” (page 5, lines 136-138). Table S7, in Supplemental Materials, reports plasma\nGb3 data by sex for participants receiving lucerastat in MODIFY and the OLE. As the information\nrequested is already included here, we have not made further changes.\nOn page 8, line 199. The statement,..”TEAEs reported with a higher frequency with placebo than\nlucerastat were abdominal distension and decreased GFR”\nIt is interesting that all the patient received Lucerstat did not have any side effects of decreasing\nGFR? Compared with 11% in placebo.\nResponse:\nWe thank the Reviewer for allowing us to clarify this point. Indeed, we consider that the fact that\nnone of the patients in the lucerastat group had side effects of decreasing GFR as compared to\n11% in the placebo group over a period of 6 months is supportive of the improvement in eGFR\nobserved in the lucerastat group overall as well as in the subgroup of subjects who already had\nrenal impairment at baseline.\nNo modification was made to the manuscript.\nOn page 9, line 228, the authors mentioned about the neuropathic pain in FD,\nHowever, some other studies, such as CPT, QST, and SSR could be done,\nIn addition, more definitely the IENFD( intraepidermal nerve fiber density ) could be done for these\ngroups of patients.\nThe neuropathic pain score could be very subjective, so the authors should mentioned about this.\nResponse:\nWe thank the Reviewer for allowing us to clarify this point. We acknowledge that additional studies\ncould be done using more objective measures, such as those quoted by the reviewer and previously\npublished (Uceyler et al. 2011, Uceyler et al. 2013). The primary endpoint of the phase 3 MODIFY\ntrial was choosen based on regulatory requirements to demonstrate a clinical effect using patient-\nreported outcome (PRO) tools such as neuropathic pain scales. Due to the subjective nature of\n11\nsuch PROs, a randomization design and comparison with a placebo arm were used in the trial. We\nhave addressed the Reviewer's concerns by mentioning the subjectivity of the neuropathic pain\nscore in the Discussion section (page 9, line 249).\nBy the way, several different types of pain in Fabry disease were noted.\nEven some showed no pain. the natural characteristic of pain in these groups of patients should be\nclassified, such as the initial pain age, the severity, the characteristics, etc.\nIn addition, the relationship between pain and plasma Gb3 could be mentioned.\nResponse:\nWe thank the Reviewer for allowing us to clarify these points. All MODIFY participants had to report\nmoderate to severe neuropathic pain over the 4 weeks prior to randomization (key inclusion criterion\nin the section Methods-participants), and a definition of Fabry neuropathic pain was included in the\nvalidated modified BPI-SF3 questionnaire.\nPatients with other diseases or conditions associated with pain components that could have\nconfounded the assessment of neuropathic pain were excluded. This has been clarified in the\nMethods-participants section (page 13, lines 355-356).\nWe have modified the text (page 5, lines 117-119) and included Table S2 in Supplemental Materials,\nwhich shows baseline characteristics of the type of neuropathic pain symptoms, time since first\nneuropathic pain symptoms, and pain symptom triggering factors.\nThe severity of neuropathic pain was assessed on a daily basis using the eDiary (Table 1), and the\nmean (SD) baseline modified BPI-SF3 score has been added to the text in “Results-Patient\npopulation and baseline characteristics” (page 5, lines 117-119).\nAs highlighted in the Discussion section, the lower plasma and urinary Gb3 levels observed after 6\nmonths of treatment were not associated with a reduction in neuropathic pain. One possible\nexplanation is that the short duration of the treatment may not be sufficient for a drug with an SRT\nmode of action, and this has been mentioned in the Discussion (page 10, lines 238-247).\nOn page 9, line 250, the authors showed the “lucerastat improved participants’ eGFR slope\ncompared to the pre-randomization slope, “\nThe important vital organ in Fabry are renal, cardiac, and CNS. So why the renal could be effective\nand no effect on heart should be mentioned and discussed, especially the mechanism . Also, from\nthis result, did the authors could give readers some suggestion in patients with FD??\nResponse:\nWe thank the Reviewer for allowing us to clarify this point. We highlight in the Discussion section that\nin this study, cardiac data were mainly collected for safety analysis as the study was considered too\nshort to evaluate changes over time, and very few patients had cardiac hypertrophy at baseline.\nNevertheless, the observation of no increase in LVMi over an 18-month period may indicate some\nstabilization. The text has been modified to clarify this (page 11, lines 288-293).\nPeer-review NCOMMS-24-45423A\nThe manuscript describes findings from a pivotal, placebo-controlled, randomized trial examining\nthe efficacy and safety of lucerastat in people with Fabry disease. The paper is well written and\ngenerally well presented. The statistical methods are reasonably robust and well-aligned with the\noriginal statistical analysis plan. Overall, the manuscript presents findings of great medical and\nscientific interest. However, the current version presents several areas for improvement. Without\nadequate clarification and justification, the paper risks spin bias and invalid claims of subgroup\neffects, among other issues.\nMajor remarks\n#1. Non-prespecified subgroup analysis. The major limitation of the manuscript refers to the\nchange of focus from the primary outcome of neuropathic pain at month 6 to a non-prespecified\nsubgroup analysis focused only on participants with abnormal renal function at baseline, defined\nas those with an eGFR <90 mL/min/1.73m2. The trial protocol included a predefined subgroup\nanalysis that compared patients based on their eGFR at the screening visit, categorizing them into\ntwo groups: those with eGFR ≥ 60 mL/min/1.73 m² and those with eGFR < 60 mL/min/1.73 m². The\nsame analysis appears in the statistical analysis plan. However, the investigators conducted a\ndifferent subgroup analysis for eGFR. Besides, the cutoff used differs substantially from what was\nplanned.\na. If possible, could the authors justify this new analysis and clearly label it in the manuscript as a\nnon-pre-specified analysis?\nResponse:\nWe thank the Reviewer for allowing us to clarify this point. The new analysis in a different subgroup\nwas performed post-hoc in replacement of the original subgroup analysis due to the low number of\nparticipants with a baseline eGFR <60 mL/min/1.73 m². Indeed, only 9 participants (7 in the\nlucerastat group and 2 in the placebo group) had a baseline eGFR <60 mL/min/1.73 m² which would\nhave limited the meaningfulness of this subgroup analysis. Table 1 has been updated to include the\nnumber of participants with a baseline eGFR <60 mL/min/1.73 m² (page 25). The post-hoc nature\nof the analysis is stated in the section “Methods-Statistical analysis” (page 16, lines 444-445).\nb. Additionally, could the authors explain the rationale behind the change in the cutoff? Was this\nadjustment based on clinical reasoning, or was it influenced by the number of participants?\nWe suggest that the main conclusions of the trial in the abstract and main text focus only on the\nprimary outcome of the randomized trial comparison.\nResponse:\nAs explained in the previous answer, the main reason for performing a new post hoc analysis was\nthe low number of participants with an eGFR<60 mL/min/1.73 m² at baseline. Instead, the\n13\nthreshold of 90 mL/min/1.73 m² was selected as this is the standard value used to distinguish\npatients with normal eGFR from those with abnormal eGFR (see (Kidney Disease: Improving Global\nOutcomes (KDIGO) CKD Work Group 2024)).\nNo modification was made to the manuscript.\n#2. Risk of an invalid claim of a subgroup effect for eGFR. There is an important limitation in the\nnon-prespecified inferences (made in Figure 3). These analyses lack support from formal\ninteraction testing that examines the interaction between treatment and eGFR ≥90 vs < 90\nmL/min/1.73 m² at baseline. Without this interaction test, the validity of the subgroup effect cannot\nbe properly assessed, potentially leading to misleading or unsupported conclusions.