Glucosylceramide Synthase (GCS) - inhibitor (IC₅₀ ~ 24 nM) [1]

Eliglustat tartrate is selective for the target enzyme and has good efficacy, with an IC50 of 24 nM [1]. Dose-dependent results were obtained by incubating K562 or B16/F10 cells with escalating concentrations of Genz-112638 (0.6-1000 nM) for 72 hours. GM1 and GM3 levels on the cell surface decreased. In K562 cells, the average IC50 value for GM1 cell surface presentation inhibition was 24 nM (range 14-34 nM), while in B16/F10 cells, the average IC50 value for GM3 inhibition was 29 nM (range 12-48 nM) [1].

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In cell-based assays using K562 (human erythroleukemic) and B16/F10 (murine melanoma) cells, Eliglustat tartrate demonstrated dose-dependent inhibition of glycosphingolipid synthesis. By measuring cell surface levels of gangliosides GM1 (on K562 cells) and GM3 (on B16/F10 cells) as surrogates for GCS activity, the mean IC₅₀ values were 24 nM (range 14-34 nM) for GM1 and 29 nM (range 12-48 nM) for GM3. No overt cellular toxicity was observed up to 1000 nM. [1]
- An alternative assay measuring intracellular glucosylceramide levels in K562 cells via high-performance thin layer chromatography (HP-TLC) yielded a mean IC₅₀ of 40 nM for inhibiting glucosylceramide synthesis, consistent with the GM1/GM3 results. [1]
- Specificity profiling showed that Eliglustat tartrate is highly selective for glucosylceramide synthase. It exhibited no detectable inhibition of intestinal glycosidases (sucrase, maltase), α-glucosidases I and II, lysosomal glucocerebrosidase (GBA1), or the glycogen debranching enzyme at concentrations up to 2500 μM. Non-lysosomal glucosylceramidase (GBA2) was weakly inhibited with an IC₅₀ of 1600 μM, representing an approximately 40,000-fold selectivity window for the target enzyme. [1]

In comparison to age-matched control animals, mice administered the medication prior to considerable substrate accumulation (10 weeks of age) displayed lower levels of glucosylceramide and fewer Gaucher cells in the liver, lungs, and spleen [1].

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In a murine model of Gaucher disease (D409V/null mice), oral administration of Eliglustat tartrate at 150 mg/kg/day for 10 weeks was well-tolerated, with no significant effects on body weight or feeding habits compared to untreated controls. [1]
- In young (10-week-old), pre-symptomatic D409V/null mice, treatment with Eliglustat tartrate (75 or 150 mg/kg/day for 10 weeks) resulted in a dose-dependent reduction of glucosylceramide levels in affected tissues. At the 150 mg/kg dose, glucosylceramide levels were reduced to 60% (liver), 40% (lung), and 75% (spleen) of those in untreated age-matched controls. Histological evaluation of the liver showed a significant reduction in the number and size of Gaucher cells compared to untreated controls. [1]
- In older (7-month-old) D409V/null mice with pre-existing pathology, treatment with Eliglustat tartrate (150 mg/kg/day for up to 10 weeks) arrested further accumulation of glucosylceramide. After 10 weeks, glucosylceramide levels were 60% lower in the liver, 50% lower in the lung, and 40% lower in the spleen than in vehicle-treated mice. Histopathological analysis confirmed that the number of Gaucher cells did not increase from baseline and was significantly lower than in untreated controls. [1]

GM1 Inhibition Assay (K562 cells): K562 cells were cultured with increasing concentrations of Eliglustat tartrate (0.6-1000 nM) for 72 hours. Cells were then harvested and incubated with FITC-conjugated cholera toxin (which binds GM1) for 30 minutes on ice. After washing, fluorescence was quantitated by flow cytometry. Non-viable cells were excluded using propidium iodide staining. Fluorescence was normalized using beads with known Molecules of Equivalent Soluble Fluorophores (MESF). [1]
- GM3 Inhibition Assay (B16/F10 cells): B16/F10 cells were cultured with increasing concentrations of Eliglustat tartrate (0.6-1000 nM) for 72 hours. Cells were harvested and incubated with an anti-GM3 monoclonal antibody for 30 minutes on ice, followed by a FITC-conjugated secondary antibody. After washing, fluorescence was quantitated by flow cytometry as described above. [1]

