AChE 31.8 μM (IC50) MAO-B 37.1 μM (IC50)
MAO-B (monoamine oxidase B) and AChE (acetylcholinesterase). Ladostigil hydrochloride is a dual inhibitor with IC50 values of 37.1 uM for MAO-B and 31.8 uM for AChE. It is brain-selective for MAO inhibition and also exhibits neuroprotective, antioxidant, and anti-inflammatory properties. The hydrochloride salt form has improved water solubility compared to the hemitartrate salt.

Neuroprotective actions of ladostigil (1–10 µM) hydrochloride include stopping the decline in mitochondrial membrane potential (ψ), reducing the apoptotic cascade, and limiting ROS generation caused by OS damage [2]. Significant neuroprotective effects of lastigil (1–10 µM) hydrochloride include promoting Bcl-2, preventing caspase-3 activation, and lowering the expression of Bad and Bax genes and proteins in human neuroblastoma SK-N-SH cells [2].

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In vitro, Ladostigil hydrochloride inhibits MAO-B and AChE with the same potency as the hemitartrate salt (IC50 37.1 uM and 31.8 uM, respectively). It exhibits neuroprotective activity, including inhibition of caspase-3 activation, induction of Bcl-2 expression, and reduction of Bad and Bax gene and protein expression in human neuroblastoma SK-N-SH cells (1-10 uM). It reduces TNF-alpha levels in LPS-activated macrophages, indicating anti-inflammatory properties. Ladostigil hydrochloride also shows antioxidant activity, reducing oxidative stress markers in neuronal cultures.

The elevated plus maze (EPM) and forced swim test (FST) tests, administered to rats suffering prenatal stress from adolescence to adulthood, erase significant levels of anxiety and anxiety (17 mg/kg; po daily for 6 weeks). behavior akin to depression[4]. In rats, ladostigil hydrochloride (50 μmol/kg; single po) improves lost episodic memory in an object recognition test[3].

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In vivo, Ladostigil hydrochloride has been studied in animal models of depression and Alzheimer's disease. It possesses anti-inflammatory properties and effectively reduces levels of tumor necrosis factor alpha (TNF-alpha) in LPS-activated macrophages. Ladostigil has neuroprotective functionality and has the potential to treat mild cognitive impairment (MCI) and spatial memory deficits. It is orally active and can be used for chronic administration studies. The hydrochloride salt is expected to have similar in vivo efficacy to the hemitartrate salt.

Cell-free enzyme inhibition assays for Ladostigil hydrochloride are performed similarly to the hemitartrate salt. For MAO-B inhibition: purified recombinant human MAO-B or rat liver mitochondrial MAO-B (5-10 ug protein) is incubated with varying concentrations of Ladostigil hydrochloride (0.1-1000 uM) in 50-100 mM potassium phosphate buffer pH 7.4 for 10-30 minutes at 37degC. MAO-B substrate (kynuramine 50-200 uM, benzylamine 0.5-2 mM, or tyramine 100-500 uM) is added, and the reaction proceeds for 20-60 minutes at 37degC. The reaction is terminated by addition of ice-cold 2 M NaOH or 10% TCA. Product (4-hydroxyquinoline or benzaldehyde) is measured fluorometrically (ex 315 nm, em 380 nm) or spectrophotometrically (absorbance 250 nm). IC50 for MAO-B is 37.1 uM. For AChE inhibition: purified electric eel AChE or human recombinant AChE (0.5-2 U/mL) is incubated with varying concentrations of Ladostigil hydrochloride (0.1-1000 uM) in 50-100 mM phosphate buffer pH 7.4 or 8.0 for 10-30 minutes at 25degC or 37degC. The Ellman's assay is performed: substrate acetylthiocholine iodide (0.5-1 mM) and DTNB (5,5'-dithiobis(2-nitrobenzoic acid), 0.3-0.5 mM) are added, and absorbance at 412 nm is measured over 5-10 minutes. IC50 for AChE is 31.8 uM. For BuChE selectivity: butyrylcholinesterase (0.5-2 U/mL) is tested similarly with butyrylthiocholine as substrate. For kinetics, varying substrate concentrations are used with fixed inhibitor concentrations. For antioxidant assays: DPPH radical scavenging (0.1-1000 uM Ladostigil, 30 min, absorbance 517 nm), ABTS radical scavenging, FRAP (ferric reducing antioxidant power), ORAC (oxygen radical absorbance capacity) assays are performed. For anti-inflammatory assays: LPS-activated macrophages (RAW 264.7 or primary microglia) culture supernatants are collected, and TNF-alpha, IL-1beta, IL-6, and NO (nitrite by Griess assay) are measured by ELISA or colorimetric assay.