\nResponse:\nWe thank the Reviewer for allowing us to clarify this point. While we found this result of interest,\nwe did not intend to claim a subgroup effect for eGFR (eGFR <90 mL/min/1.73m2). For transparency,\nwe have added the results of the complementary subgroup (eGFR ≥90 mL/min/1.73m2) and also added\nthat the interaction test was not statistically significant (p=0.09) (page 6, lines 143-148).\n#3. Fixed-sequence testing and reported P-values. The trial used fixed-sequence testing (page 51 in\nthe statistical analysis plan). If the null hypothesis is not rejected for an endpoint in the sequence,\nno claims of statistical significance can be made for the subsequent endpoints. Hence, if the null\nwas not rejected for the primary outcomes, can the authors:\na. Justify why P-values have been reported for the remaining secondary outcomes.\nAlso, please clarify in the main text that the fixed-sequence testing was pre-specified.\nResponse:\nWe thank the Reviewer for allowing us to clarify these points. We have explained that the fixed-\nsequence testing was prespecified in the section “Methods – Statistical analyses” (page 15, lines\n426-429). We also added that “If a hypothesis in the sequence was not rejected, p-values for the\nsubsequent hypotheses were to be considered exploratory”. We propose still reporting these p-\nvalues as readers may (or may not) use them as indicators of the probability that the result is a\nchance finding.\n#4. Outcomes not reported. All outcomes that were initially planned should be reported. For\nexample, could the authors clarify why quality of life, depression, and Patient’s rating of change in\ndisease severity outcomes were not included?\nResponse:\nsecondary endpoints of MODIFY and several longer-term outcomes observed in the OLE. To reduce\nthe amount of information presented in the manuscript, not all outcomes were reported. However,\nQoL, depression, and Patient’s rating of change may be reported in a future publication.\nNo modification was made to the manuscript.\n#5. In general, we suggest that the authors avoid causal terminology when describing results of\nanalyses that are not the main analysis of the primary outcome. These are generally exploratory, not\nproperly powered analyses.\nResponse:\nWe agree with the Reviewer and have removed the few instances of causal terminology in the\nmanuscript.\n#6. Please provide further information to allow proper interpretation of your primary outcome and\nsome of the secondary outcomes. For instance, what is the possible range of scores and what do\nhigher values mean for your primary outcome?\nResponse:\nWe thank the Reviewer for allowing us to clarify this point. The text has been modified to show that\nboth neuropathic pain and gastrointestinal pain were rated by the patients on a scale from 0-10\n(higher values corresponding to more severe pain) (page 14, lines 411-415). More details can be\nfound in Wanner 2022.\n#7. Could the authors please discuss how relevant is the between-group difference in the\nbiomarkers? Generally, a moderate to large between-group difference in biomarkers would be\nneeded so that a minimally important between-group difference can be observed downstream in\nclinically relevant outcomes.\nResponse:\nWe thank the Reviewer for allowing us to clarify this point. The between-group differences at Month\n6 reported for plasma Gb3 (-873.53), plasma lysoGb3 (-5.88) and urine Gb3 (-8.32) correspond to\neffect sizes (i.e., difference divided by standard deviation) of approximately 1.5, 0.4, and 0.7,\nrespectively, which could be considered as large, medium and large, respectively. However, it is\nacknowledged that effects on these biomarkers did not translate into effects on the primary and\nsecondary efficacy endpoints, neuropathic pain, or abdominal pain, the main MODIFY endpoints\nconsidered in this paper.\nNo modification was made to the manuscript.\n15\n#8. Can the authors make an explicit statement about blinding of outcome assessors in the trial?\nBecause the primary outcome is patient reported, it can be considered that outcome assessor is\nalso blinded. But it would be also important that the authors clarify this for other outcomes, not\nreported by patients, in this trial.\nResponse:\nWe thank the Reviewer for allowing us to clarify this point. In the section “Methods-Randomization\nand masking” we state that “Participants, investigators, site personnel, and sponsor personnel\ninvolved in the study conduct were unaware of study treatment allocation. In the event of a\nmedical emergency, investigators were permitted to initiate the unmasking process; no unmasking\nevents occurred in the MODIFY study.”\nNo modification was made to the manuscript.\nMinor remarks\n#9. Use of standard errors to convey uncertainty. It would be more appropriate to use 95%\nconfidence intervals rather than standard error, which has been used inconsistently throughout the\nmanuscript (SE or SEM).\nResponse:\nWe thank the Reviewer for highlighting this issue. To achieve consistency, we have removed the\nstandard errors from the text (see also our response to comment #10) and kept only the confidence\nintervals.\n#10. Use of standard errors combined with inappropriate wording of statistical inferences. In the\nsentence below (page 6), the phrase \"numerically higher increase\" gives a misleading impression\nof the statistical and clinical superiority of lucerastat compared to placebo.\nResponse:\nWe thank the Reviewer for highlighting this issue. Although the phrase “numerically higher” does not\nimply statistical significance, we have reworded the sentence to clarify that “numerically higher”\ndoes not mean it is statistically significant (page 6). We have also removed the standard errors to\navoid any suggestion of statistical analysis.\n#11. We were able to replicate the sample size calculation assuming that a 2:1 randomization was\ntaken into consideration.\nCan the authors explicitly state this in their sample size considerations?\nResponse:\nWe thank the Reviewer for highlighting this issue. We have repeated in the statistical analysis\nsection on page 15, lines 422-423 that the randomization was 2:1.\n16\n#12. ANCOVA was not “adjusted” by the treatment group, because treatment group is the main\nfactor of interest. Please rephrase it accordingly.\nResponse:\nWe thank the Reviewer for highlighting this issue and have adjusted the text on Page 15 (lines 435-\n437) according to the reviewer’s suggestion.\nReferences\nBiegstraaten, M, Arngrímsson, R, Barbey, F, et al. (2015). \"Recommendations for initiation and\ncessation of enzyme replacement therapy in patients with Fabry disease: the European\nFabry Working Group consensus document.\" Orphanet J Rare Dis 10: 36. doi:\n10.1186/s13023-015-0253-6.\nGermain, DP (2010). \"Fabry disease.\" Orphanet J Rare Dis 5: 30. doi: 10.1186/1750-1172-5-30.\nGuérard, N, Oder, D, Nordbeck, P, et al. (2018). \"Lucerastat, an Iminosugar for Substrate Reduction\nTherapy: Tolerability, Pharmacodynamics, and Pharmacokinetics in Patients With Fabry\nDisease on Enzyme Replacement.\" Clin Pharmacol Ther 103(4): 703-11. doi:\n10.1002/cpt.790.\nKidney Disease: Improving Global Outcomes (KDIGO) CKD Work Group (2024). \"KDIGO 2024\nClinical Practice Guideline for the Evaluation and Management of Chronic Kidney Disease.\"\nKidney Int 105(4s): S117-s314. doi: 10.1016/j.kint.2023.10.018.\nUceyler, N, He, L, Schonfeld, D, et al. (2011). \"Small fibers in Fabry disease: baseline and follow-up\ndata under enzyme replacement therapy.\" J Peripher Nerv Syst 16(4): 304-14. doi:\n10.1111/j.1529-8027.2011.00365.x.\nUceyler, N, Kahn, AK, Kramer, D, et al. (2013). \"Impaired small fiber conduction in patients with\nFabry disease: a neurophysiological case-control study.\" BMC Neurol 13: 47. doi:\n10.1186/1471-2377-13-47.\nWelford, RWD, Mühlemann, A, Priestman, D, et al. (2017). \"Lucerastat, an iminosugar for substrate\nreduction therapy in Fabry disease: preclinical evidence.\" Mol Genet Metab 120(1): S139-\nS40. doi: https://doi.org/10.1016/j.ymgme.2016.11.369.\n17"}, {"round": 2, "reviewer_comments": {"reviewer_1": "Overall, the authors have addressed the revisions thoroughly and significantly improved the manuscript. I find the current version to be well-structured, scientifically sound, and suitable for publication in its present form.", "reviewer_2": "The authors have responded satisfactorily and comprehensively to the concerns raised in the initial review. The article is improved and would endorse its acceptance for publication.", "reviewer_3": "It is an important manuscript about the treatment of Fabry Disease with Lucerastate.