Use of Ambroxol as Therapy for Gaucher Disease

Key Points Question What are the biochemical changes associated with ambroxol repurposing among patients with Gaucher disease who have not received any disease-specific treatment? Findings In this case series of 28 unselected participants, significant improvements in hematologic parameters, visceral volumes, and plasma biomarkers were observed for 26 patients after a mean (SD) treatment duration of 2.6 (1.7) years. Meaning Long-term treatment with ambroxol was associated with patient improvement; improvements in hematologic parameters, visceral volumes, and plasma biomarkers were larger among patients with relatively mild symptoms of Gaucher disease and patients who received initial treatment at younger ages.


Introduction
Gaucher disease (GD) is one of the most common lysosomal storage disorders caused by deficiency in the enzyme β-glucocerebrosidase because of variants in the GBA1 (OMIM 606463) gene, resulting in glucosylceramide accumulation.Three main clinical types of GD are generally characterized based on the absence or presence of central nervous system involvement: nonneuronopathic type 1 (GD1), acute neuronopathic type 2 (GD2), and chronic neuronopathic type 3 (GD3).The incidence of GD in the general population is 1 in 50 000 to 100 000. 1 The pilot newborn screening for GD in Shanghai reported that the incidence in east China is approximately 1 in 80 855. 2 Two therapeutic approaches have been approved for treating GD: enzyme replacement therapy (ERT) and substrate reduction therapy (SRT). 3Visceral and hematologic abnormalities were significantly improved with ERT. 4,5Unfortunately, the effectiveness of ERT for neurologic symptoms of GD is almost negligible due to the inability of the enzyme to cross the blood-brain barrier (BBB). 6,7glustat and eliglustat could cross the BBB but failed to show significant benefits for neurologic manifestations. 8,9Health insurance for ERT is not fully covered in the People's Republic of China, and some patients for whom ERT is indicated must pay for it themselves.Some patients cannot receive efficacious treatment due to the prohibitive costs of ERT or SRT.
Unlike the current drugs used to treat GD, which are very expensive and do not cross the BBB, new small-molecule therapies that enhance glucocerebrosidase are being developed to overcome these limitations. 10Ambroxol is a widely used cough suppressant and mucolytic with an excellent safety profile. 11,12In 2009, ambroxol was identified in a high-throughput screening of a regulatoryapproved compound collection as an enhancer of stability and residual activity of several misfolded glucocerebrosidase variants. 13Later, a pilot open-label study with 12 adult patients with GD1 naive to any disease-specific treatment showed some improvements in disease parameters after the patients received ambroxol for 6 months. 14broxol has been shown to cross the BBB. 11,15Based on this evidence, in several countries where ambroxol is approved and available, it has been prescribed off label with ERT or SRT for patients with neuronopathic GD (nGD). 11,16,17With no specific treatment for nGD and the aforementioned encouraging reports, off-label use of high-dose ambroxol has become a realistic option for managing nGD as an adjunct to ERT or for GD among patients without access to ERT or SRT.This study investigated the observational data of ambroxol repurposing in a large case series of patients without GD-specific treatment and its association with hematologic parameters, visceral volumes, and biomarkers of GD.