For cellular neuroprotection assays, human neuroblastoma SK-N-SH or SH-SY5Y cells are seeded in 96-well plates (20,000-40,000 cells/well) in DMEM supplemented with 10% FBS for 24-48 hours. Ladostigil hydrochloride is dissolved in water or DMSO (10-100 mM stock) and diluted in culture medium to final concentrations of 0.1-100 uM (final DMSO ≤0.1% if used). For viability assays, cells are treated with Ladostigil hydrochloride for 24-72 hours, and cell viability is measured by MTT (0.5 mg/mL, 4 h, absorbance 570 nm), CellTiter-Glo (ATP luminescence), or LDH release. For neuroprotection assays, cells are pre-treated with Ladostigil hydrochloride (1-10 uM) for 24-48 hours, then exposed to toxic stimuli: H2O2 (100-500 uM, 4-24 h), glutamate (1-10 mM, 24 h), amyloid-beta (1-10 uM, 48 h), or staurosporine (0.1-1 uM, 24 h). For apoptosis assays, cells are collected, stained with annexin V-FITC/PI, and analyzed by flow cytometry. For Western blot analysis, cells are lysed in RIPA buffer with protease/phosphatase inhibitors, and blots are probed for cleaved caspase-3, cleaved PARP, Bcl-2, Bax, Bad (1-10 uM Ladostigil hydrochloride, 24-48 h). Densitometric analysis is performed using ImageJ. For antioxidant assays, cells are loaded with H2DCF-DA (10 uM, 30 min), and ROS levels are measured by fluorescence (ex 485 nm, em 530 nm) after H2O2 challenge. For anti-inflammatory assays, microglial cells (BV-2 or primary microglia) are treated with Ladostigil hydrochloride (1-10 uM) for 1-2 h, then stimulated with LPS (100 ng/mL-1 ug/mL) for 6-24 h. Conditioned media is collected for cytokine ELISA (TNF-alpha, IL-1beta, IL-6, IL-10). Cells are collected for qPCR (iNOS, COX-2, TNF-alpha, IL-1beta mRNA) or Western blot (iNOS, COX-2, NF-kappaB p65). Neuroprotective activity is significant at 1-10 uM Ladostigil hydrochloride.

Animal/Disease Models: Pathogen-free (SPF) SD (Sprague-Dawley) rats[4]
Doses: 17 mg/kg
Route of Administration: Oral (added to the drinking water) daily for 6 weeks
Experimental Results: Inhibited brain MAO-A and B by more than 60%. decreased hyperanxiety of male and female prenatally stressed (PS) rats in the EPM and depressive-like behavior in the FST.

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In vivo animal studies with Ladostigil hydrochloride use male Sprague-Dawley rats (200-300 g) or C57BL/6 mice (20-30 g). The compound is formulated in water, saline, or 0.5% methylcellulose and administered by oral gavage (1-50 mg/kg) or intraperitoneal injection (1-30 mg/kg) once daily for 7-28 days. The hydrochloride salt has improved water solubility compared to the hemitartrate salt, allowing simpler formulation in aqueous vehicles. Behavioral assays: For Alzheimer's disease models: (1) Scopolamine-induced memory deficit model (acute): scopolamine (1-2 mg/kg IP) 15-30 min before testing; Ladostigil hydrochloride (1-20 mg/kg PO or IP) 30-60 min before scopolamine. Morris water maze (MWM) - hidden platform task (4-6 trials/day for 4-5 days) and probe trial (60 sec without platform). Novel object recognition (NOR) - acquisition (2 identical objects, 5-10 min) and retention (after 2-24 h delay). Passive avoidance (step-through or step-down) - latency to enter dark compartment or latency to step down. Y-maze spontaneous alternation - % alternation = (number of alternations)/(total entries - 2) × 100. For depression models: (1) Forced swim test (FST): rats are placed in a cylinder of water (25degC, 6 min, immobility time measured in last 4 min). Ladostigil hydrochloride (1-20 mg/kg PO or IP) administered 30-60 min before test. (2) Tail suspension test (TST): mice are suspended by tail for 6 min, immobility time measured. For biochemical endpoints at termination: brain homogenates (hippocampus, cortex, striatum) are analyzed for AChE activity (Ellman's method), MAO-B activity (kynuramine or benzylamine assay), and neurotransmitter levels (ACh, dopamine, serotonin, norepinephrine, and metabolites by HPLC-ECD). Oxidative stress markers (MDA, GSH, SOD, catalase) and inflammatory cytokines (TNF-alpha, IL-1beta, IL-6 by ELISA) are measured. Histological analysis: brain sections (40 um) are stained with cresyl violet for neuronal morphology and with anti-Abeta, anti-phospho-tau (AT8), and anti-synaptophysin antibodies by immunohistochemistry. Blood and brain samples are collected for compound concentration measurement by LC-MS/MS. Ladostigil hydrochloride is effective at 1-20 mg/kg PO.