\n#1. In the supplemental materials, the authors showed a good presentation (Figures S6) of the relationship between important factors ( such as GB3, LysoBG3 and urine GB3) and the 3 subgroups,( Placebo MODIFY, Lucerastat MODIFY / OLE and Lucerastat OLE) . It is also important to show the relationship between GFR (or changed from baseline in eGFR) and the 3 subgroups ( Placebo MODIFY, Lucerastat MODIFY / OLE and Lucerastat OLE), on the line chart figures with the longer duration ( OLE to 18 months). In the line chart, the statistical significance should be presented.\n#2. With the important results, the results on males and females who receive Lucerastat showed totally different. The figure 3 should include the Figure S4, and the important meanings on different gender. In addition, the figure 3 should include the 3 subgroups (Placebo MODIFY, Lucerastat MODIFY/OLE, Lucerastat OLE) with longer duration. With the extended figures, the readers could understand the long-term relation of renal function and Lucerastat more clearly.", "reviewer_4": "We appreciate the authors' efforts to accommodate some of our remarks. We believe the manuscript improved slightly.\nHowever, the spin bias persists. Overall, the updated manuscript still has strong causal conclusions, which are not supported by the data. Below, we describe each remark in more detail:\nMajor remarks\n#1. Spin bias. Our main concern with the updated version of the manuscript continues to be with the spin bias. As pointed out in our first round of reviews, the authors changed their narrative from neuropathic pain (primary outcome) to renal function. Besides, the claim of lucerastat’s superiority over placebo on renal function lacks statistical support and could have very low credibility, as it is based on a secondary outcome derived from a subgroup analysis with a non-prespecified cutoff.\nWe believe the strong causal statements (e.g., abstract: “eGFR increased with lucerastat and decreased with placebo”) should be more circumspect, since the results shown in Figure 3 are very consistent with the null hypothesis. The authors did not report an effect size (95% confidence interval), but, in the results section, they mentioned: “In the 27 participants on lucerastat, mean eGFR increased from baseline to Month 6 by 3.8 (SEM 2.2) mL/min/1.73m2, while in the 13 participants on placebo, mean eGFR decreased by -1.6 (SEM 4.0) mL/min/1.73m2 (Figure 3).”\nThese results can be used to perform a simple t-test for independent samples, which indicates a mean difference of 5.40\nmL/min/1.73m2 (95% CI, -3.11 to 13.91 mL/min/1.73m2), yielding a two-sided P = 0.21. Thus, the results presented in Figure\n3 remain remarkably consistent with the null hypothesis.\nOverall, we believe the manuscript provides suboptimal evidence of benefit of lucerastat over placebo for renal function,\neither among participants with normal renal function or those with impaired renal function at baseline. We acknowledge that\nthe interaction test (with a P = 0.09) is markedly underpowered, but based on the reported data, there is no convincing\nevidence on subgroup effects. All causal statements regarding the benefits of lucerastat on renal function should be\nremoved and replaced with more cautious and circumspect terms.\nOpportunities for improvement:\n#2. Ensuring that all non-prespecified analyses are clearly labelled as such. The authors insist on focusing on the results of\na small subsample of 40 participants with renal impairment at baseline. Although the subgroup analysis had been pre-\nspecified, the authors used a different cutoff (eGFR <90 mL/min/1.73m2 versus eGFR <60 mL/min/1.73m2 in the protocol).\nThe authors explained in the rebuttal letter that this change was necessary due to the low number of participants. If possible,\nall these critical changes to the protocol, which impact how the study's conclusions are drawn, should be clearly mentioned\nand labelled in the results section and all display items.\n#3. eGFR subgroup analyses . It would be important to provide readers with a table showing baseline characteristics for\nthose eGFR subgroups. Imbalances in ARB/ACE inhibitor co-treatments could have, at least to some extent, influenced the\neGFR mean differences toward apparently “more positive” values in the lucerastat group. Adjustment for co-treatments\nknown to provide renal protection would be essential for these subgroup analyses.\n#4. Hypothesis generating results. If other trials are available, the potential association between lucerastat and\nimprovements in renal function over time could be better assessed through an individual patient data meta-analysis, which\nwould provide greater power to evaluate both main effects and interaction effects. The current trial should be considered\nsolely as a hypothesis-generating study and all renal subgroup results should be clearly labelled as exploratory outcomes.\n#5. Interaction tests. Testing for interaction in randomized trials requires a systematic approach to ensure valid and\ninterpretable results. We recommend that the authors follow the guidance provided in the relevant literature [1-4], and revisit\nthe statistical methodology and reporting practices used to assess interaction in future submissions. It will be essential to\nprovide a rationale for why an interaction might be anticipated, supported by a sound theoretical/clinical justification.\nIdeally, it is essential to guide readers through the analysis by explaining the steps taken and presenting all the data, both\noverall and by subgroups, in a forest/caterpillar plot. This allows readers to draw their own conclusions about whether non-\nsignificant interactions should be considered definitive evidence of no interaction.\nReferences guiding subgroup analyses and interaction testing in randomized trials\n1. Moher D, Hopewell S, Schulz KF, Montori V, Gøtzsche PC, Devereaux PJ, Elbourne D, Egger M, Altman DG. CONSORT\n2010 explanation and elaboration: updated guidelines for reporting parallel group randomised trials. BMJ. 2010 Mar\n23;340:c869 (Specially item Item 12b, page 14)\n2. Lagakos SW. The challenge of subgroup analyses--reporting without distorting. N Engl J Med. 2006 Apr 20;354(16):1667-\n9. doi: 10.1056/NEJMp068070. Erratum in:N Engl J Med. 2006 Aug 3;355(5):533. PMID: 16625007.\n3. Yusuf S, Wittes J, Probstfield J, Tyroler HA. Analysis and interpretation of treatment effects in subgroups of patients in\nrandomized clinical trials. JAMA. 1991 Jul 3;266(1):93-8. PMID: 2046134.\n4. Wallach JD, Sullivan PG, Trepanowski JF, Sainani KL, Steyerberg EW, Ioannidis JP. Evaluation of Evidence of Statistical\nSupport and Corroboration of Subgroup Claims in Randomized Clinical Trials. JAMA Intern Med. 2017 Apr 1;177(4):554-\n560.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "REVIEWERS' COMMENTS\nReviewer #3 (Remarks to the Author):\nWe appreciate the authors' efforts to address our remarks.\nThe manuscript has significantly improved and is now satisfactory.\nWe endorse its acceptance for publication.\nReviewer #4 (Remarks to the Author):\nThe authors have addressed the requested revisions, resulting in a clearer and more robust manuscript. The use of causal\nlanguage has been appropriately refined, with more circumspect interpretations of the the findings. We have no further\ncomments.\nReviewer #5 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.\n\n======================================================================\nATTACHED REVIEWER REPORT — IMPORTANT\nThe text below is a complete reviewer report submitted as a PDF\nattachment. It appears after the Creative Commons license block.\nAssign it to whichever reviewer wrote 'see pdf' or had no inline text.\n======================================================================\n\nNature Communications manuscript: NCOMMS-24-45423A\nWe wish to thank the Reviewers for their interest in our manuscript and the valuable and\nconstructive comments we have received. As you will see, we have revised our manuscript based\non these comments, with changes in the manuscript text file highlighted in yellow. We also provide\na clean copy for your convenience.\nPlease find our point-by-point responses below.\nWe hope these modifications have sufficiently addressed the referees’ comments and look forward\nto your final decision.