JAMA Network Open | Pediatrics
Use of Ambroxol as Therapy for Gaucher Disease

Patients
The local institutional research ethics committees approved the study at Xinhua Hospital, affiliated with Shanghai Jiao Tong University School of Medicine, Shanghai, China.Written informed consent was obtained from all participants aged 18 years or older and from parents of patients younger than 18 years.This study followed the Appropriate Use and Reporting of Uncontrolled Case Series in the Medical Literature reporting guideline for case series studies.
Patients with GD who could not afford ERT due to the prohibitive cost were enrolled and received oral ambroxol from May 6, 2015, to November 9, 2022.All patients were confirmed to have GD by measuring leukocyte lysosomal acid β-glucosidase activity within the affected range and GBA1 biallelic pathogenic variants.Patients were recommended to be followed up once every 6 months.
Patients were assessed at baseline and scheduled follow-up visits, including disease status inquiry, physical examination, recording adverse events, routine blood tests, and measuring chitotriosidase activity and glucosylsphingosine level.Spleen and liver volumes were derived from volumetric magnetic resonance imaging if available and measured in multiples of normal (MN).
Ambroxol administration was initiated with a starting dose of 5 mg/kg/d divided into 3 equal doses.It was suggested to increase the doses gradually according to clinical outcomes of ambroxol at follow-up visits, no more than the maximal dose (25 mg/kg).Patients received an escalating dose of oral ambroxol (mean [SD] dose, 12.7 [3.9] mg/kg/d) at the last visits.
We evaluated the achievement of the following therapeutic goals: spleen volume, 8 MN or less or a 50% or more decrease from baseline; liver volume, 1.5 MN or less or a 30% or more decrease from baseline; hemoglobin, 11.0 g/dL or more for women and 12.0 g/dL or more for men (to convert to grams per liter, multiply by 10.0); and platelet count, 100 × 10 3 /μL or more or double the baseline value for patients with a platelet count less than 60 × 10 3 /μL (to convert to ×10 9 per liter, multiply by 1.0) at baseline. 18

Glucosylsphingosine Assay
Dried blood spot glucosylsphingosine level was measured for 1 patient, while plasma glucosylsphingosine level was monitored throughout treatment for the other patients.
Glucosylsphingosine was measured on ultra-performance liquid chromatography using a Xevo TQ S detector (Waters).The glucosylsphingosine standard and the internal standard that was used were purchased from Matreya Inc. Plasma samples were collected in heparin tubes and frozen at −80 °C.In brief, in a 1.5-mL vial, 30 μL of plasma sample was mixed with 200 μL of internal standard solution.
Samples were vortexed and centrifuged (12 000g for 5 minutes), and then the supernatant was transferred to a 96-well plate.
Dried blood spot samples were collected on filter cards and frozen at −80 °C.One 5-mm blood spot and each dried blood spot calibrator, control, and sample were punched into a 96-well plate.
Then 200 μL of the internal standard working solution was added to each vial.The plate was sealed with aluminum foil tape and shaken for 30 minutes at room temperature in an incubator-shaker unit.
The supernatant was transferred to a 96-well plate.
Analytes were separated on an Acquity BEH Amide column (column dimensions, 2.1 × 100 mm; particle size, 1.7 μm; Waters).Mass spectrometry was performed in the positive ionization mode using an electrospray ionization source, with the following transitions: the mass number of an ion

JAMA Network Open | Pediatrics
Use of Ambroxol as Therapy for Gaucher Disease

Study Cohort
Thirty-two patients with GD, including 29 with GD1, 2 with GD3, and 1 with GD intermediate type 2-3, were enrolled and treated with ambroxol.Patients with a follow-up duration of fewer than 6 months were excluded from the analysis.Four of 32 patients with GD1 (cases 29-32) were lost to follow-up and were excluded from analyses (eFigure 1 in Supplement 1

SI conversion factors:
To convert hemoglobin to grams per liter, multiply by 10.0; platelet count to ×10 9 per liter, multiply by 1.0; and white blood cell count to ×10 9 per liter, multiply by 0.001.
a Excludes 4 patients lost to follow-up.
d Twelve patients with no chitotriosidase activity were excluded.
e Eleven patients with no chitotriosidase activity were excluded.
f Ten patients with no chitotriosidase activity were excluded.
g Two patients with no chitotriosidase activity were excluded.
h Five patients with no chitotriosidase activity were excluded.
i Two patients with no chitotriosidase activity were excluded.
j Normal ranges, less than 1.1 ng/mL in plasma sample, less than 10 ng/mL in dried blood spot sample.

