Phase 1 Healthy Volunteer Study of AL01211, an Oral, Non‐brain Penetrant Glucosylceramide Synthase Inhibitor, to Treat Fabry Disease and Type 1 Gaucher Disease

Glycosphingolipid (GSL) storage diseases are caused by deficiencies in the enzymes that metabolize different GSLs in the lysosome. Glucosylceramide synthase (GCS) inhibitors reduce GSL production and have potential to treat multiple GSL storage diseases. AL01211 is a potent, oral GCS inhibitor being developed for the treatment of Type 1 Gaucher disease and Fabry disease. AL01211 has minimal central nervous system penetration, allowing for treatment of peripheral organs without risking CNS‐associated adverse effects. AL01211 was evaluated in a Phase 1 healthy volunteer study with single ascending dose (SAD) and multiple ascending dose (MAD) arms, to determine safety, pharmacokinetics including food effect, and pharmacodynamic effects on associated GSLs. In the SAD arm, AL01211 showed a Tmax of approximately 3.5 hours, mean clearance (CL/F) of 130.1 L/h, and t1/2 of 39.3 hours. Consuming a high‐fat meal prior to dose administration reduced exposures 3.5‐5.5‐fold, indicating a food effect. In the MAD arm, AL01211 had an approximately 2‐fold accumulation, reaching steady‐state levels by 10 days. Increasing exposure inversely correlated with a decrease in GSL with plasma glucosylceramide and globotriacylceramide reduction from baseline levels, reaching 78% and 52% by day 14, respectively. AL01211 was generally well‐tolerated with no AL01211 associated serious adverse events, thus supporting its further clinical development.

2][3][4] GCS catalyzes the addition of glucose to ceramide to produce glucosylceramide (GL1) (Figure S1).Glucosylceramide can be further glycosylated by other synthetic enzymes to form a variety of GSL derivatives.These GSLs are present in the plasma membrane (and intracellular membrane structures) and play diverse roles in cell biology. 4The degradation of each of these GSLs is dependent on specific enzymes located in the lysosome.Several genetic GSL storage diseases are caused by loss-of-function mutations in the genes that encode the enzymes that degrade individual classes of GSLs. 5 For example, Gaucher disease (GD) is caused by mutations in the gene encoding the enzyme glucocerebrosidase, 6,7 which degrades GL1, and Fabry disease (FD) is caused by mutations in the gene encoding the enzyme alpha-galactosidase A (αGal), which de-grades the GSL globotriaosylceramide (GL3). 8,9These mutations lead to accumulation of GL1 and GL3 in the lysosomes of different cell types, resulting in multiple organ disease and organ failure in GD and FD, respectively.
GSL storage diseases are classified as either neuronopathic, where the disease affects the central nervous system (CNS), and non-neuronopathic, where the disease does not directly affect the CNS.FD and Type 1 Gaucher disease (GD1) is primarily non-neuronopathic while Type II and III Gaucher disease (GD2 & GD3) and GM2 and GM1 gangliosidosis are neuronopathic GSL storage disorder resulting in severe infantile to later onset neurodegenerative disease. 41][12][13] ERT works by providing affected patients with functional enzymes through intravenous infusion.Although effective, ERT has some limitations due to incomplete penetration of the enzyme into all affected tissues. 14,15Chaperone therapy for FD targets folding and maturation of the mutant αGal enzyme.Migalastat is a small molecule pharmacological chaperone approved for FD patients having amendable mutant forms of αGal. 16However, only approximately 35% of Fabry patients have amenable GLA mutations. 17liglustat and Miglustat are GCS inhibitors that have been approved for the treatment of GD1. 6 These marketed GCS inhibitors are generally well tolerated, safe, and effective at treating GSL storage diseases by reducing the synthesis of the GSLs that accumulate in these diseases. 18,19This therapeutic approach is referred to as substrate reduction therapy (SRT) because it reduces the synthesis of the molecules that directly contribute to these different storage diseases.][22][23][24][25][26] Venglustat is a brain penetrant GCS inhibitor that is being developed for FD, GD3, and GM2 gangliosidosis.Lucerastat is an imino-sugar analog that inhibits GCS at micromolar concentrations and is being developed for Fabry disease.
AL01211 is a novel GCS inhibitor that is being developed to treat the non-neuronopathic GSL storage disorders, GD1 and FD (Figure 1).A series of in vitro and in vivo preclinical studies were conducted to characterize the pharmacological properties of AL01211, which will be published in another manuscript.AL01211 inhibits GCS enzyme activity in vitro with an IC50 of 5.3 nM.8][29] In vivo excretion studies in rats have shown that AL01211 is primarily excreted in feces via hepatobiliary routes and has low levels of renal clearance.In vitro studies have shown that AL01211 is primarily metabolized by CYP3A4.Caco2 studies have shown that AL01211 is a substrate for the P-glycoprotien (Pgp) transporter, which may explain the low level of CNS penetration.The in vivo human metabolism of AL01211 and any clinical drug-drug interactions will be assessed in future clinical studies.
A Phase 1, first-in-human, randomized, doubledblind, placebo-controlled study of AL01211 was conducted in healthy adult participants.The study included a single ascending dose (SAD) arm with a crossover food effect cohort, followed by a multiple ascending dose (MAD) arm.The results of this study showed that the pharmacokinetics (PK), pharmacodynamics (PD), and safety and tolerability findings support the further clinical development of AL01211.These studies also revealed a significant food effect when AL01211 was given immediately after a high fat meal.