\nKindest regards,\nProf. Hughes\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThis manuscript evaluates the effects of lucerastat, a substrate reduction therapy, in patients with\nFabry disease. The primary objective was to determine the effect of lucerastat on neuropathic pain,\nwith secondary objectives assessing gastrointestinal symptoms, FD biomarkers, and\nsafety/tolerability. The results indicate that lucerastat failed to improve neuropathic pain,\nabdominal pain, or diarrhea at six months. However, in an interim analysis of the open-label\nextension, lucerastat showed promising effects in improving eGFR in participants with reduced\nbaseline kidney function.\nThe finding of an improvement in eGFR with lucerastat is particularly interesting and warrants\nfurther investigation. Below are my comments and suggestions to enhance the quality and impact\nof the manuscript:\n1. Fabry disease is a progressive disorder. What is the rationale for discontinuing effective and\napproved treatments, such as enzyme replacement therapy (ERT), in this study? Were there\nmeasures to ensure disease progression did not compromise patient outcomes during the trial?\nClarification of the ethical and clinical justifications is necessary.\nResponse:\nWe thank the Reviewer for their comments and for allowing us to clarify our rationale for\ndiscontinuing effective and approved treatments. During the MODIFY study, the temporary\ninterruption of ERT in subjects with persisting neuropathic pain despite ERT was considered\nacceptable for three reasons:\n1\n1. Fabry is a slowly progressive disease, and no significant clinical worsening in subjects who\ndiscontinued ERT was expected within the time frame of the study.\n2. Patients with an advanced stage of disease and those at high risk of developing clinical signs of\norgan involvement within the time frame of the study were excluded.\n3. One of the criteria for discontinuing ERT, as recommended by the European Fabry Working\nGroup, includes the lack of clinical response when the sole “indication” for ERT is neuropathic\npain and the patient requests to stop ERT (Biegstraaten et al. 2015).\nIn addition, both the MODIFY and its open-label extension protocols allowed the re-start of ERT if this\nwas judged medically indicated by a significant disease progression. Of the 118 randomized\nparticipants, only 3 (re-)initiated ERT during the 6-month double-blind placebo-controlled period of\nMODIFY as reported in “Results – Patient population and baseline characteristics”.\nNo modification was made to the manuscript.\n2. The manuscript reports that mean eGFR increased from baseline to Month 6 in both the\nlucerastat and placebo groups. How do the authors explain this improvement, particularly in the\nplacebo group?\nResponse:\nThis is an interesting point, and we thank the Reviewer for highlighting it. Overall, there was indeed\nno mean decrease in eGFR in the placebo group, and instead, a small increase (+1.3 mL/min/1.73m2)\nwas observed. However, of the 118 participants randomized in MODIFY, 78 had no renal impairment\nat baseline (a small increase in renal function was observed in these participants for both lucerastat\nand placebo), and 40 had renal impairment. Notably, in participants with abnormal kidney function\nat baseline, eGFR decreased on placebo during MODIFY, as shown in Figure 3 and described on page\n6.\nIn Fabry disease, renal deterioration is not a linear process, and patients with a normal range of renal\nfunction can remain stable for a long time, even classic male patients, and then experience\nproteinuria and a rapid deterioration (Germain 2010). No deterioration but a small improvement was\nobserved on placebo in participants without renal impairment at baseline or in the overall\npopulation, with unclear cause.\nWe have included a sentence in the Discussion to explain the small improvement in eGFR in the\noverall placebo group (page 10, lines 266-268).\n3. A significant proportion of participants had the classic subtype of Fabry disease, yet the number\nof patients with cardiac involvement appears limited. This could significantly restrict the evaluation\nof left ventricular mass index (LVMi). Do the authors believe this limitation influenced their ability to\ndetect changes in cardiac outcomes?\nResponse:\nLVMi was limited by the absence of cardiac hypertrophy at baseline. We have added a sentence in\nthe Discussion to clarify this point (page 11, line 288-290).\n4. Fabry disease typically manifests renal involvement in approximately 80% of patients with the\nclassic phenotype around the age of 40. However, Table 1 indicates a relatively low median UACR\nvalue. Could the authors provide an explanation for this finding? Was it influenced by study\ninclusion criteria or prior treatments?\nResponse:\nThe Reviewer raises an interesting point here. The relatively low median UACR in the study\npopulation with a mean age of 40 years can be explained by several factors:\n- Patients with severe renal insufficiency (eGFR <30 mL/min/1.73 m2) or on dialysis for the\ntreatment of chronic kidney disease were excluded.\n- More than 50% of the patients enrolled were female, in which the progression of the disease is\nusually slower than in males.\n- The female participants were, on average, 10 years older than the male participants. Despite\nthis, the median UACR was much lower in females than males (20.0 vs 28.6 mg/g).\n- Approximately 60% of the patients were ERT-naïve or pseudo-naïve. One can assume that\nthese patients were in a less advanced stage of the disease than patients who required ERT.\nNo modification was made to the manuscript.\n5. Due to mosaicism, disease manifestations can differ significantly between male and female\npatients. Were there any notable differences in the effect of lucerastat on eGFR between genders?\nResponse:\nWe thank the Reviewer for allowing us to clarify this point. No notable differences in the effect of\nlucerastat on eGFR between male and female participants were observed. The annual eGFR slope\nLS mean improved on lucerastat in both sex by more than 50% (from -2.84 to -1.04 mL/min/1.73 m2\nin 46 female participants and from -4.07 to -1.94 mL/min/1.73 m2 in 47 male participants).\nNo modification was made to the manuscript.\n6. Fabry disease is a slow-progressing condition, and six months might not be sufficient to\ndemonstrate the full therapeutic potential of lucerastat. What are the authors' thoughts on this\nlimitation?\nResponse:\nWe agree with the Reviewer. This is why we integrated the interim analysis of the open-label extension\nwhere lucerastat patients were continued on lucerastat and where we see this amelioration of eGFR\nslope in patients who were rapidly deteriorating. The assessment of symptoms such as neuropathic\npain and gastrointestinal symptoms based on patient-reported outcomes are subjective by nature\n3\nand require the use of a placebo arm comparator. The placebo-controlled period in MODIFY was\nlimited to 6 months for ethical reasons. In the Discussion, we acknowledge that the inhibition of Gb3\nsynthesis by lucerastat may not have caused sufficient substrate reduction in the dorsal root ganglia\nin the 6-month treatment period (page 9, lines 241-244). The open-label extension was designed to\nassess the long-term effect of lucerastat on objective parameters such as eGFR slope in comparison\nto the slopes observed in those patients prior to their inclusion in the MODIFY study.\nNo modification was made to the manuscript.\n7. Given the significant renal findings, it may be beneficial to revise the title to better reflect the\nstudy’s impact on kidney outcomes.\nResponse:\nWe agree with the reviewer and have modified the title as suggested. The revised title is now:\nEffect of lucerastat on kidney function in Fabry disease: Results from a pivotal phase 3 study\nand its open-label extension\nReviewer #2 (Remarks to the Author):\nThe manuscript by Nordbeck et al. reports on a phase 3 RCT of lucerastat along with an open label\nextension (OLE) study for the treatment of Fabry Disease. The main study showed no benefit of\nlucerastat, a substrate reduction therapy, compared with placebo on the primary outcome of\nneuropathic pain reduction. Although the primary outcome was negative, there were some\nunexpected secondary findings of improvement or stabilization in kidney function with lucerastat,\nparticularly in those with evidence of more rapid progression prior to study entry. The study appears\nto have been well conducted, reported and adequately powered with regards to the main outcome.