Animals:** D409V/null Gaucher mice (both sexes) were used. Mice were acclimated to oral gavaging with water for one week prior to treatment initiation. [1]
- **Drug Preparation and Administration:** Eliglustat tartrate was dissolved in Water For Injection (WFI). Drug or vehicle was administered once daily by oral gavage at a volume of 10 mL/kg. A dose escalation scheme was used, starting at 75 mg/kg/day and increasing to 150 mg/kg/day over nine days (3 days at each dose, with 25 mg/kg/day increments), followed by continued dosing at 150 mg/kg/day. [1]
- **Monitoring and Sample Collection:** Mice were weighed three times per week to monitor health. At the end of the study, animals were euthanized by carbon dioxide inhalation. Tissues (liver, lung, spleen) were harvested immediately. Half of each tissue was snap-frozen for biochemical analysis, and the other half was fixed for histological examination. [1]

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Animals: D409V/null Gaucher mice (both sexes) were used. Mice were acclimated to oral gavaging with water for one week prior to treatment initiation. [1]
- Drug Preparation and Administration: Eliglustat tartrate was dissolved in Water For Injection (WFI). Drug or vehicle was administered once daily by oral gavage at a volume of 10 mL/kg. A dose escalation scheme was used, starting at 75 mg/kg/day and increasing to 150 mg/kg/day over nine days (3 days at each dose, with 25 mg/kg/day increments), followed by continued dosing at 150 mg/kg/day. [1]
- Monitoring and Sample Collection: Mice were weighed three times per week to monitor health. At the end of the study, animals were euthanized by carbon dioxide inhalation. Tissues (liver, lung, spleen) were harvested immediately. Half of each tissue was snap-frozen for biochemical analysis, and the other half was fixed for histological examination. [1]

Absorption, Distribution and Excretion
In patients with non-end-stage renal disease, the Cmax of agarsidase α was 3710 ± 855 U/mL, and the AUC was 256,958 ± 63,499 minU/mL. Following non-specific proteolysis, amino acids in the protein drug can be reused for protein synthesis or further broken down and excreted via the kidneys. In patients with non-end-stage renal disease, the steady-state volume of distribution is approximately 17% of body weight, regardless of sex. At doses of 0.007–0.2 mg/kg, the clearance rate was 2.66 mL/min/kg in males and 2.10 mL/min/kg in females. Metabolism/Metabolites Data on the metabolism of agarsidase α are currently unavailable. However, protein drugs are expected to be degraded into smaller peptides and amino acids by proteases and other catalytic enzymes.

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Biological half-life
The elimination half-life for males is 108 ± 17 minutes, and for females it is 89 ± 28 minutes.

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In single and repeat oral dose ADME studies in rats and dogs using ¹⁴C-radiolabeled compound, Eliglustat tartrate was readily metabolized and cleared. Both the parent compound and its metabolites were effectively cleared within 24 hours. [1]
- Serum concentrations at and above the in vitro IC₅₀ (24-40 nM) were readily achievable with oral doses below the maximum tolerated level. [1]

Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation
Since there is no published experience regarding the use of eligliflozin during lactation, alternative medications may be preferred, especially when breastfeeding newborns or premature infants.
◉ Effects on Breastfed Infants
No published information found as of the revision date.
◉ Effects on Lactation and Breast Milk
No published information found as of the revision date.

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Protein Binding
Agarsidase α is not expected to bind to proteins in circulation.

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In the D409V/null mouse model, daily oral administration of Eliglustat tartrate at the effective dose of 150 mg/kg/day for up to 10 weeks was well-tolerated. There were no observable gastrointestinal issues and no significant difference in body weight between treated and control groups. [1]
- In vitro specificity assays showed no inhibition of lysosomal glucocerebrosidase (the enzyme deficient in Gaucher disease) at concentrations up to 2500 μM, suggesting that Eliglustat tartrate would not interfere with residual enzyme activity or co-administered enzyme replacement therapy. [1]

[1]. A specific and potent inhibitor of glucosylceramide synthase for substrate inhibition therapy ofGaucher disease. Mol Genet Metab. 2007 Jul;91(3):259-67.