Phase 1 Study design
This Phase 1 study is a first-in-human, double-blind, randomized, placebo-controlled study comprised of a single ascending dose (SAD) arm, including a crossover food effect cohort, followed by a multiple ascending dose (MAD) arm (NCT04908462, Figure S2).The study was reviewed and approved in writing by the study's Human Research Ethics Committee (HREC): the Alfred Hospital Ethics Committee.An Australian Therapeutic Goods Administration acknowledgement with CTN number of CT-2021-CTN-01489-1 was obtained.The study was conducted at a single site by Nucleus Network, Melbourne, AUS.
In the single ascending dose (SAD) arm, the study evaluated 5 cohorts consisting of single, orally administered doses of 2, 5, 15, 30, and 60 mg under fasting conditions.The intended enrollment was n = 8 per cohort with 6 receiving AL01211 and 2 receiving placebo.Actual enrollment is described in the results section.A group of 2 sentinel participants, randomized 1 AL01211:1 placebo, were dosed first for each cohort.After a favorable review of available safety data 24 hours post dose by the Principal Investigator (PI), the remaining subjects were dosed (Figure S2).The remaining subjects in each cohort were enrolled and randomized in a double-blind manner to a single dose of active or placebo treatment (5 AL01211:1 placebo).After the completion of the single dose in fasting conditions in Cohort 3 (15 mg), and after a 7-day washout period, the same subjects received another single oral dose of AL01211 or placebo after a standard high-fat (approximately 50% of total caloric content) and high-calorie (approximately 800-1000 calories) breakfast.
In the MAD arm, the study evaluated 4 cohorts with a target enrollment of 8 participants each and randomized to receive multiple, orally administered doses of 2, 5, 15, and 30 mg of AL01211 or placebo (Cohorts B1 to B4, respectively).At each dose level, subjects were randomized in a double-blind manner in a 3:1 ratio to receive daily doses of active or placebo treatment for 2 weeks (6 AL01211: 2 placebo).The actual number enrolled and treated with AL01211 in each cohort is described in the results section.All participants in Part B were confined to the clinical research unit (CRU) from Day -1 (predose) until completion of the postdose assessments on Day 15.Participants were administered AL01211 or placebo following an overnight fast beginning at midnight, every 24 hours for 14 days.Participants were evaluated for safety and plasma samples were collected for assessment of PK and PD at predefined time points.Participants returned to the CRU for Follow-up Visits on Days 16, 18, and 21 for safety assessments and collection of PK and PD samples.
Oversight of the study was provided by a safety monitoring committee (SMC) comprising the principal investigator (PI), a local Medical Monitor, and AceLink's Medical Monitor.For all SAD cohorts, the decision to escalate the dose was determined by the SMC following a review of the 7-day safety data from the preceding cohort.Dosing of the first cohort in the MAD study commenced after the SMC reviewed at least 7 days of safety data and 2 days of PK data (required), and PD (GL-1) data (if available) from participants at the 2, 5, and 15 mg dose levels in the SAD study (Cohorts A1, A2, and A3).Escalation of dosing in Cohorts B2 to B5 was not to commence unless 7 days of safety data and 2 days of PK data were available from preceding SAD cohorts, as well as PK data and 21 days of safety data from preceding MAD cohorts.