JAMA Network Open | Pediatrics
Use of Ambroxol as Therapy for Gaucher Disease To further analyze the response of ambroxol among patients, the baseline and final hemoglobin, platelet, and biomarker levels were analyzed in subgroups according to the age at starting treatment:

Hematologic and Visceral Outcomes
An increase in hemoglobin level was observed 1 year after the initiation of ambroxol.After a mean (SD) of 2.6 (1.7) years of ambroxol, the mean (SD) hemoglobin concentration improved by 14

Biomarkers in Plasma Before and During Ambroxol Treatment
Reductions in glucosylsphingosine level and chitotriosidase activity occured after the initiation of ambroxol, and the reductions were maintained (Figure 3A and C

JAMA Network Open | Pediatrics
Use of Ambroxol as Therapy for Gaucher Disease  3F).Larger decreases in chitotriosidase activity and glucosylsphingosine level were observed among patients who received treatment at a younger age.

Discussion
Previous studies reported that ambroxol had a promising outcome when used with ERT or SRT for patients with GD. 11,16,17,19 However, to our knowledge, there have been few reports on the clinical benefits of ambroxol alone for patients with GD. 14,20 Our laboratory previously reported the early beneficial outcomes of ambroxol on skeletal and hematologic manifestations in a child with GD1. 20re, we report observational data on ambroxol repurposing among patients without GD-specific treatments.
Some GBA1 pathogenic variants are associated with relatively mild symptoms.2][23][24] Most of the 26 individuals in the study who were considered good responders to ambroxol carried 1 or 2 mild variants.Previous research 21,22,25 showed variants L483P, RecNcil, R535C, and R202X, and gene conversion with GBAP (OMIM 606463) N227S, N227K, and V230G were associated with nGD or severe GD1 phenotype.Some patients with 1 severe variant and 1 moderate variant, such as L483P heterozygote with another mild variant (cases 2-4 and case 21), as well as 1 patient with RecNcil/G85E (case 19), responded favorably to ambroxol.We hypothesized that there could be benefits associated with ambroxol among patients with GBA1 variants associated with some degree of residual β-glucocerebrosidase activity.
Case 28 had a severe R535C-R202X genotype and was classified as having GD intermediate type 2-3.Case 11 had genotype gene conversion with GBAP N227S, N227K, V230G, R535H, and R87Q and received a diagnosis of severe GD1 phenotype.These 2 patients were at significantly advanced stages of GD at baseline (eg, case 11 had a spleen volume of 41 MN; and poor hematologic parameters;

