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| Targets |
Biperiden is a potent inhibitor of mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1), a key mediator of NF-κB signaling [4].
MALT1 contains a paracaspase domain that catalyzes arginine-specific protein cleavage and is a key mediator of upstream NF-κB signaling [4]. |
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| ln Vitro |
In silico protein-ligand docking studies revealed that biperiden binds to the allosteric-inhibitory pocket of MALT1 with favorable binding energy (-72.5 kcal/mol for orientation 3), forming hydrophobic interactions with residues in the binding pocket [4].
In a fluorogenic activity assay with recombinant human MALT1 (339-719) protein, biperiden significantly decreased MALT1 activity. Compared to the positive control Z-VRPR-fmk (set as 100%), MALT1 activity inhibited by biperiden was reduced by 33% [4]. In human PDAC cell lines (Panc-1, Panc-2, BxPC3, L3.6pl wt, and gemcitabine-resistant L3.6pl-Res), treatment with biperiden (29.6 μg/ml) for 24 hours resulted in loss of cell adherence, morphological changes indicative of apoptosis, and inability to re-adhere after reseeding [4]. Cleaved Caspase-3 ELISA showed that biperiden treatment (29.6 μg/ml) induced significant apoptosis in Panc-1 and L3.6pl-Res cells compared to DMSO control [4]. MALT1 paracaspase activity in Panc-1 cells was significantly reduced after 24 hours of treatment with biperiden (29.6 μg/ml) compared to DMSO control (p < 0.001) [4]. MTT proliferation assays demonstrated that biperiden inhibited proliferation of PDAC cells in a dose- and time-dependent manner. At the highest dose (29.6 μg/ml), cell proliferation was nearly completely impeded after 72 hours of incubation. In contrast, human pancreatic ductal epithelial (HPDE) cells showed only minor reduction in proliferation at the same dose [4]. In MALT1 knockdown cells (Panc-1-MALT1\(^{KD}\)), biperiden treatment did not significantly affect proliferation, confirming that the drug acts through MALT1 [4]. Western blot analysis of cell fractions showed that biperiden treatment reduced nuclear translocation of c-Rel in Panc-1 cells, with the c-Rel/Lamin B ratio in the nucleus diminished by at least 0.5 after treatment [4]. |
| ln Vivo |
In a subcutaneous xenograft mouse model using Panc-1 human PDAC cells, mice treated daily with biperiden (10 mg/kg i.p.) showed an average tumor size reduction of 83% compared to untreated controls (factor 0.17; 95% CI [0.02, 1.60]; p = 0.112) [4].
Neurological assessment using standardized tests (challenging beam traversal, adhesive removal, spontaneous activity in cylinder) showed that biperiden treatment had tolerable side effects on motor coordination and balance. Beam traversal time and step count were significantly different between the biperiden group and control group (p < 0.001), but fine motor coordination (adhesive removal) was not adversely affected [4]. |
| Enzyme Assay |
A fluorogenic MALT1 activity assay was performed using recombinant human MALT1 (339-719) protein expressed in E. coli and purified via Ni-NTA affinity chromatography. The recombinant protein was present in bioactive dimeric structure. MALT1 activity was measured upon incubation with biperiden (29.6 μg/ml), mepazine (25 μM), thioridazine (reference substance), and Z-VRPR-fmk (positive control). All substances significantly decreased MALT1 activity compared to untreated recombinant hMALT1 [4].
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| Cell Assay |
For MALT1 activity cell assays, PDAC cells were incubated with biperiden (29.6 μg/ml) or mepazine (25 μM) for 24 hours. Cellular MALT1 protease activity was measured using a modified version of the protocol of Nagel et al. [4].
For apoptosis assays, cleaved caspase-3 was measured by Sandwich ELISA according to manufacturer's instructions after 24-hour treatment with biperiden (29.6 μg/ml) or mepazine (25 μM) [4]. For proliferation assays, cells were seeded in 96-well plates and treated with increasing concentrations of biperiden (7.4, 14.8, and 29.6 μg/ml). MTT assays were performed over 5 days to monitor metabolic activity and proliferation rates [4]. For Ki67 staining, cells on coverslips were fixed, incubated with anti-Ki67 antibody overnight, followed by secondary antibody for 2 hours, and mounted with Fluoroshield containing DAPI [4]. For cell fractionation, Nuclear and Cytoplasmic Extraction Reagents were used according to manufacturer's instructions to assess nuclear c-Rel translocation by western blot [4]. |
| Animal Protocol |
A subcutaneous xenograft mouse model was generated using Pfp-/-Rag2-/- double-knockout mice lacking NK cell function, mature T and B lymphocytes. 10⁶ viable Panc-1 cells suspended in 200 μl RPMI-1640 medium were injected subcutaneously between the mouse scapulae. Twelve days after tumor cell injection, mice were randomized and treated daily with either biperiden (10 mg/kg i.p.), mepazine (16 mg/kg i.p.), or no treatment (control group). Treatment was conducted under standardized conditions: Day 1 - drug injection and body weight determination; Day 2 - drug injection and tumor measurement with caliper; Day 3 - drug injection and neurological scoring. Mice were sacrificed when tumors in the control group reached ~10 mm in diameter or ulcerated [4].