Key Inclusion/exclusion criteria
Inclusion criteria included: healthy male or female volunteers aged between 18 and 55 years with body mass index (BMI) between 18.5 and 35.0 kg/m 2 (inclusive) and body weight > 48 kg.Females must have been nonpregnant and nonlactating and agreed to use an acceptable, highly effective double contraception from screening until at least 30 days poststudy drug administration, and males must have been surgically sterile or using a contraception method from screening until at least 90 days post study drug administration.
Exclusion criteria included: history or symptoms of psychiatric disease, history or evidence of significant hepatic or renal disease or impairment, evidence of an active or suspected cancer or a history of malignancy for at least 5 years, uncontrolled hypertension of > 140/90 mmHg despite optimal therapy, abnormal ECG findings, any hepatic laboratory abnormality, cortical cataract of greater than one-quarter of the lens circumference or a posterior subcapsular cataract > 2 mm, or currently receiving potentially cataractogenic medications.

Drug substance
AL01211 was a capsule manufactured at strengths of 1, 5, or 30 mg.The oral placebo consisted of the same size capsules filled with a dry powder formulation with matching colors to the 3 corresponding AL01211 strengths (1, 5, or 30 mg).AL01211 provided for this study was manufactured under current Good Manufacturing Practices (cGMP) and was suitable for human use.Starting dose were based on ICH guidelines and discussion with the Australian Therapeutic Goods Administration.
Adverse events (AEs) were monitored up to day 90 after the final dose with most assessments completed by day 7 after the last dose.On day −1, all participants underwent physical examination, vital signs measurements, drug and alcohol screen, clinical laboratory test, and a pregnancy test.Complete physical examinations, vital signs, included general appearance, head, ears, eyes, nose, throat, dentition, thyroid, chest (heart, lungs), abdomen, skin, neurological, extremities, back, neck, musculoskeletal, and lymph nodes.Symptom-directed physical examination included head, ears, eyes, nose, throat, chest (heart, lungs), abdomen, skin, musculoskeletal, and lymph nodes, and any pertinent system based on any prior findings.
In addition to routine safety monitoring in this first-in-human study, particular attention was paid to ophthalmological examinations and cardiac monitoring due to the observations made during preclinical toxicology studies.Lens opacities and lens fiber degeneration were observed in toxicology studies in rats.Therefore, all SAD arm participants underwent ophthalmology exams at baseline, day 7, and a final ophthalmology follow-up visit 90 days after the first dose.All MAD arm participants underwent ophthalmology exams at baseline, day 21, and a final ophthalmology follow-up visit 90 days after the first dose.In addition, a small but statistically significant increase in the PR interval of the ECG was observed in both the 28-day and 13-week dog toxicology studies.Therefore, in this clinical study, besides routine baseline and end of treatment ECGs, 24hour Holter monitoring on Day 1 through Day 14 was included in MAD B3 (15 mg) and B4 (30 mg) cohorts.

Pharmacokinetics
AL01211 concentrations in plasma with actual sampling times were evaluated using Phoenix WinNonlin software, version 8.3 (Pharsight Corporation, USA), using noncompartmental analysis.All AUC calculations were determined using the linear trapezoidal rule.In the SAD arm (Part A, Cohorts 1-5), AL01211 concentrations were evaluated up to 96 hours postdose.In the MAD arm (Part B, Cohorts 1-4), AL01211 concentrations were evaluated up to 24 hours postdose on Day 1 and Day 14. Trough levels were measured predose on Days 3, 4, 7, 10, and Day 14.On day 14, additional sample collections were made up to 168 hours postdose.
AL01211 concentration in plasma was determined using a validated LC-MS/MS method by 360Biolabs, Melbourne, AUS.AL01211 was processed from 50 μL of plasma by precipitation of the protein using an acetonitrile and methanol mixture containing the internal standard (diclofenac).The resultant suspension was filtered by centrifugation and analyzed by LC-MS/MS.The lower limit of quantitation for AL01211 was 0.25 ng/mL and the range was 0.25-1000 ng/mL.Mobile phase A was 0.1% formic acid and 2 mM ammonium acetate in 5% acetonitrile in water and mobile phase B was 0.1% formic acid and 2 mM ammonium acetate in 95% acetonitrile in water.The UHPLC column was Zorbax RRHD Eclipse Plus Phenyl-Hexyl, 2.1×50 mm, 1.8 μm with guard (Agilent, Santa Clara, CA).The gradient started at 60% A, 40% B, ramping for 3.5 minutes, 95% B at 3.75-4.74minutes, then back to 40% B at 4.75 minutes and stopped at 5.0 minutes.A Triple Quadrupole Mass Spectrometer (Model G6460C) was used for detection monitoring m/z ratios of 542.16/84.1 for AL01211 and 296.03/214.03 for diclofenac.The interassay and intra-assay precision and accuracy parameters met the validation criteria with CVs that were ≤15%.