\nThe limitations described are appropriate. This is a multicentric and large study for a rare disease,\nwhich provides important insight on the use of substrate reduction therapy for clinically relevant\noutcomes. It provides valid information on the primary outcome of neuropathic pain. There are\nsome issues that should be addressed by the authors:\n1. Due to the COVID pandemic several amendments were made, including a switch of the primary\noutcome from a binary to continuous measure for analysis, and combination of the OLE part of the\nstudy with the main trial for some of the analysis. The OLE was also lengthened from 24 to 72\nmonths. When the primary outcome was negative, there was very good participation in the\nextension study. Were participants informed of this result and allowed or informed that they could\nresume their enzyme replacement therapy during the OLE? Would this not have been considered\nstandard of care?\nResponse:\nwas unblinded, the participants were informed of the results, which were included in an updated\nversion of the patient information leaflet. Participants were asked to re-consent to continue\nparticipating in the OLE study.\nFurthermore, irrespective of the MODIFY results, both MODIFY and OLE study protocols included the\noption to re-start ERT if judged clinically needed by the investigator (this is also described in the\nmanuscript in “Methods-Procedures” page 14, lines 385-387). This information was included in the\npatient information leaflet and the consent process since the start of the studies.\nNo modification was made to the manuscript.\n2. The improvement of GFR is quite interesting. It would be helpful for the authors to propose a\nbiologic mechanism to understand this effect. As the median baseline eGFR was quite preserved,\nare the authors suggesting that lucerastat had a favourable (disease-modifying) effect on the\nkidneys as early as 2-3 months. Could this be a hemodynamic effect or hyperfiltration? Did any pre-\nclinical or earlier phase studies show similar effects? Was there a washout period after the main\nstudy or after the OLE where eGFR was measured again to assess if the GFR improvement was\nsustained?\nResponse:\nWe agree with the reviewer and have modified the Discussion to include a proposed mechanism of\naction to explain the effect of lucerastat on eGFR, as follows (page 10, lines 274-279):\n“The mechanism of action of lucerastat on eGFR is assumed to be primarily based on the reduction\nof lipid inclusions in the kidney cells. A pre-clinical study indicated that, compared with untreated\ncontrols, lucerastat treatment significantly reduced the storage of α-GalA substrates, including Gb3,\nin the kidneys of Fabry mouse models after 20 weeks (Welford et al. 2017). In the previous clinical\ntrial in patients with Fabry disease, the duration of lucerastat treatment was too short (12 weeks) to\nmake any meaningful conclusion on the effect of lucerastat on renal function (Guérard et al. 2018)”.\nThe Introduction has also been revised to include the observations on the effect of lucerastat in the\nkidneys of Fabry mice (page 4, line 83).\nAs there was no withdrawal period after the main study or the OLE, we were unable to assess if the\neGFR improvement was sustained.\n3. If lucerastat is proposed to have a renoprotective benefit, was there any impact on albuminuria\nthroughout the study?\nResponse:\nThis is an interesting point, and we thank the Reviewer for highlighting it. As mentioned by Reviewer\n#1, UACR was low at baseline, with a median of 23.0 mg/g (Table 1). There was no difference in the\nchange in UACR from baseline to Month 6 between lucerastat and placebo in the MODIFY study, as\ndetermined by an additional post-hoc analysis of UACR on log-transformed data, which yielded a\ndifference between treatment groups.\nNo modification was made to the manuscript.\n4. The authors use the pre-study eGFR decline to understand better those that had a renal benefit\nwith lucerastat. Although this is interesting, it is a measure that was not in the original study\nprotocol, and was not collected systematically. The authors do show in the supplement that the\nmajority had at least 3-5 creatinine values over time, but it is unclear if these represent values when\na patient was stable or if they were done for some cause. This raises the concern for assessment or\ndetection bias. I would recommend that the description of eGFR decline as compared to placebo\nbe compared only after study randomization.\nResponse:\nRespectfully, we disagree with the Reviewer. The collection of available serum creatinine data from\nthe period preceding randomization was planned in the original protocol of MODIFY. Specifically, in\norder to build a pre-randomization slope, at least 2 values per year, 6 months apart, during the last\ntwo years before randomization were to be collected (if available). We do agree, however, that we\ncannot exclude some bias in the timing of creatinine measurements, and this point is mentioned as\na limitation in the Discussion (page 11, lines 296-299). However, the overall slope should not be\naffected by the selected time points.\nNo modification was made to the manuscript.\n5. It appears that only a few participants were not included in the OLE. Were any placebo treated\nparticipants excluded due to the eGFR <30 criteria? If so, removing these individuals may result in the\nappearance of more stability of mean eGFR in the OLE cohort.\nResponse:\nWe thank the Reviewer for allowing us to clarify this point. After randomization in MODIFY,\nparticipants had to discontinue study treatment if eGFR was <15 mL/min/1.73m2. Hence,\nparticipants with an eGFR >15 mL/min/1.73m2 and <30 mL/min/1.73m2 at the end of MODIFY could\nstill be eligible to enroll in the OLE. This has been clarified in the “Methods-Participants” section\n(page 13, lines 361-363).\nAs shown in Figure 1, of the 37 placebo-treated participants in MODIFY, 34 completed the 6-month\ntreatment, and all were enrolled in the OLE. Among the 3 participants who did not complete the\ntreatment in MODIFY, 1 was lost to follow-up, and 2 participants prematurely discontinued due to\nadverse events. These participants had eGFR values ≥90 mL/min/1.73m2 throughout their\nparticipation in MODIFY. Of note, an eGFR <30 mL/min/1.73m2 was only an exclusion criterion for the\nMODIFY trial.\n6\n6. An improvement in eGFR could be related to a reduction in NSAID use. Although there was no\nreduction in pain, was there any reduction in NSAID use?\nResponse:\nThe Reviewer raises an interesting point here. The use of NSAIDs, which was recorded daily in the\neDiary, was fluctuating as NSAIDs were used PRN by the patient. We, therefore, did not look at the\nuse of NSAIDs in MODIFY, as daily fluctuations of such treatment over a 6-month period would\nrender any analysis of the potential impact on eGFR difficult to interpret.\nNo modification was made to the manuscript.\nReviewer #3 (Remarks to the Author):\nIt is an important manuscript about the treatment of Fabry Disease with Lucerastate\nHow, some suggest and revision should be done.\nPage3 line 73.\nIn this part, the authors should not mention about the ERT disadvantage,\nSome bias could be noted,\nIf they want to do this, they should put the important part on the discussion for detailed comparison\nof different treatment strategies such as ERT and Lucerastat.\nResponse:\nWe agree with the Reviewer and have deleted the sentence on the pitfalls of ERT and migalastat.\nOn page 4, line 77\nOn the section of introduction, mostly mentioned about the kidney function, but they did not\nmentioned about the heart function, brain function, etc.\nHowever, if only focus on the kidney function, should we say the heart function is not important?\nSo the manuscript must be more fair for all the important vital organ function statement.\nResponse:\nWe agree with the Reviewer and have added a sentence on other organ involvement (page 4, lines\n78-79).\nOn the page 4, line 101, the authors mention about the “surprising effect of lucerastat on kidney\nfunction.”\nThis result should be more neutral in the introduction part.\nSuch as “ in this study, we try to see the kidney function after lucerastat.\nResponse:\nwhich showed a surprising effect of lucerastat on kidney function.”\nOn page 5, line 127\n“with a numerically smaller reduction with lucerastat versus placebo (LSM\ndifference 0.42 [95% confidence interval (CI) -0.40 to 1.23], p=0.32; Figure 2).”