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| 100mg |
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| Targets |
The primary target of velnacrine maleate is acetylcholinesterase (AChE), which it inhibits reversibly. Similar to its parent compound tacrine, velnacrine acts by inhibiting acetylcholinesterase, thereby reducing the hydrolysis of acetylcholine in the synaptic cleft and increasing acetylcholine levels in the central nervous system to improve cognitive function in patients with Alzheimer's disease. Studies have also shown that velnacrine may exhibit nonselective blocking actions on potassium channels in motor nerve terminals at high concentrations, though this effect is primarily observed at elevated concentrations.
Velnacrine maleate targets cholinesterase enzymes, primarily acetylcholinesterase (AChE). By inhibiting AChE, the compound increases the levels of acetylcholine in the synaptic cleft, enhancing cholinergic neurotransmission. This mechanism is the basis for its potential use in Alzheimer's disease, where cholinergic deficits are a hallmark of the disease. As a metabolite of tacrine, Velnacrine maleate likely shares similar cholinesterase inhibitory activity with the parent compound, though it may have a different potency and safety profile. |
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| ln Vitro |
: In vitro studies demonstrate that velnacrine maleate acts primarily as an anticholinesterase agent. In experiments on isolated nerve-muscle preparations, velnacrine, along with tacrine, augmented responses of chick biventer cervicis preparations to nerve stimulation and increased responses to exogenously applied acetylcholine, indicating its classical anticholinesterase activity. In mouse diaphragm preparations, velnacrine reversed twitch block induced by tubocurarine or low calcium solutions, further confirming its ability to enhance neuromuscular transmission. In toxicity mechanism studies, in vitro hepatocyte culture experiments showed that velnacrine alone did not cause prominent cytotoxicity, but induced cytotoxic responses under conditions of glutathione depletion.
In vitro, Velnacrine maleate acts as a cholinesterase inhibitor. Its activity is typically assessed using colorimetric assays (e.g., Ellman's method) that measure the hydrolysis of acetylthiocholine by AChE. The compound's IC50 for AChE inhibition is determined by incubating the enzyme with various concentrations of the compound and substrate, and measuring the rate of product formation. As a metabolite of tacrine, Velnacrine maleate may exhibit similar or different potency compared to the parent compound. Standard in vitro assays also include assessment of butyrylcholinesterase inhibition for selectivity profiling. |
| ln Vivo |
In vivo studies demonstrate that velnacrine maleate can slow cognitive decline in patients with Alzheimer's disease. A 24-week double-blind, placebo-controlled Phase III clinical trial (n=449) showed that cognitive behavior and memory components of the Alzheimer's Disease Assessment Scale deteriorated in the placebo-treated group (P<.05) but not in velnacrine-treated groups, with the 225 mg/day dose demonstrating superior efficacy to the 150 mg/day dose (P<.05). However, the drug was associated with dose-dependent liver function abnormalities in clinical trials. Pharmacokinetic studies showed that following oral administration of [¹⁴C]-labeled velnacrine to rats and dogs, drug-related material was well absorbed, with the majority of the dose recovered in the urine and eliminated primarily within 24 hours.
In vivo, Velnacrine maleate has been studied in animal models of cognitive impairment, including aged rats and scopolamine-induced memory deficit models. In these models, the compound's ability to improve learning and memory is assessed using behavioral tests such as the Morris water maze, passive avoidance, or radial arm maze. The compound's cholinesterase inhibitory activity in brain tissue can be confirmed by measuring AChE activity in brain homogenates ex vivo. However, comprehensive in vivo efficacy data from published literature are limited. |
| Enzyme Assay |
Enzyme Source Preparation: Use human erythrocyte-derived acetylcholinesterase or purified acetylcholinesterase from electric eel.
Substrate Preparation: Using the Ellman colorimetric method, prepare acetylthiocholine iodide as substrate with DTNB in 0.1 M phosphate buffer (pH 7.4).
Inhibitor Incubation: Pre-incubate varying concentrations of velnacrine maleate (e.g., 0.1-100 µM) with the enzyme in buffer for 5-10 minutes at 25°C.
Reaction Initiation and Detection: Initiate the reaction by adding substrate, continuously monitor absorbance changes at 412 nm, and record the reaction rate.
Data Analysis: Calculate IC₅₀ values and inhibition constant Ki through enzyme kinetic analysis, plotting Lineweaver-Burk double reciprocal plots to determine the inhibition type.