Statistical analysis
The sample size of approximately 8 participants per dose cohort (6 AL01211: 2 placebo per dose level) in Parts A and B reflects customary sample sizes employed in early development dose escalation studies of similar design and objectives, and it was expected to allow clinical judgment of safety and tolerability and assessment of PK profile.PK parameters were estimated from the plasma concentrations of AL01211 by noncompartmental method using Phoenix WinNonlin software (Version 8.3).The decrease from Baseline in plasma GL-1 levels are presented using summary statistics for the results at baseline and each scheduled postbaseline visit, as well as the change from baseline and percentage change from baseline values (continuous descriptive analysis).Descriptive statistics included the number of nonmissing values (n), arithmetic mean, standard deviation (SD), median, minimum, and maximum values.Graphs represent mean and standard error.In the PD studies significance from control group are represented as follows: * = P≤.05,** = P≤.01,*** = P≤.001,**** = P≤.0001.A 2-way repeated measures ANOVA with Tukey correction for multiple comparisons was used for GL1 (on log-transformed) and GL3 data (not transformed).Error bars for these GSL measurements represent standard errors.
To evaluate the correlation between AL01211 exposure and pharmacodynamic effect, the plasma GL1 (as % of baseline levels) were plotted against the Log transformed C max , AUC 0-24h , and C trough .The data was fit to a nonlinear regression (sigmoidal dose response) model using GraphPad Prism software.Placebo values were set to 0.1 ng/mL, 1 h•ng/mL, and 0.03 ng/mL for C max , AUC 0-24h , and C trough , respectively, to allow for the placebo data to be log transformed, included on the log transformed plots, and to better define the top of the sigmoidal curve.The bottom of the curve was constrained to 0. Using this model, the exposure level for half-maximal effect was 13.8 ng/mL, 150.7 h•ng/mL, and 3.7 ng/mL, the R 2 values were 0.80, 0.83, and 0.84, and the Hill slopes were −1.1, −1.1, and −1.1, for C max , AUC 0-24h , and C trough , respectively.

Subject Disposition and Demographics
Thirty-seven subjects were included in the SAD arm (n = 6 in Cohort A1; n = 7 in Cohort A4; n = 8 in Cohorts A2, A3, and A5), including 16 females and 21 males (Table S1).One participant in the placebo group was lost to follow-up and did not complete the study.By race, most participants were White (30 [81.1%] of 37 participants), 6 (16.2%) were Asian, and 1 (2.7%) was American Indian or Alaska Native.The mean BMI of participants was similar across all treatment cohorts, with an overall mean (SD) of 24.72 (3.69) kg/m 2 .
Thirty-two subjects were included in the MAD arm (n = 8 in Cohort B1; n = 6 in Cohort B2; n = 11 in Cohort B3; n = 7 in Cohort B4), including 25 males and 7 females (Table S2).All subjects completed the study as planned with the exception of 4 early terminations in MAD Cohort B3 (15 mg).Two participants withdrew due to nontreatment related AEs.Two additional subjects withdrew, 1 after Day 11 due to a family emergency and 1 subject after Day 1 due to poor venous access.By race, most participants were White (20 [62.5%] of 32 participants), 11 (34.4%)participants were Asian, and 1 (3.1%) participant was Black.The mean BMI of participants was similar across all cohorts, with an overall mean (SD) of 24.37 (3.09) kg/m 2 .