Baseline Ambroxol treatment
Liver and spleen volume data before and after treatment were available for only 8 patients.Patient 1 was splenectomized.Due to the expense and time spent on testing, some patients did not undergo abdominal imaging to monitor spleen and liver volumes throughout therapy.
case 28 had a spleen volume of 87 MN; and horizontal gaze palsy), with deterioration of hematologic parameters and biomarkers, showed no improvement in clinical presentation, and ambroxol treatment was considered nonefficacious.We believe that in the clinical course of GD, there is a critical threshold beyond which ambroxol therapy may not be beneficial as previously described for nGD. 26Thus, early initiation of ambroxol therapy will be critical for the progression of the disease.
Another therapeutic modality may act synergistically to better control disease progression.our study, platelets responded to ambroxol less favorably and showed a smaller increase than hemoglobin.The relatively poor platelet responses might be associated with the large spleen volumes.As shown in Figure 2, for almost all patients, despite substantial improvement from baseline, spleen volumes continued to exceed 10 MN.
Due to the expense and time spent on testing, some patients did not undergo abdominal imaging to monitor spleen and liver volumes.In a large cohort of patients with type 1 GD treated with imiglucerase for 2 years, mean spleen and liver volumes were reduced by 49% and 29% from baseline, respectively, according to retrospective research from the International Collaborative Gaucher Group Gaucher Registry. 28Although ambroxol therapy did not improve visceral outcomes as significantly as ERT, it was still encouraging.
Glucosylsphingosine, a downstream metabolic product of glucosylceramide, is increasingly recognized as a sensitive biomarker of GD with direct involvement in disease pathogenesis. 29nitoring chitotriosidase activity in patients with GD1 has limitations since it is subject to genetic heterogeneity. 30Nearly 40% of patients in this study had no chitotriosidase activity.
Glucosylsphingosine level could be used to assess the efficiency of ambroxol in patients with no chitotriosidase activity.A significant decrease in glucosylsphingosine level was observed in the patients.However, it was reported that glucosylsphingosine level decreased more substantially in patients treated with ERT or SRT. 31,32The median glucosylsphingosine level (327.0 ng/mL [range, 123.6 to 482.6 ng/mL]) was reduced by 85.7% in patients with GD1 who completed 209 weeks of velaglucerase alfa treatment. 31In a study of patients with GD1 receiving eliglustat therapy for 8 years, glucosylsphingosine was decreased approximately 60% from baseline at 1 year and 92% from baseline at 8 years. 32The degree of decrease in glucosylsphingosine level might be associated with the overall disease severity of the patients.Several reports demonstrated an additional decrease in glucosylsphingosine level after ambroxol therapy was started for patients treated with long-term ERT. 16,33tients with younger ages at the start of treatment had larger improvements in hematologic parameters and plasma biomarkers.Early administration of ambroxol might achieve better outcomes for patients with early-stage GD.Consequently, earlier intervention is advisable for patients with GD.
In rodent and primate models, ambroxol increases intracellular β-glucocerebrosidase activity in tissues, including the brain. 34,35In our study, neurologic stability or improvement was observed in 2 patients with GD type 3 (cases 25 and 26; eTable in Supplement 1), which suggests that the therapeutic outcomes of ambroxol might delay the rapid natural progression of neurologic manifestations.However, due to sampling difficulty, we did not have data on changes in glucosylsphingosine level in the cerebrospinal fluid, which has previously been reported to be decreased considerably in patients with GD3 after ambroxol treatment. 11,15A recent nonrandomized, noncontrolled trial of ambroxol for patients with Parkinson disease demonstrated that ambroxol could cross the BBB and have a modulatory association with the β-glucocerebrosidase enzyme. 15broxol increases β-glucocerebrosidase enzyme protein levels and cerebrospinal fluid α-synuclein levels, thus explaining at least part of the clinical improvement observed in patients with nGD.
Two patients in our study (case 3 and case 20) who experienced a stable clinical course without deterioration had no significant improvement in biomarkers during early ambroxol therapy.After continuous ambroxol treatment for 4 years, a marked reduction in glucosylsphingosine level and chitotriosidase activity was observed.This evidence indicates that ambroxol may take longer to reverse the pathologic glycosphingolipid accumulation in GD.Therefore, it is important that patients take their medication consistently.
Most patients in this cohort were from provinces other than Shanghai.It is difficult for some patients to visit our hospital regularly due to time and cost reasons, particularly for patients living in remote rural areas.Clinical improvements were observed in 4 patients (cases 5, 6, 16, and 17) with mild gene variants, but biomarkers were volatile or did not improve significantly at follow-up visits.

JAMA Network Open | Pediatrics
Use of Ambroxol as Therapy for Gaucher Disease

Figure 2 .
Figure 2. Absolute Spleen and Liver Volume Changes in Individual Patients

Figure 3 .
Figure 3. Changes From Baseline in Disease-Related Biomarkers During Ambroxol Treatment
Splenectomized patients had a robust treatment response.Of the 5 splenectomized patients (cases 3, 13, 18, 24, and 27), case 13 had a comparatively mildly elevated glucosylsphingosine level (73.7 ng/mL) at baseline and showed relatively stable glucosylsphingosine levels over the treatment period, with −42.7% liver volume.Biomarker improvement was noted among all other splenectomized patients, with decreased glucosylsphingosine levels ranging from −16.9% to −77.9% and decreased chitotriosidase activity ranging from −24.2% to −55.4%.