For non-cell-based receptor binding assays, Velnacrine maleate can be evaluated using purified acetylcholinesterase or butyrylcholinesterase enzymes. Enzyme activity assays are performed using the Ellman method, where the enzyme is incubated with the substrate acetylthiocholine and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB). Increasing concentrations of the test compound are added, and the rate of thiocholine production (measured by absorbance at 412 nm) is monitored. IC50 values are calculated from the inhibition curves. This assay does not involve receptor binding but rather enzyme inhibition kinetics. |
| Cell Assay |
Cell Culture: Use rat primary hepatocytes or human hepatoma cell lines (e.g., HepG2) for in vitro cytotoxicity studies. Culture cells in medium containing 10% fetal bovine serum at 37°C in a 5% CO₂ incubator.
Glutathione Depletion Treatment: Pre-treat cells with diamide (0.5 mM) for 30 minutes to deplete intracellular glutathione, or use combination treatment with diamide and t-butyl hydroperoxide to induce oxidative stress.
Drug Treatment: Add varying concentrations of velnacrine maleate (e.g., 1-50 µg/mL) and incubate for 4-24 hours.
Cytotoxicity Detection: Assess cytotoxicity using lactate dehydrogenase leakage assay, neutral red uptake assay, and MTT reduction assay.
Mechanistic Studies: Detect intracellular reactive oxygen species levels and calcium influx using fluorescent probes.
Data Analysis: Compare cell viability metrics and cytotoxicity markers between treatment and control groups.
For in vitro cellular assays, neuroblastoma cells or primary neurons can be used to assess the compound's effects on acetylcholine levels and neuronal function. Cells are treated with Velnacrine maleate and extracellular acetylcholine levels are measured using HPLC or biosensors. For cytotoxicity assessment, cell viability is measured using MTT or LDH assays. For neuronal function studies, electrophysiological recordings or calcium imaging may be performed to assess the compound's effects on neuronal activity. The compound's effects on cholinergic signaling are evaluated in the context of Alzheimer's disease research. |
| Animal Protocol |
Animal Selection and Handling: Use male Sprague-Dawley rats and Beagle dogs for pharmacokinetic and metabolism studies. Administer [¹⁴C]-labeled velnacrine maleate to animals via intravenous injection or oral gavage.
Pharmacokinetic Sampling: Collect blood samples at specified time points (0-72 hours) after administration; house some animals in metabolic cages to collect urine and feces.
Radioactivity Detection: Measure radioactivity in plasma, urine, and feces by liquid scintillation counting to calculate pharmacokinetic parameters (AUC, Cmax, Tmax, half-life, etc.).
Metabolite Identification: Separate metabolites by thin-layer chromatography and identify their structures in dog urine by GC/MS and proton NMR.
Tissue Distribution Studies: Euthanize animals at various time points after administration, collect tissues including brain, liver, kidney, heart, and lung, and measure radioactivity distribution in these tissues.
Data Analysis: Compare pharmacokinetic profiles and metabolic characteristics across different species.
For in vivo animal studies, Velnacrine maleate is typically administered orally to rodents. In cognitive impairment models (e.g., aged rats, scopolamine-induced amnesia models, or transgenic Alzheimer's disease models), behavioral tests (Morris water maze, novel object recognition, passive avoidance) are performed following compound administration. Brain tissue is collected post-mortem for measurement of AChE activity and acetylcholine levels. Cholinergic markers may be assessed by immunohistochemistry. Dosing regimens vary depending on the specific model. |
| ADME/Pharmacokinetics |
Velnacrine maleate is well absorbed after oral administration. In rats and dogs, pharmacokinetic parameters for plasma radioactivity elimination following oral dosing are similar to those following intravenous dosing, suggesting high oral bioavailability. Drug-related material is primarily eliminated via urine, with fecal elimination accounting for the remainder; most radioactivity is eliminated within 24 hours. Velnacrine is extensively metabolized, with approximately 33%, 19%, and 10% of the dose excreted unchanged in the urine of rats, dogs, and humans, respectively. The main metabolic pathway involves hydroxylation of the tetrahydroaminoacridine ring, producing mono-hydroxylated and di-hydroxylated metabolites, as well as two dihydrodiol metabolites. Phase II metabolism does not appear to be a significant pathway.