Pharmacokinetic Results
A summary of selected PK parameters for the AL01211 SAD cohorts is provided in Table 1 and plasma concentrations of AL01211 versus time profiles are displayed in Figure 2A.Across all dose levels evaluated in the SAD cohorts, AL01211 absorption was characterized by a median T max of 3.5 hours and disposition was characterized by a mean t 1/2 of 39.3 hours (excluding the 2 mg and 5 mg cohorts due to low levels of AL01211).Clearance values (CL/F) were calculated from the 15, 30, and 60 mg cohorts and had a mean value of 130 L/h.Mean AL01211 C max increased in an approximately dose proportional manner from 2 to 60 mg (28.9-fold) and mean AL01211 exposure (AUC 0-24h ) also increased in a dose-proportional manner from 5 to 60 mg (13.7fold).While overall, exposure increased in a dose proportional manner, the AL01211 exposure at 30 and 60 mg were similar (C max and AUC 0-24h ).The 60 mg cohort was 23% heavier than the 30 mg cohort (84.8 vs 68.7 kg, respectively).To evaluate if this lack of dose proportionality between 30 and 60 mg was due to variation in weight between cohorts, the C max and AUC 0-24h were plotted after the dose level was normalized to kilogram weight of each participant (Figure S3A & B).After normalizing the dose levels to individual weights, the C max and AUC 0-24h were dose proportional as determined by the Power-Law model with the slope   AUC, area under the time-concentration curve; AUC 0-24h , AUC from time 0-24 hours post dose; AUC 0-last , AUC from time 0 to the last measurable concentration; C max , maximum plasma concentration; T max , time to C max a T max values are shown as median (minimum, maximum).being 1.2 and for C max and AUC 0-24h , respectively, with the 95% confidence intervals including 1.
Ingesting a high fat meal prior to dose administration resulted in a reduction in exposure with C max and AUC 0-last having fed/fasted geometric mean ratios of 0.2 (90% CI: 0.13-0.31)and 0.36 (90% CI: 0. 26-0.51),respectively, indicating a food effect (Table 2 and Figure 2B).The median T max was increased from 3.5 hours in the fasted state to 6 hours in the fed state.
For the MAD cohorts the PK parameters on Days 1 and 14 are shown in Table 3 and plasma concentrations of AL01211 versus time profiles are displayed in Figure 3A.AL01211 absorption was characterized by a median T max of 3-3.5 hours on Days 1 and 14.The mean Day 14 plasma CL/F values (119.4L/h) were similar to those determined on Day 1 in the SAD study (130 L/h).Mean AL01211 C max on day 1 and 14 increased in a slightly greater than dose proportional manner from 2 to 30 mg (25.3-and 22.8-fold, respectively) and mean AL01211 exposure (AUC 0-24 ) on day 14 also increased in a slightly greater than dose-proportional manner from 2 to 30 mg (25.8-fold).For the 30 mg cohort, an apparent plateau in exposure was reached by day 10 (Figure 3B).By Day 14, the accumulation ratio (AR), as indicated by AR (C max ) and AR (AUC 0-24h ) was approximately 2-fold across all AL01211 doses (2-30 mg) (Table 3).

Pharmacodynamic Results
The PD response to AL01211 was determined by measuring the concentration of GL1 in plasma.A validated liquid chromatography with tandem mass spectrometry (LC-MS/MS) method to detect C16-GL1 (glucosylceramide with a 16-carbon acyl chain length) was used to quantify the levels of GL1 in plasma.In the SAD portion of the study, the maximum reduction from baseline in GL1 levels occurred at the 30 mg (37.6%) and 60 mg (38.6%) dose levels and was achieved at 48-72 hours postdosing, respectively (Figure 4A).GL1 levels returned toward baseline values after the maximum reduction was achieved.
In the MAD portion of the study, there was a time and dose-dependent decrease in plasma GL1 with increasing dose level (Figure 4B).Relative to baseline, the 15 mg dose produced a 46.6% reduction in GL1 on Day 14 while a 30 mg dose produced a 78.0%reduction in GL1 on Day 14, which was the maximum reduction in GL1.GL1 levels in placebo-treated individuals remained similar to baseline throughout the study.On an individual participant basis the percent reduction in GL1 on Day 15 correlated well with the individual Day 14 C max , C trough , and AUC 0-24h for all 4 doses (Figure 5A-C).The exposure level for half-maximal effect was calculated to be 13.8 ng/mL, 150.7 h•ng/mL, and 3.7 ng/mL for C max , AUC 0-24h , and C trough , respectively.
To determine whether plasma GL3 levels, an important biomarker for Fabry disease, are reduced by treatment with AL01211, we quantified plasma C16-GL3 levels (globotriacylceramide with a 16-carbon acyl chain length) from the 30 mg and placebo cohorts.Other dose levels were not measured.The results showed that once-a-day oral treatment with AL01211 resulted in 25.5% reduction of GL3 from baseline on Day 7 of treatment and 53.1% reduction on Day 14 of treatment, while the plasma GL3 levels did not change significantly in the placebo group (Figure 4C).

