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| Targets |
glucosylceramide synthase[1]
Glucosylceramide (GlcCer) synthase (GCS, also called UDP‑glucose:ceramide glucosyltransferase). D-threo-PPMP is a potent inhibitor of GCS, blocking the transfer of glucose from UDP‑glucose to ceramide. By inhibiting GCS, the compound reduces the biosynthesis of glycosphingolipids, including gangliosides, globosides, and lactosylceramides. This inhibition affects membrane organization, cell signaling, and cell adhesion. |
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| ln Vitro |
In KB-V0.01 cells, D-threo-PPMP (10 μM; 72 h) hydrochloride reduces MDR1 expression by 70%[1].
In vitro, D-threo-PPMP hydrochloride potently inhibits glucosylceramide synthase activity with IC50 values in the low nanomolar range (reported IC50 approx. 20‑30 nM). In MDCK (Madin‑Darby canine kidney) epithelial cells, treatment with D-threo-PPMP (1‑10 uM) blocks cyst formation and induces cyst regression in three‑dimensional culture models of polycystic kidney disease (PKD). The compound also blocks nuclear division (karyokinesis) and reduces the size and number of cysts. By inhibiting GSL synthesis, D-threo-PPMP affects cell‑cell and cell‑matrix interactions. |
| ln Vivo |
In vivo, D-threo-PPMP has been evaluated in mouse models of polycystic kidney disease (PKD). In the PKD1 mutant mice (a model of autosomal dominant PKD), D-threo-PPMP (2 mg/kg/day) given orally or intraperitoneally reduced kidney cyst formation, preserved renal function, and prolonged survival. The compound reduces the accumulation of glycosphingolipids in the kidneys. It also has been studied in models of cancer, where reducing GSLs affects tumor growth and metastasis. Detailed in vivo activity parameters are not fully published.
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| Enzyme Assay |
Standard cell‑free glucosylceramide synthase (GCS) assays use rat liver microsomes or recombinant human GCS expressed in insect cells. Assays are performed in 100 mM HEPES buffer (pH 7.2) containing 20 mM KCl, 10 mM MgCl2, 5 mM DTT, 0.1% Tween‑20, and 0.1% BSA. Microsomes (50‑100 ug protein) or recombinant GCS are incubated with varying concentrations of D-threo-PPMP (0.01‑1000 nM) for 10‑15 min at 37degC. The reaction is initiated by adding 50 uM C6‑NBD‑ceramide (a fluorescent ceramide analog) and 0.5‑1 mM UDP‑glucose. After 30‑60 min at 37degC, the reaction is terminated by adding 200 uL of chloroform:methanol (2:1). Lipids are extracted, separated by thin layer chromatography (TLC) on silica gel plates using chloroform:methanol:water (65:25:4) as the mobile phase. The fluorescent GlcCer band (C6‑NBD‑glucosylceramide) is quantified by fluorescence imaging (ex 470 nm, em 535 nm) or by scraping the silica and extracting the product. IC50 values are calculated from inhibition curves. For radiolabeled assays, [3H]UDP‑glucose (0.5 uCi/assay) is used as a donor, and the product [3H]GlcCer is quantified by liquid scintillation counting. For selectivity, D-threo-PPMP does not inhibit other glycosyltransferases at concentrations up to 10 uM.
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| Cell Assay |
For cellular assays, MDCK cells (Madin‑Darby canine kidney cells) are seeded in 3D collagen‑I or Matrigel cultures (5000 cells/well in 96‑well plates) to induce cyst formation. Cells are cultured in DMEM/10% FBS for 10‑14 days until cysts form (size ≥50 um). D-threo-PPMP is dissolved in DMSO and added to the culture medium at final concentrations of 0.1‑20 uM (final DMSO ≤0.1%). After 1‑6 days, cyst size (diameter) is measured using an inverted microscope with imaging software (ImageJ). Cyst number, lumen area, and total cyst volume are quantified. For assays of proliferation, cells are seeded in 96‑well plates (5,000 cells/well) in 2D culture. D-threo-PPMP (0.1‑20 uM) is added for 24‑72 hours. Viability is measured by MTT or CellTiter‑Glo. For apoptosis assays, cells are stained with annexin V‑FITC/PI and analyzed by flow cytometry. For GSL analysis, cells are treated with D-threo-PPMP for 48 hours, lipids are extracted with chloroform:methanol, and GSLs (glucosylceramide, lactosylceramide, gangliosides GM1, GM2, GM3) are resolved by TLC and visualized by orcinol or resorcinol staining. GM1 levels can also be measured by cholera toxin B‑FITC binding and flow cytometry. For cell cycle analysis, cells are fixed in 70% ethanol and stained with propidium iodide. For Western blot analysis, lysates are probed for markers of proliferation (PCNA, Ki‑67), apoptosis (cleaved caspase‑3, PARP), and cell adhesion (E‑cadherin, beta‑catenin). D-threo-PPMP inhibits GSL synthesis, reduces cyst formation, and blocks mitogenesis at 1‑10 uM in MDCK cells.