Neurological side effects were monitored every third day with a test battery including challenging beam traversal (beam traversal time, step count, errors), spontaneous activity in the cylinder (rear count, grooming time), and adhesive removal test (time to sticker removal) [4]. A subcutaneous xenograft mouse model was generated using Pfp-/-Rag2-/- double-knockout mice lacking NK cell function, mature T and B lymphocytes. 10⁶ viable Panc-1 cells suspended in 200 μl RPMI-1640 medium were injected subcutaneously between the mouse scapulae. Twelve days after tumor cell injection, mice were randomized and treated daily with either biperiden (10 mg/kg i.p.), mepazine (16 mg/kg i.p.), or no treatment (control group). Treatment was conducted under standardized conditions: Day 1 - drug injection and body weight determination; Day 2 - drug injection and tumor measurement with caliper; Day 3 - drug injection and neurological scoring. Mice were sacrificed when tumors in the control group reached ~10 mm in diameter or ulcerated [4]. Neurological side effects were monitored every third day with a test battery including challenging beam traversal (beam traversal time, step count, errors), spontaneous activity in the cylinder (rear count, grooming time), and adhesive removal test (time to sticker removal) [4]. |
| ADME/Pharmacokinetics |
Biperiden is an anticholinergic drug that has been in clinical use for more than 60 years. It binds to muscarinic receptors (M1), leading to repression of acetylcholine and enhanced dopamine signaling in the central nervous system [4].
The dosage used in experiments (29.6 μg/ml in vitro; 10 mg/kg i.p. in vivo) was adapted to the known bioavailability and therapeutic range of the drug [4]. |
| Toxicity/Toxicokinetics |
Biperiden is a well-tolerated drug even at high dosages, with only mild adverse effects, especially when compared to conventional chemotherapeutics. Common adverse effects of chemotherapeutic drugs lead to symptoms of immune system deficiency with high infection susceptibility and impaired cell regeneration, whereas biperiden does not show such effects [4].
In the xenograft mouse model, biperiden treatment resulted in only minor motor side effects that were acceptable in light of the severity of the illness and the common adverse effects of chemotherapy. Beam traversal time and step count showed significant differences from controls, but these effects were tolerable [4]. In contrast, mepazine treatment resulted in three mice dying due to intraabdominal hemorrhages, possibly due to neutropenia, agranulocytosis, or thrombocytopenia. No such effects were reported for biperiden [4]. In vitro assays with human pancreatic ductal epithelial (HPDE) cells showed only minor reduction in cell proliferation when treated with high-dose biperiden (29.6 μg/ml) after 72 hours, suggesting that healthy pancreatic tissue should not be significantly affected by the drug [4]. |
| References | |
| Additional Infomation |
Bellidifolin is a flavonoid compound, a product of belitidin with a methyl group substituted at the O-3 position. It is a natural product, primarily found in Swertia chirata and Gentianella campestris. Bellidifolin has multiple functions, including acting as an EC 3.1.1.7 (acetylcholinesterase) inhibitor, a hypoglycemic agent, and a metabolite. It belongs to the flavonoid and polyphenol classes and is functionally related to belitidin. Bellidifolin has been reported in Gentiana algida, Gentiana thunbergii, and other organisms with relevant data.
Biperiden is an antiparkinsonian drug of the anticholinergic type that has been in clinical use for decades. It is regularly used to treat neurological side effects of phenothiazines, like extrapyramidal symptoms [4]. The study identifies biperiden as a novel MALT1 inhibitor with even less pharmacological side effects than mepazine. By compromising MALT1 activity, nuclear translocation of c-Rel is prevented, which is critical for NF-κB-dependent inhibition of apoptosis [4]. MALT1 is expressed in the majority of pancreatic ductal adenocarcinomas (PDACs) but absent from normal exocrine pancreatic tissue, making it a specific target in cancer cells. Immunohistochemistry of a tissue microarray containing 213 primary PDACs showed MALT1 expression in 75.1% of tumors [4]. The study concludes that off-label use of biperiden represents a promising new therapeutic approach to PDAC treatment, with reduced proliferation and increased apoptosis in PDAC cells in vitro and in vivo, and tolerable side effects [4]. Biperiden is an FDA-approved prescription drug (ATC code N04AA02) with a well-established safety profile based on decades of clinical use [4]. |
| Molecular Formula |
C14H10O6
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|---|---|
| Molecular Weight |
274.228
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| Exact Mass |
274.047
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| CAS # |
2798-25-6
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| PubChem CID |
5281623
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| Appearance |
Light yellow to yellow solid powder
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| Density |
1.6±0.1 g/cm3
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| Boiling Point |
580.2±50.0 °C at 760 mmHg
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| Melting Point |
265-267ºC
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| Flash Point |
228.0±23.6 °C
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| Vapour Pressure |
0.0±1.7 mmHg at 25°C
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| Index of Refraction |
1.714
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| LogP |
0.9
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
1
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| Heavy Atom Count |
20
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| Complexity |
386
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
JDIORNFCMMYMLF-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C14H10O6/c1-19-6-4-9(17)11-10(5-6)20-14-8(16)3-2-7(15)12(14)13(11)18/h2-5,15-17H,1H3
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| Chemical Name |
1,5,8-trihydroxy-3-methoxyxanthen-9-one
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| Synonyms |
Bellidifolin CCRIS 5471 BRN 0288441
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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 (e.g. under nitrogen), avoid exposure to moisture and light. |
| 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 | 3.6466 mL | 18.2329 mL | 36.4657 mL | |
| 5 mM | 0.7293 mL | 3.6466 mL | 7.2931 mL | |
| 10 mM | 0.3647 mL | 1.8233 mL | 3.6466 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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