Safety Results
A summary of treatment emergent adverse events (TEAEs) in the SAD study is provided in Table S3.Overall, the frequency of TEAEs were mild to moderate and similar in frequency in the placebo and treated groups.There was one Grade 3 TEAE, which was an event of hypertension in a subject administered a single 30 mg dose, which occurred 7 days after dose administration, spontaneously resolved without treatment, and was considered unrelated to AL01211.One subject in the 15 mg dose group AUC, area under the time-concentration curve; AUC 0-24h = AUC from time 0-24 hours postdose; AUC 0-last , AUC from time 0 to the last measurable concentration; AUC 0-∞ , AUC from time 0 extrapolated to infinity; C max , maximum plasma concentration; C min , minimum plasma concentration; CL/F, apparent total clearance from plasma; MAD, multiple ascending dose; NA, not available; PK, pharmacokinetic; SD, standard deviation; t 1/2 , terminal half-life; T max , time to C max ; AR, accumulation ratio based on AUC 0-24h , Day 14 / AUC 0-24h , Day 1 and C max , Day 14/ C max Day 1.
(food effect) and another subject in the placebo group developed Grade 1 tachycardia as defined by their pulse rates transiently increasing slightly above 100 beats per minute (increasing to 112 and 105 bpm).Both events resolved spontaneously within 1 hour and were not considered by the PI to be related to AL01211.An overall summary of TEAEs in the MAD study is provided in Table S4 with most being mild to moderate and seen at similar frequency in placebo and treated groups.In the 15 mg dose group, during continuous cardiac monitoring, 2 participants were reported to have isolated single, 3 and 5 beat, episodes of cardiac ectopy, which were considered unrelated to AL01211 treatment.
Overall, for the SAD and MAD cohort there were no clinically significant findings on physical examination, vital signs, or ophthalmology assessments for any participant.There were no severe adverse events (SAEs), life-threatening events, or fatal events reported.