\nThis should add on without statistically significant.\nResponse:\nWe agree with the Reviewer and have clarified the sentence to show that it was not statistically\nsignificant. We have also deleted the P value.\nOn page 5, line 127\n“The median number of days on significant rescue pain therapy was 0 in both treatment groups, “\nOn Table S3, “Days on significant rescue pain therapy” on both treatment groups and placebo all\nshowed 0\nBut in this sentence, the result just showed 0 on both treatment group.\nThe authors should clearly showed the both group is MODY and placebo? MODY and OLE??\nMODY/OLE and placebo\nHowever is there statistical significance noted relative shorter mean days on the Lucerastat group\nshould be calculated and showed the results.\nIn addition, the pain characeterisitic change? The number of patients with pain-control medication,\nor other important parameters for pain should be mentioned to see the pain change after\nLucerastat group,\nBy the way, the S3 just mentioned about the MODIFY study( for 6 months) , but the OLE (extended\nstudy) should also be presented.\nResponse:\nWe thank the Reviewer for their comments and for allowing us to clarify this information. Significant\nrescue pain therapy was only collected in the eDiary in MODIFY, as it is a variable that would be\ndifficult to evaluate in the OLE in the absence of a placebo control arm. The “Methods-Procedures”\nsection and the Results section have been updated to make this clearer (page 14, lines 383-385).\nNo statistical difference was noted relative to the numerically smaller mean number of days with\nsignificant rescue pain medication in the lucerastat group compared with the placebo group. This\nhas been updated in the “Results-primary and secondary endpoints” (page 5, lines 130-133).\nThe study focused on assessing the worst intensity of neuropathic pain using the validated tool\nmodified BPI-SF3 rather than evaluating the change in pain characteristics. The pain questionnaire\nand the definition of neuropathic pain have been included in Supplemental Materials Table S10.\nAt the time of screening, each patient was provided with an Individual Pain Management Plan\n(IPMP) by the investigator. The IPMP included customized instructions regarding the use of pain\nmedication during the study including the use of rescue pain medication in case of unbearable\n8\npain. The section “Procedures” has been amended to reflect the use of IPMP during the study (page\n13, lines 380-387). In total, 79% of the patients were using pain medication at the time of\nenrollment in MODIFY, as stated in section “Patient population and baseline characteristics” (page\n5, lines 115-116), with more details provided in Table 1 (page 26).\nOn page 5 Line 1333, “LSM difference”\nSome showed “LSM” , some showed “LS mean” , the manuscript should be corrected carefully.\nResponse:\nWe agree with the Reviewer and have changed “LS mean” to “LSM” throughout the manuscript.\nOn page 6, line 137, the study showed “ in both treatment groups”,\nThe authors should clearly say the both groups meaning. The MODY vs Placebo”?,\nThe MODY vs OLE( extended study)?. In addition, the result of MODY and OLE ( extended group)\nshould be presented here, not just MODY and placebo.\nResponse:\nWe thank the Reviewer for their comments and for allowing us to clarify this information. Indeed, in\nthe section “Renal function endpoints,” we report results for MODIFY (6 months placebo-\ncontrolled) (first and second paragraphs) and the MODIFY+open-label extension (third paragraph\nstarting with “In the 114 participants exposed to lucerastat in MODIFY/OLE,….”.)\nWe have also clarified the definition of each group (in terms of renal impairment at baseline) in the\nfirst sentence of the second paragraph of this section.\nOn page 6, line 140, the statement: “mean eGFR increased from baseline to Month 6 by 3.8 (SEM\n2.2) mL/min/1.73m2””\nIn the result, the authors showed the increased GFR per year, but totally, the decreasing GFR/year\nwas noted ( page 6, line 143),\nIs this mean that the normal GFR patient received Lucerastat showed a rapid decreasing GFR???\nIn this short period, how many years be calculaged.\nResponse:\nWe thank the Reviewer for their comments and for allowing us to clarify this information. The first\nstatement (“mean eGFR increased (..)”) referred to change from baseline to Month 6 in the Modify\ntrial, whereas the second statement (“decreasing eGFR/year”) referred to the eGFR slope in the\nMonth-18 interim analysis of the longer Modify/OLE study, which was indeed negative. This slope\nwas, however, less negative than the historical eGFR slope.\nNo modification was made to the manuscript.\nThe figures of MODY/OLE/Placebo patients with GFR < 90 , and normal GFR should be presented.\nthe comparison with placebo should be important.\nResponse:\nWe thank the Reviewer for their comments and for allowing us to clarify this information. The figure\nshowing the change from baseline over time in eGFR in MODIFY in participants without renal\nimpairment has been included in the Supplemental material (Figure S3). The results in this\nsubgroup have been added to the Renal function endpoints section (page 6, lines 143-148).\nIn addition, the Fabry disease showed an X-linked disease. So the results of renal function in male\nand female groups should be presented separately.\nWe also very interested in the different improvements in male and female groups.\nResponse:\nWe thank the Reviewer for their comments and for allowing us to clarify this information. The\nfigures showing the change from baseline over time in eGFR in MODIFY in males and female have\nbeen included in the Supplemental material (Figure S4). The results are described in section “Renal\nfunction endpoints,” (page 6, lines 149-151).\nOn page 6, line 148, some showed LSM, and some showed LS mean, please correct it.\nResponse:\nWe agree with the Reviewer and have changed “LS mean” to “LSM” throughout the manuscript.\nOn page 6 , line 153, the Figure 4, should also showed the results of lucerastat and placebo group.\nResponse:\nWe respectfully disagree with the Reviewer. Figure 5 (previously Figure 4) displays the long-term\neffect of lucerastat on annualized eGFR slope versus the pre-randomization annualized eGFR slope\nusing data from MODIFY/OLE. In the OLE, there was no placebo arm.\nNo modification was made to the manuscript.\nON page 6, line 162, the statement “ lucerastat treatment stopped the eGFR decline. In these\npatients”\nWith the results, the authors should showed the results of all the GFR decline in GFR >90, GFR<90,\nMODY group, OLE group, placebo group.\nIn addition, results in male and female groups are also important.\nResponse:\nWe thank the Reviewer for allowing us to clarify this comment. The results of eGFR slopes for the\nsubgroups of subjects with baseline eGFR >=90, <90, and classic males and females can be found\nin Supplemental Materials Figure S53. We have also added Supplemental Materials Figure S4\nshowing the mean change from baseline over time in eGFR in male and female participants in\nMODIFY.\nResponse:\nWe agree with the Reviewer and have changed “LS mean” to “LSM” throughout the manuscript.\nOn page 7, line 181, the authors showed the decreased plasma Gb3,…;\nThe results in MODY and OLE should be presented.\nIt is interesting in elevated the plasma GB3 in female. So the male data and female data should be\npresented.\nResponse:\nWe thank the Reviewer for allowing us to clarify this point. In MODIFY, the change in plasma Gb3\nwas one of the secondary endpoints, and results are described in the section “Results- Primary and\nsecondary endpoints” (page 5, lines 136-138). Table S7, in Supplemental Materials, reports plasma\nGb3 data by sex for participants receiving lucerastat in MODIFY and the OLE. As the information\nrequested is already included here, we have not made further changes.\nOn page 8, line 199. The statement,..”TEAEs reported with a higher frequency with placebo than\nlucerastat were abdominal distension and decreased GFR”\nIt is interesting that all the patient received Lucerstat did not have any side effects of decreasing\nGFR? Compared with 11% in placebo.