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| Animal Protocol |
In vivo studies are performed in male C57BL/6 or PKD1 mutant mice (e.g., Pkd1flox/flox; Ksp‑Cre mice). D-threo-PPMP hydrochloride is formulated in 0.5% methylcellulose or 10% DMSO/40% PEG300/5% Tween‑80/45% saline and administered by oral gavage (1‑10 mg/kg) or intraperitoneal injection (0.5‑5 mg/kg) daily for 4‑12 weeks. In the PKD model, treatment starts at 4 weeks of age and continues for 8 weeks. Kidney function is assessed by measuring blood urea nitrogen (BUN) and serum creatinine. At termination, kidney weight is measured, and the cyst index (percentage of kidney area occupied by cysts) is determined by H&E histology. Immunohistochemistry is performed for PCNA (proliferation), cleaved caspase‑3 (apoptosis), and E‑cadherin (tubular integrity). Glycosphingolipid levels in kidney tissue are measured by TLC or mass spectrometry. For survival studies, mice are treated for up to 6 months, and the Kaplan‑Meier survival curve is plotted. For PK/PD correlation, plasma and kidney tissue concentrations of D-threo-PPMP are measured by LC‑MS/MS. D-threo-PPMP reduces cyst formation and preserves renal function in PKD models.
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| ADME/Pharmacokinetics |
D-threo-PPMP hydrochloride (MW 619.85, C38H72Cl3N3O3) is the active enantiomer of the racemic mixture. It has moderate oral bioavailability (estimated 30‑50% in rodents). The compound is metabolized by CYP3A4 in the liver. Plasma half‑life in rats is approximately 3‑6 hours. Peak plasma concentrations occur within 2‑4 hours after oral administration. The compound is soluble in DMSO (≥25 mg/mL) and has low solubility in water. Plasma protein binding is high (>95%). Storage: powder at -20degC for 3 years.
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| Toxicity/Toxicokinetics |
Preclinical toxicity data are limited. In mouse studies, oral doses up to 30 mg/kg produce no overt signs of toxicity or mortality. D-threo-PPMP at 10 mg/kg/day for 8 weeks in mice is well‑tolerated, with no significant changes in body weight or liver enzymes (ALT, AST). At higher doses (>50 mg/kg), gastrointestinal distress (diarrhea) may occur due to inhibition of GSL synthesis in the intestinal mucosa. No genotoxicity or carcinogenicity data are available. D-threo-PPMP is for research use only, not FDA‑approved.
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| References |
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| Additional Infomation |
D-threo-PPMP hydrochloride (CAS 139889-65-9) is a potent and selective inhibitor of glucosylceramide synthase (GCS). It is the active enantiomer of DL-threo-PPMP. By blocking GSL biosynthesis, it reduces cyst formation in polycystic kidney disease models and blocks mitosis. The compound is used as a research tool to study the role of GSLs in cell proliferation, differentiation, and disease. It has not entered clinical trials. Storage: desiccated at -20degC.
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| Molecular Formula |
C29H51CLN2O3
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|---|---|
| Molecular Weight |
511.18
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| Exact Mass |
510.358
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| CAS # |
139889-65-9
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| Related CAS # |
D-threo-PPMP;139889-53-5
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| PubChem CID |
49837889
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| Appearance |
White to off-white solid powder
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
19
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| Heavy Atom Count |
35
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| Complexity |
490
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| Defined Atom Stereocenter Count |
2
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| SMILES |
CCCCCCCCCCCCCCCC(=O)N[C@H](CN1CCOCC1)[C@@H](C2=CC=CC=C2)O.Cl
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| InChi Key |
ORVAUBQYJOFWFY-ZHESDOBJSA-N
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| InChi Code |
InChI=1S/C29H50N2O3.ClH/c1-2-3-4-5-6-7-8-9-10-11-12-13-17-20-28(32)30-27(25-31-21-23-34-24-22-31)29(33)26-18-15-14-16-19-26;/h14-16,18-19,27,29,33H,2-13,17,20-25H2,1H3,(H,30,32);1H/t27-,29-;/m1./s1
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| Chemical Name |
N-[(1R,2R)-1-hydroxy-3-morpholin-4-yl-1-phenylpropan-2-yl]hexadecanamide;hydrochloride
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| HS Tariff Code |
2934.99.9001
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| Storage |
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. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.9563 mL | 9.7813 mL | 19.5626 mL | |
| 5 mM | 0.3913 mL | 1.9563 mL | 3.9125 mL | |
| 10 mM | 0.1956 mL | 0.9781 mL | 1.9563 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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