Discussion
AL01211 is a novel GCS inhibitor that was selected based on its potency, low off-target activity, and tissue distribution properties for treating non-neuronopathic GSL storage diseases, such as Fabry disease and type 1 Gaucher disease.8][29] A key feature of AL01211 is its high penetration into peripheral tissues without significant CNS penetration.These properties allow for maximizing GCS inhibition in the key peripheral organs that are affected in GD1 and FD without the potential risk of adverse CNS activity from either on or off target activity.The preclinical characterization and drug properties of AL01211 will be published in a separate manuscript.
AL01211 was tested in a phase 1 SAD and MAD study to determine safety, tolerability, food effects, PK and PD in male and female healthy volunteers.The PK profile of AL01211 from both SAD (2 to 60 mg) and MAD (2 to 30 mg) arms showed that the concentration of AL01211 increases with dose level in a nearly linear but slightly greater than dose proportional fashion.AL01211 underwent rapid absorption reaching C max at approximately 3.5 hours and there was an approximately 2-fold accumulation of AL01211 with repeated once-daily dosing.Nominally, 5 times the terminal half-life is considered adequate for the establishment of steady state.Based on the terminal half-life of approximately 40 hours, steady state is predicted to be reached in approximately 200 hours or approximately 8 days.Consistent with this calculation, the 30 mg cohort resulted in an apparent plateau in exposure (AUC 0-24h ) from Day 10 onwards.At the 30 mg dose level, the C trough levels of AL01211 at steady state were above the in vitro IC 50 for inhibition of GCS by day 3.
Feeding a high fat meal to participants prior to an oral dose of AL01211 produced significant lower exposure with an accompanying longer T max compared to the fasted state.Food effects on bioavailability after a high fat meal are common and could be due to a number of factors including changing gastric pH or interactions with bile salts and lipid particles. 30The reason for this food effect is unknown, although given that AL01211 is more soluble in acidic conditions, it is possible the high fat meal may increase gastric pH resulting in lower AL01211 solubility.Alternatively, AL01211 interactions with the high fat food particles could delay and prevent dissolution and/or absorption.Further food effect studies will be needed to clarify the impact of food on AL01211 exposure and future formulation work may help reduce this food effect.In the meantime, for upcoming phase 2 clinical studies, AL01211 will be taken under fasted condition to ensure consistent exposure.
The PD effects of AL01211 were monitored by measuring the levels of GL1 and GL3 in plasma after a single or multiple doses of AL01211.At baseline, these subjects had normal level of GSLs so all reported effects reduce these levels below normal physiological levels.After a single dose of AL01211, GL1 was reduced by 38% compared to baseline after 48-72 hours at 30 and 60 mg.With repeated daily dosing at 30 mg, GL1 was reduced by 75% of baseline levels by day 10 and reached a maximum reduction of 78% by day 14.Once AL01211 dosing was stopped on day 15, the levels of GL1 increased back toward baseline levels.The % reduction of GL1 correlated well with the C max , C trough , or AUC 0-24h of AL01211 (Figure 5 A-C).
The half-maximal effect level for AL01211 plasma C trough concentrations was calculated from these dose response curves to be 3.7 ng/mL (corresponds to 6.8 nM AL01211), which is similar to the in vitro IC50 values in GCS enzyme assays.GL3, the glycolipid that accumulates in Fabry disease, was also measured from the placebo group and the 30 mg MAD cohort and showed a maximum of 53% reduction by day 14.The % reduction from baseline in GL3 on Day 14 was less than the effect on GL1 on day 14, likely due to the slower kinetics of GL3 turnover relative to GL1.
2][23][24] The daily oral dose of AL01211 needed to achieve 78% reduction in plasma GL1 is greater compared to venglustat likely due to a lower oral bioavailability of AL01211.However, the bioavailability of AL01211 is sufficient to reduce GL1 to the same extent as venglustat with similar percent variation in exposure at the effective dose levels. 20L01211 is a more potent inhibitor of GCS when compared to venglustat.8][29] Consistent with this greater potency in vitro , AL01211 can achieve the same level of GL1 reduction as venglustat with less systemic drug exposure (6-fold lower) compared to venglustat.For example, the mean AUC 0-24h , C max , and C trough for AL01211 associated with 78% GL1 reduction is 382.8 h•ng/mL, 31.0 ng/mL, and 8.3 ng/mL, respectively.These levels can be compared to venglustat, which produces a similar level of GL1 reduction in a Phase 1 healthy volunteer study with mean AUC 0-24h , C max , and C trough of 2420 h•ng/mL, 142 ng/mL, and 73.3 ng/mL, respectively. 20n this Phase 1 study, AL01211 was shown to be generally well tolerated up to a single dose of 60 mg or multiple daily doses of up to 30 mg AL01211 for up to 14 days.Overall, the frequency of TEAEs in both the SAD and MAD studies was similar to placebo.While the maximum dose level of AL01211 tested with repeated dosing in this study was 30 mg, a higher dose of 60 mg will be evaluated in phase 2 studies to determine if an even larger reduction in GL1 can be safely achieved and produce greater clinical benefit.
Two participants in the 15 mg cohort of the SAD study developed tachycardia (Grade 1) as defined by an increase in heart rate above 100 BPM, which was mild and resolved spontaneously.Of note, 1 participant with tachycardia was in the placebo group and neither AE was considered related to AL01211.In the 15 mg MAD cohort, 2 participants receiving AL01211 had asymptomatic cardiac ectopy as defined by isolated single, 3 and 5 beat, episodes.Similar cardiac ectopy was not seen in the 30 mg MAD cohort.Transient occurrences of these asymptomatic, nonsustained, and isolated ectopic beats are often detected in healthy individuals during frequent or continuous cardiac monitoring.For example, similar transient events have been reported in up to 3% of healthy participants with prolonged cardiac monitoring 31 and exercise-induced events were recorded in nearly 4% of asymptomatic adult volunteers. 32Therefore, the incidence of these episodes in this study reflects that of a population of healthy adults and is unlikely related to AL01211 administration.Furthermore, the recording of such spontaneous events in a healthy individual does not necessarily have prognostic significance.
Due to the observation of lens fiber degeneration and accompanying lens opacities in rat toxicology studies, ophthalmology exams were conducted in both SAD and MAD arms of the study to monitor any adverse effects on the lens.However, the occurrence of lens opacities was not seen in any individuals even after careful monitoring with slit lamp ophthalmological exams that were conducted out to 90 days after the last dose.Notably, lens fiber degeneration was also observed in rats in preclinical toxicology studies of venglustat, suggesting this finding may be a rat specific class effect of GCS inhibitors. 222][23][24] Preclinical toxicology studies of AL01211 (and venglustat) that were conducted in dogs, including longer term chronic toxicology studies, did not show lens opacities, suggesting these findings are specific to rats and are possibly due to the different lipid composition of the rodent lens. 22o significant GI adverse effects have been seen for AL01211.GI effects have been reported from other GCS inhibitors including the imino-sugar derived GCS inhibitors miglustat and lucerastat. 26These adverse GI effects occur due to the inhibition of intestinal glycohydrolases like lactase and sucrase-isomaltase by these simple sugar analog-based GCS inhibitors.AL01211 is not structurally related to these imino sugar molecules and is less likely to have the same effects on intestinal glycohydrolases.These imino-sugar related GCS inhibitors have also been shown to be potent inhibitors of the enzyme glucosylceramidase beta 2 (GBA2).[29]