\nResponse:\nWe thank the Reviewer for allowing us to clarify this point. Indeed, we consider that the fact that\nnone of the patients in the lucerastat group had side effects of decreasing GFR as compared to\n11% in the placebo group over a period of 6 months is supportive of the improvement in eGFR\nobserved in the lucerastat group overall as well as in the subgroup of subjects who already had\nrenal impairment at baseline.\nNo modification was made to the manuscript.\nOn page 9, line 228, the authors mentioned about the neuropathic pain in FD,\nHowever, some other studies, such as CPT, QST, and SSR could be done,\nIn addition, more definitely the IENFD( intraepidermal nerve fiber density ) could be done for these\ngroups of patients.\nThe neuropathic pain score could be very subjective, so the authors should mentioned about this.\nResponse:\nWe thank the Reviewer for allowing us to clarify this point. We acknowledge that additional studies\ncould be done using more objective measures, such as those quoted by the reviewer and previously\npublished (Uceyler et al. 2011, Uceyler et al. 2013). The primary endpoint of the phase 3 MODIFY\ntrial was choosen based on regulatory requirements to demonstrate a clinical effect using patient-\nreported outcome (PRO) tools such as neuropathic pain scales. Due to the subjective nature of\n11\nsuch PROs, a randomization design and comparison with a placebo arm were used in the trial. We\nhave addressed the Reviewer's concerns by mentioning the subjectivity of the neuropathic pain\nscore in the Discussion section (page 9, line 249).\nBy the way, several different types of pain in Fabry disease were noted.\nEven some showed no pain. the natural characteristic of pain in these groups of patients should be\nclassified, such as the initial pain age, the severity, the characteristics, etc.\nIn addition, the relationship between pain and plasma Gb3 could be mentioned.\nResponse:\nWe thank the Reviewer for allowing us to clarify these points. All MODIFY participants had to report\nmoderate to severe neuropathic pain over the 4 weeks prior to randomization (key inclusion criterion\nin the section Methods-participants), and a definition of Fabry neuropathic pain was included in the\nvalidated modified BPI-SF3 questionnaire.\nPatients with other diseases or conditions associated with pain components that could have\nconfounded the assessment of neuropathic pain were excluded. This has been clarified in the\nMethods-participants section (page 13, lines 355-356).\nWe have modified the text (page 5, lines 117-119) and included Table S2 in Supplemental Materials,\nwhich shows baseline characteristics of the type of neuropathic pain symptoms, time since first\nneuropathic pain symptoms, and pain symptom triggering factors.\nThe severity of neuropathic pain was assessed on a daily basis using the eDiary (Table 1), and the\nmean (SD) baseline modified BPI-SF3 score has been added to the text in “Results-Patient\npopulation and baseline characteristics” (page 5, lines 117-119).\nAs highlighted in the Discussion section, the lower plasma and urinary Gb3 levels observed after 6\nmonths of treatment were not associated with a reduction in neuropathic pain. One possible\nexplanation is that the short duration of the treatment may not be sufficient for a drug with an SRT\nmode of action, and this has been mentioned in the Discussion (page 10, lines 238-247).\nOn page 9, line 250, the authors showed the “lucerastat improved participants’ eGFR slope\ncompared to the pre-randomization slope, “\nThe important vital organ in Fabry are renal, cardiac, and CNS. So why the renal could be effective\nand no effect on heart should be mentioned and discussed, especially the mechanism . Also, from\nthis result, did the authors could give readers some suggestion in patients with FD??\nResponse:\nWe thank the Reviewer for allowing us to clarify this point. We highlight in the Discussion section that\nin this study, cardiac data were mainly collected for safety analysis as the study was considered too\nshort to evaluate changes over time, and very few patients had cardiac hypertrophy at baseline.\nNevertheless, the observation of no increase in LVMi over an 18-month period may indicate some\nstabilization. The text has been modified to clarify this (page 11, lines 288-293).\nPeer-review NCOMMS-24-45423A\nThe manuscript describes findings from a pivotal, placebo-controlled, randomized trial examining\nthe efficacy and safety of lucerastat in people with Fabry disease. The paper is well written and\ngenerally well presented. The statistical methods are reasonably robust and well-aligned with the\noriginal statistical analysis plan. Overall, the manuscript presents findings of great medical and\nscientific interest. However, the current version presents several areas for improvement. Without\nadequate clarification and justification, the paper risks spin bias and invalid claims of subgroup\neffects, among other issues.\nMajor remarks\n#1. Non-prespecified subgroup analysis. The major limitation of the manuscript refers to the\nchange of focus from the primary outcome of neuropathic pain at month 6 to a non-prespecified\nsubgroup analysis focused only on participants with abnormal renal function at baseline, defined\nas those with an eGFR <90 mL/min/1.73m2. The trial protocol included a predefined subgroup\nanalysis that compared patients based on their eGFR at the screening visit, categorizing them into\ntwo groups: those with eGFR ≥ 60 mL/min/1.73 m² and those with eGFR < 60 mL/min/1.73 m². The\nsame analysis appears in the statistical analysis plan. However, the investigators conducted a\ndifferent subgroup analysis for eGFR. Besides, the cutoff used differs substantially from what was\nplanned.\na. If possible, could the authors justify this new analysis and clearly label it in the manuscript as a\nnon-pre-specified analysis?\nResponse:\nWe thank the Reviewer for allowing us to clarify this point. The new analysis in a different subgroup\nwas performed post-hoc in replacement of the original subgroup analysis due to the low number of\nparticipants with a baseline eGFR <60 mL/min/1.73 m². Indeed, only 9 participants (7 in the\nlucerastat group and 2 in the placebo group) had a baseline eGFR <60 mL/min/1.73 m² which would\nhave limited the meaningfulness of this subgroup analysis. Table 1 has been updated to include the\nnumber of participants with a baseline eGFR <60 mL/min/1.73 m² (page 25). The post-hoc nature\nof the analysis is stated in the section “Methods-Statistical analysis” (page 16, lines 444-445).\nb. Additionally, could the authors explain the rationale behind the change in the cutoff? Was this\nadjustment based on clinical reasoning, or was it influenced by the number of participants?\nWe suggest that the main conclusions of the trial in the abstract and main text focus only on the\nprimary outcome of the randomized trial comparison.\nResponse:\nAs explained in the previous answer, the main reason for performing a new post hoc analysis was\nthe low number of participants with an eGFR<60 mL/min/1.73 m² at baseline. Instead, the\n13\nthreshold of 90 mL/min/1.73 m² was selected as this is the standard value used to distinguish\npatients with normal eGFR from those with abnormal eGFR (see (Kidney Disease: Improving Global\nOutcomes (KDIGO) CKD Work Group 2024)).\nNo modification was made to the manuscript.\n#2. Risk of an invalid claim of a subgroup effect for eGFR. There is an important limitation in the\nnon-prespecified inferences (made in Figure 3). These analyses lack support from formal\ninteraction testing that examines the interaction between treatment and eGFR ≥90 vs < 90\nmL/min/1.73 m² at baseline. Without this interaction test, the validity of the subgroup effect cannot\nbe properly assessed, potentially leading to misleading or unsupported conclusions.\nResponse:\nWe thank the Reviewer for allowing us to clarify this point. While we found this result of interest,\nwe did not intend to claim a subgroup effect for eGFR (eGFR <90 mL/min/1.73m2). For transparency,\nwe have added the results of the complementary subgroup (eGFR ≥90 mL/min/1.73m2) and also added\nthat the interaction test was not statistically significant (p=0.09) (page 6, lines 143-148).\n#3. Fixed-sequence testing and reported P-values. The trial used fixed-sequence testing (page 51 in\nthe statistical analysis plan). If the null hypothesis is not rejected for an endpoint in the sequence,\nno claims of statistical significance can be made for the subsequent endpoints. Hence, if the null\nwas not rejected for the primary outcomes, can the authors:\na. Justify why P-values have been reported for the remaining secondary outcomes.