The pharmacokinetic properties of Velnacrine maleate have not been extensively characterized. As an orally active compound, it is expected to have good oral absorption. The compound has a molecular weight of 330.33. As a tacrine metabolite, its pharmacokinetic profile may be similar to that of tacrine, which has a half-life of 2-4 hours and is metabolized by CYP1A2. Comprehensive ADME studies would be needed for full pharmacokinetic characterization, including assessment of oral bioavailability, half-life, protein binding, and tissue distribution. |
| Toxicity/Toxicokinetics |
The primary target organ of velnacrine maleate toxicity is the liver. In Phase III clinical trials, 30% of patients in the 150 mg/day group and 24% in the 225 mg/day group discontinued treatment due to reversible liver function abnormalities (ALT/AST elevations more than 5 times the upper limit of normal), compared to only 3% in the placebo group. This liver injury presents as reversible hepatocellular injury, analogous to the hepatotoxicity profile reported for tacrine. In vitro mechanistic studies suggest that oxidative stress and glutathione depletion may enhance the hepatotoxic potential of velnacrine. Other common adverse events in clinical trials included diarrhea (28-30% incidence), though this rarely led to treatment discontinuation. Researchers developed a statistical model called PROPP (Physician Reference Of Predicted Probabilities) to predict the risk of hepatotoxicity in individual patients prior to drug exposure.
The toxicity profile of Velnacrine maleate is an important consideration, as tacrine (the parent compound) is associated with hepatotoxicity. Velnacrine maleate (hydroxytacrine) may have a different safety profile due to the hydroxylation modification. Potential adverse effects may include gastrointestinal disturbances and hepatotoxicity, similar to other cholinesterase inhibitors. The compound was investigated for Alzheimer's disease but may not have received regulatory approval. Standard toxicological evaluation would include acute and repeated-dose toxicity studies, as well as liver function monitoring. |
| References | |
| Additional Infomation |
See also: Velnacrine (has active moiety).
Velnacrine maleate (HP 029; Hydroxytacrine maleate) is an orally active cholinesterase inhibitor investigated for the research of Alzheimer's disease. It is a major active metabolite of tacrine, formed via hydroxylation by CYP1A1 and CYP1A2. The compound is also known as 9-amino-1,2,3,4-tetrahydroacridin-1-ol maleate. Velnacrine maleate has a molecular weight of 330.33 and was studied as a potential alternative to tacrine with potentially improved safety profile. It is available for research purposes only. |
| Molecular Formula |
C17H18N2O5
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|---|---|
| Molecular Weight |
330.34
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| Exact Mass |
330.121
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| Elemental Analysis |
C, 61.81; H, 5.49; N, 8.48; O, 24.22
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| CAS # |
118909-22-1
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| Related CAS # |
124027-47-0
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| PubChem CID |
5702293
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| Appearance |
Light yellow to khaki solid powder
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| Melting Point |
171-173ºC
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| LogP |
2.479
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
24
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| Complexity |
377
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1CC(C2=C(C3=CC=CC=C3N=C2C1)N)O.C(=C\C(=O)O)\C(=O)O
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| InChi Key |
NEEKVKZFYBQFGT-BTJKTKAUSA-N
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| InChi Code |
InChI=1S/C13H14N2O.C4H4O4/c14-13-8-4-1-2-5-9(8)15-10-6-3-7-11(16)12(10)13;5-3(6)1-2-4(7)8/h1-2,4-5,11,16H,3,6-7H2,(H2,14,15);1-2H,(H,5,6)(H,7,8)/b;2-1-
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| Chemical Name |
9-amino-1,2,3,4-tetrahydroacridin-1-ol;(Z)-but-2-enedioic acid
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| Synonyms |
Velnacrine maleate; Velnacrine; 124027-47-0; 9-amino-1,2,3,4-tetrahydroacridin-1-ol; 1-hydroxytacrine; 1-Acridinol, 9-amino-1,2,3,4-tetrahydro-; 118909-22-1; Mentane; HP-029; HP 029;
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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) |
DMSO: 30 mg/mL (90.82 mM)
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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 | 3.0272 mL | 15.1359 mL | 30.2718 mL | |
| 5 mM | 0.6054 mL | 3.0272 mL | 6.0544 mL | |
| 10 mM | 0.3027 mL | 1.5136 mL | 3.0272 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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