Conclusions
Phase 1 SAD and MAD studies have shown that AL01211 was generally safe and well tolerated with no severe adverse events (SAEs) at single doses up to 60 mg or 14 days of once-daily oral doses up to 30 mg.AL01211 systemic exposure increased with dose in a nearly dose proportional manner for 2-60 mg in the SAD study or 2-30 mg in the MAD study.Repeated doses of AL01211 at 30 mg resulted in a 78% reduction in GL1 on day 14.The safety, PK, and PD results in this study support the further development of AL01211 as a SRT therapy for FD and GD1.

Figure 2 .
Figure 2. (A) Mean (+/-standard error) AL01211 plasma concentrations in the single ascending-dose study.(B) Concentration-time Curves of SAD cohort 3 (15 mg) in the fed versus fasted state.Mean (+/-) standard error AL01211 plasma concentrations in the single ascending-dose study for SAD cohort 3 (15 mg) in the fed versus fasted state.Values below the lower limit of quantitation (LLOQ) were considered 0 for this graph.Dashed line represents values for participants that received a high fat, high calory meal and the solid line represents fasted values.

Figure 3 .
Figure 3. (A) Mean (+/− standard error) AL01211 plasma concentrations in the multiple ascending-dose study.Dashed line represents day 1 and the solid line represents day 14 values.Measurements below the lower limit of quantitation (LLOQ) were considered 0 for both graphs.(B) Plasma Ctrough concentrations of AL01211 from the MAD arm.Mean (+/− standard error) AL01211 plasma concentrations predose on day 1, 2, 3, 4, 7, 10, and 14 and 24 hours postdose on day 14.The dashed line represents the IC50 of AL01211 in a GCS enzyme assay.

Figure 5 .
Figure 5.Correlation between individual PK parameters (C max , AUC 0-24h , and C trough ) with PD response as measured by GL1 reduction relative to baseline on day 15 of the MAD arm.The lines on the graph represent the log transformed data fit to a nonlinear regression (sigmoidal dose response) model.Using this model, the exposure level for half-maximal effect was 13.8 ng/mL, 150.7 h•ng/mL, and 3.7 ng/mL, the R 2 values were 0.80, 0.83, and 0.84 for C max , AUC 0-24h , and C trough , respectively.

Table 1 .
Summary of AL01211 Pharmacokinetic Parameters Under Fasted Conditions in the SAD Arm

Table 2 .
Part A: Key Pharmacokinetic Parameters Following Oral Administration of AL01211 at 15 mg in the Fed and Fasted State

Table 3 .
Summary of AL01211 Pharmacokinetic Parameters in the MAD Arm