Eliglustat tartrate is a tartrate salt, specifically the hemitartrate salt of ligorupstat. It is a ceramide glucosyltransferase inhibitor (in its tartrate form) used to treat Gaucher disease. It is an EC 2.4.1.80 (ceramide glucosyltransferase) inhibitor. It contains ligorupstat (1+). Agardilase Alpha is a recombinant human α-galactosidase A, similar to agarsidase Beta. While patients generally do not experience a significant difference in efficacy between the two drugs, some patients may benefit more from agarsidase Beta. Following a contamination incident in 2009, the use of agarsidase Beta declined in Europe, and agarsidase Alpha was adopted instead. Agardilase Alpha was approved by the EMA on August 3, 2001. See also: Eliglustat (with the active moiety); Agardilase Beta (note moved to).

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Drug Indications
Agarsidase Alpha is indicated for the treatment of Fabry disease.

Repula is indicated for long-term enzyme replacement therapy in patients diagnosed with Fabry disease (α-galactosidase A deficiency).
Mechanism of Action
α-galactosidase A is taken up by cells via mannose-6-phosphate receptors. Agardase α hydrolyzes globular trihexosylceramide and other glycosphingolipids, which are normally hydrolyzed by endogenous α-galactosidase A. Preventing the accumulation of glycosphingolipids can prevent or alleviate symptoms of Fabry disease, such as renal failure, cardiomyopathy, or cerebrovascular events.
Pharmacodynamics
Agardase α is a recombinant human α-galactosidase A used as enzyme replacement therapy for Fabry disease. It is characterized by a long duration of action and a wide therapeutic index. Patients should be informed of the risks of infusion-related reactions and hypersensitivity reactions.

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Eliglustat tartrate (Genz-112638) is a synthetic small molecule inhibitor of glucosylceramide synthase, designed as a structural homologue of D-threo-ethylenedioxyphenyl-2-palmitoylamino-3-pyrrolidino-propanol and formulated as a tartrate salt. It was developed for substrate inhibition therapy (also called substrate reduction therapy) for Gaucher disease. [1]
- The therapeutic strategy aims to reduce the synthesis of glucosylceramide, the substrate that accumulates pathologically in Gaucher disease due to deficiency of glucocerebrosidase. By balancing substrate synthesis with the impaired catabolic rate, this approach can prevent further accumulation and potentially allow residual enzyme activity to reduce existing storage. [1]
- The study demonstrates that Eliglustat tartrate is effective in both preventing disease progression in pre-symptomatic mice and arresting further pathology in mice with established disease, suggesting its potential as a maintenance therapy or in combination with enzyme replacement therapy. [1]
- Because glucosylceramide synthesis is the first step in the glycosphingolipid biosynthetic pathway, inhibition with Eliglustat tartrate may also have therapeutic potential for other glycosphingolipid storage disorders such as Fabry disease. [1]

2[C23H36N2O4].C4H6O6

959.173

958.551

928659-70-5

Eliglustat;491833-29-5;Eliglustat-d15 tartrate;1884556-84-6

52918379

White to off-white solid powder

6.298

8

16

25

68

617

6

CCCCCCCC(=O)N[C@H](CN1CCCC1)[C@@H](C2=CC3=C(C=C2)OCCO3)O.CCCCCCCC(=O)N[C@H](CN1CCCC1)[C@@H](C2=CC3=C(C=C2)OCCO3)O.[C@@H]([C@H](C(=O)O)O)(C(=O)O)O

KUBARPMUNHKBIQ-VTHUDJRQSA-N

InChI=1S/2C23H36N2O4.C4H6O6/c2*1-2-3-4-5-6-9-22(26)24-19(17-25-12-7-8-13-25)23(27)18-10-11-20-21(16-18)29-15-14-28-20;5-1(3(7)8)2(6)4(9)10/h2*10-11,16,19,23,27H,2-9,12-15,17H2,1H3,(H,24,26);1-2,5-6H,(H,7,8)(H,9,10)/t2*19-,23-;1-,2-/m111/s1

N-[(1R,2R)-1-(2,3-dihydro-1,4-benzodioxin-6-yl)-1-hydroxy-3-pyrrolidin-1-ylpropan-2-yl]octanamide;(2R,3R)-2,3-dihydroxybutanedioic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~104.26 mM)
H2O : ≥ 50 mg/mL (~52.13 mM)

Solubility in Formulation 1: ≥ 2.75 mg/mL (2.87 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 27.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.75 mg/mL (2.87 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 27.5 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.75 mg/mL (2.87 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 27.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 150 mg/mL (156.39 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)