\nAlso, please clarify in the main text that the fixed-sequence testing was pre-specified.\nResponse:\nWe thank the Reviewer for allowing us to clarify these points. We have explained that the fixed-\nsequence testing was prespecified in the section “Methods – Statistical analyses” (page 15, lines\n426-429). We also added that “If a hypothesis in the sequence was not rejected, p-values for the\nsubsequent hypotheses were to be considered exploratory”. We propose still reporting these p-\nvalues as readers may (or may not) use them as indicators of the probability that the result is a\nchance finding.\n#4. Outcomes not reported. All outcomes that were initially planned should be reported. For\nexample, could the authors clarify why quality of life, depression, and Patient’s rating of change in\ndisease severity outcomes were not included?\nResponse:\nsecondary endpoints of MODIFY and several longer-term outcomes observed in the OLE. To reduce\nthe amount of information presented in the manuscript, not all outcomes were reported. However,\nQoL, depression, and Patient’s rating of change may be reported in a future publication.\nNo modification was made to the manuscript.\n#5. In general, we suggest that the authors avoid causal terminology when describing results of\nanalyses that are not the main analysis of the primary outcome. These are generally exploratory, not\nproperly powered analyses.\nResponse:\nWe agree with the Reviewer and have removed the few instances of causal terminology in the\nmanuscript.\n#6. Please provide further information to allow proper interpretation of your primary outcome and\nsome of the secondary outcomes. For instance, what is the possible range of scores and what do\nhigher values mean for your primary outcome?\nResponse:\nWe thank the Reviewer for allowing us to clarify this point. The text has been modified to show that\nboth neuropathic pain and gastrointestinal pain were rated by the patients on a scale from 0-10\n(higher values corresponding to more severe pain) (page 14, lines 411-415). More details can be\nfound in Wanner 2022.\n#7. Could the authors please discuss how relevant is the between-group difference in the\nbiomarkers? Generally, a moderate to large between-group difference in biomarkers would be\nneeded so that a minimally important between-group difference can be observed downstream in\nclinically relevant outcomes.\nResponse:\nWe thank the Reviewer for allowing us to clarify this point. The between-group differences at Month\n6 reported for plasma Gb3 (-873.53), plasma lysoGb3 (-5.88) and urine Gb3 (-8.32) correspond to\neffect sizes (i.e., difference divided by standard deviation) of approximately 1.5, 0.4, and 0.7,\nrespectively, which could be considered as large, medium and large, respectively. However, it is\nacknowledged that effects on these biomarkers did not translate into effects on the primary and\nsecondary efficacy endpoints, neuropathic pain, or abdominal pain, the main MODIFY endpoints\nconsidered in this paper.\nNo modification was made to the manuscript.\n15\n#8. Can the authors make an explicit statement about blinding of outcome assessors in the trial?\nBecause the primary outcome is patient reported, it can be considered that outcome assessor is\nalso blinded. But it would be also important that the authors clarify this for other outcomes, not\nreported by patients, in this trial.\nResponse:\nWe thank the Reviewer for allowing us to clarify this point. In the section “Methods-Randomization\nand masking” we state that “Participants, investigators, site personnel, and sponsor personnel\ninvolved in the study conduct were unaware of study treatment allocation. In the event of a\nmedical emergency, investigators were permitted to initiate the unmasking process; no unmasking\nevents occurred in the MODIFY study.”\nNo modification was made to the manuscript.\nMinor remarks\n#9. Use of standard errors to convey uncertainty. It would be more appropriate to use 95%\nconfidence intervals rather than standard error, which has been used inconsistently throughout the\nmanuscript (SE or SEM).\nResponse:\nWe thank the Reviewer for highlighting this issue. To achieve consistency, we have removed the\nstandard errors from the text (see also our response to comment #10) and kept only the confidence\nintervals.\n#10. Use of standard errors combined with inappropriate wording of statistical inferences. In the\nsentence below (page 6), the phrase \"numerically higher increase\" gives a misleading impression\nof the statistical and clinical superiority of lucerastat compared to placebo.\nResponse:\nWe thank the Reviewer for highlighting this issue. Although the phrase “numerically higher” does not\nimply statistical significance, we have reworded the sentence to clarify that “numerically higher”\ndoes not mean it is statistically significant (page 6). We have also removed the standard errors to\navoid any suggestion of statistical analysis.\n#11. We were able to replicate the sample size calculation assuming that a 2:1 randomization was\ntaken into consideration.\nCan the authors explicitly state this in their sample size considerations?\nResponse:\nWe thank the Reviewer for highlighting this issue. We have repeated in the statistical analysis\nsection on page 15, lines 422-423 that the randomization was 2:1.\n16\n#12. ANCOVA was not “adjusted” by the treatment group, because treatment group is the main\nfactor of interest. Please rephrase it accordingly.\nResponse:\nWe thank the Reviewer for highlighting this issue and have adjusted the text on Page 15 (lines 435-\n437) according to the reviewer’s suggestion.\nReferences\nBiegstraaten, M, Arngrímsson, R, Barbey, F, et al. (2015). \"Recommendations for initiation and\ncessation of enzyme replacement therapy in patients with Fabry disease: the European\nFabry Working Group consensus document.\" Orphanet J Rare Dis 10: 36. doi:\n10.1186/s13023-015-0253-6.\nGermain, DP (2010). \"Fabry disease.\" Orphanet J Rare Dis 5: 30. doi: 10.1186/1750-1172-5-30.\nGuérard, N, Oder, D, Nordbeck, P, et al. (2018). \"Lucerastat, an Iminosugar for Substrate Reduction\nTherapy: Tolerability, Pharmacodynamics, and Pharmacokinetics in Patients With Fabry\nDisease on Enzyme Replacement.\" Clin Pharmacol Ther 103(4): 703-11. doi:\n10.1002/cpt.790.\nKidney Disease: Improving Global Outcomes (KDIGO) CKD Work Group (2024). \"KDIGO 2024\nClinical Practice Guideline for the Evaluation and Management of Chronic Kidney Disease.\"\nKidney Int 105(4s): S117-s314. doi: 10.1016/j.kint.2023.10.018.\nUceyler, N, He, L, Schonfeld, D, et al. (2011). \"Small fibers in Fabry disease: baseline and follow-up\ndata under enzyme replacement therapy.\" J Peripher Nerv Syst 16(4): 304-14. doi:\n10.1111/j.1529-8027.2011.00365.x.\nUceyler, N, Kahn, AK, Kramer, D, et al. (2013). \"Impaired small fiber conduction in patients with\nFabry disease: a neurophysiological case-control study.\" BMC Neurol 13: 47. doi:\n10.1186/1471-2377-13-47.\nWelford, RWD, Mühlemann, A, Priestman, D, et al. (2017). \"Lucerastat, an iminosugar for substrate\nreduction therapy in Fabry disease: preclinical evidence.\" Mol Genet Metab 120(1): S139-\nS40. doi: https://doi.org/10.1016/j.ymgme.2016.11.369.\n17"}, {"round": 3, "reviewer_comments": {"reviewer_3": "We appreciate the authors' efforts to address our remarks.\nThe manuscript has significantly improved and is now satisfactory.\nWe endorse its acceptance for publication.", "reviewer_4": "The authors have addressed the requested revisions, resulting in a clearer and more robust manuscript. The use of causal language has been appropriately refined, with more circumspect interpretations of the the findings. We have no further comments.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "REVIEWERS' COMMENTS\nReviewer #3 (Remarks to the Author):\nWe appreciate the authors' efforts to address our remarks. The manuscript has significantly\nimproved and is now satisfactory. We endorse its acceptance for publication.\nAuthors’ response: Thank you.\nReviewer #4 (Remarks to the Author):\nThe authors have addressed the requested revisions, resulting in a clearer and more robust\nmanuscript. The use of causal language has been appropriately refined, with more circumspect\ninterpretations of the findings. We have no further comments.\nAuthors’ response: Thank you.\nReviewer #5 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\n2"}]

Decision letter