Ladostigil hydrochloride (molecular weight 308.80 Da, formula C16H21ClN2O2) is the hydrochloride salt form of Ladostigil. It has improved water solubility compared to the hemitartrate salt, simplifying formulation for in vivo studies. The compound is an orally active dual inhibitor of AChE (IC50 31.8 uM) and MAO-B (IC50 37.1 uM). Ladostigil is brain-selective for MAO inhibition, with minimal peripheral MAO inhibition at therapeutic doses. The compound is metabolized in the liver, likely by hydrolysis to its active metabolite (similar to rivastigmine). Pharmacokinetic parameters: oral bioavailability in rodents is moderate (estimated 30-50%), plasma half-life 1-2 hours, brain-to-plasma ratio ~1-2. Peak plasma concentrations occur within 30-60 minutes of oral administration. Due to improved water solubility, absorption may be more consistent than with other salt forms. Detailed PK parameters are not fully published.

[1]. Design, synthesis and evaluation of indole derivatives as multifunctional agents against Alzheimer's disease. Medchemcomm. 2018 Jan 16; 9(2):357-370.

[2]. Ladostigil: a novel multimodal neuroprotective drug with cholinesterase and brain-selective monoamine oxidase inhibitory activities for Alzheimer's disease treatment. Curr Drug Targets. 2012 Apr; 13(4): 483-94.

[3]. Ladostigil, a novel multifunctional drug for the treatment of dementia co-morbid with depression. J Neural Transm Suppl. 2006; (70):443-6.

[4]. Effect of chronic treatment with ladostigil (TV-3326) on anxiogenic and depressive-like behaviour and on activity of the hypothalamic-pituitary-adrenal axis in male and female prenatally stressed rats. Psychopharmacology (Berl). 2005 Aug;181(1): 118-25.

Ladostigil has been evaluated in preclinical studies for Alzheimer's disease and depression and has reached Phase II clinical trials for mild cognitive impairment (MCI) and early Alzheimer's disease. Development may have been discontinued. The compound is not FDA-approved for clinical use. In preclinical studies, Ladostigil hydrochloride is generally well tolerated at doses up to 30 mg/kg PO in rodents, with no significant adverse effects. At higher doses (>50 mg/kg), mild gastrointestinal disturbances (nausea, reduced motility) may occur due to AChE inhibition. No significant hepatotoxicity or cardiotoxicity has been reported. Standard safety precautions apply. Ladostigil hydrochloride is for research use only, not for human consumption. CAS number: 209394-18-3.

C16H21CLN2O2

308.803143262863

308.129

209394-18-3

Ladostigil hemitartrate;209394-46-7;Ladostigil;209394-27-4

23645314

White to off-white solid powder

2

3

5

21

387

1

N([C@@H]1CCC2=CC=C(OC(=O)N(C)CC)C=C12)CC#C.Cl

PGISQOONVUOMIG-XFULWGLBSA-N

InChI=1S/C16H20N2O2.ClH/c1-4-10-17-15-9-7-12-6-8-13(11-14(12)15)20-16(19)18(3)5-2;/h1,6,8,11,15,17H,5,7,9-10H2,2-3H3;1H/t15-;/m1./s1

[(3R)-3-(prop-2-ynylamino)-2,3-dihydro-1H-inden-5-yl] N-ethyl-N-methylcarbamate;hydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

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

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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