The primary mechanism of action for levamisole as an anthelmintic is its agonistic activity at the L-subtype nicotinic acetylcholine receptors (nAChRs) located on nematode muscle cells. These receptors are ligand-gated ion channels. When levamisole binds to and activates these receptors, it causes a sustained depolarization of the muscle cell membrane, leading to spastic paralysis of the worm. This paralysis prevents the worm from maintaining its attachment to the host's intestinal wall, leading to its expulsion. The L-subtype receptor is distinct from the P- and M-subtype receptors targeted by other anthelmintics like pyrantel and morantel. Levamisole's immunomodulatory mechanism is complex and not fully understood. It appears to act as a positive allosteric modulator (PAM) for certain subtypes of nAChRs, including α3β2 and α3β4, with EC50 values of 300 µM and 100 µM, respectively. However, its immunomodulatory effects are believed to be mediated through other mechanisms as well. It is thought to restore depressed immune function rather than stimulate it above normal levels. It can stimulate the formation of antibodies to various antigens, enhance T-cell responses by stimulating T-cell activation and proliferation, potentiate monocyte and macrophage functions (including phagocytosis and chemotaxis), and increase neutrophil mobility, adherence, and chemotaxis. These diverse effects on the immune system have made it a useful, though now less commonly used, immunomodulatory agent.

In vitro studies have extensively characterized levamisole's activity on both parasitic and immune cells. Its anthelmintic activity is typically assessed using nematode motility assays, where the compound is added to cultures of parasitic worms, and the paralysis and death of the worms are monitored. Levamisole is a potent agonist of the L-subtype nAChR, demonstrating high efficacy in inducing spastic paralysis. Its immunomodulatory effects are studied in vitro using immune cell cultures. Levamisole has been shown to stimulate T-cell activation and proliferation, as measured by increased incorporation of tritiated thymidine or by the expression of activation markers like CD25. It also potentiates the phagocytic activity of monocytes and macrophages and enhances their chemotactic response to chemoattractants. Furthermore, it increases the mobility and chemotaxis of neutrophils. These effects are observed in vitro at specific concentrations, confirming its direct action on immune cells. Levamisole's activity as a positive allosteric modulator (PAM) of nAChRs (α3β2 and α3β4) has also been demonstrated in vitro using electrophysiological techniques, where it enhances the response to acetylcholine.

In mice given a high-fat diet, (S)-(-)-Levamisole (Levamisole) (50 μg/ml and 200 μg/ml; oral; 30 days) inhibits weight gain [2].

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In vivo studies have validated levamisole's efficacy as an anthelmintic and immunomodulator in animal models. As an anthelmintic, it is highly effective against a wide range of gastrointestinal nematodes in livestock. In vivo efficacy is typically measured by fecal egg count reduction (FECR) tests. Levamisole has been shown to achieve a 95.46% FECR, demonstrating its high efficacy. Its immunomodulatory effects have been studied in various animal models of disease. For example, in cancer models, levamisole has been shown to enhance the immune response against tumors, although its efficacy as a standalone agent is limited. Its primary clinical use in vivo is in combination with fluorouracil for the adjuvant treatment of Dukes' stage C colon cancer. In this context, the exact mechanism of its anti-cancer activity is unknown, but it is believed to be related to its immunomodulatory effects, restoring the patient's immune function to help fight the cancer. Levamisole is orally active, making it convenient for administration. Overall, in vivo studies have confirmed its utility as an anthelmintic and its potential as an immunomodulatory adjuvant.

The in vitro receptor binding assays for levamisole are primarily focused on its interaction with nicotinic acetylcholine receptors (nAChRs). A common technique is radioligand binding assays. In these assays, membrane preparations from cells expressing specific nAChR subtypes (such as the L-subtype from nematodes or mammalian subtypes like α3β2 and α3β4) are incubated with a radiolabeled ligand (e.g., [³H]epibatidine or [³H]nicotine) that binds to the receptor. Increasing concentrations of unlabeled levamisole are then added to compete with the radiolabeled ligand for binding. After incubation, the bound and free ligand are separated (e.g., by filtration), and the radioactivity is measured. The data is used to calculate the binding affinity (Ki) of levamisole for the receptor. For functional assays, electrophysiological techniques are used. For example, two-electrode voltage-clamp or patch-clamp electrophysiology can be performed on oocytes or mammalian cells expressing nAChRs. Levamisole is applied to the cells, and the resulting ion current is measured. This allows researchers to determine if levamisole acts as an agonist, antagonist, or positive allosteric modulator. For example, levamisole has been shown to act as a positive allosteric modulator (PAM) for the α3β2 and α3β4 subtypes, enhancing the response to acetylcholine. These assays provide a detailed pharmacological profile of levamisole's interaction with its molecular targets.

In vitro cell-based assays for levamisole are used to study both its anthelmintic and immunomodulatory activities. For its anthelmintic activity, the primary assay is the nematode motility assay. In this assay, live parasitic worms (e.g., Caenorhabditis elegans or Haemonchus contortus) are placed in a multi-well plate containing a buffer or culture medium. Levamisole is added at various concentrations, and the motility of the worms is monitored over time using a microscope or an automated imaging system. The endpoint is the concentration that causes paralysis or death of the worms. For its immunomodulatory activity, assays are performed using primary immune cells or cell lines. T-cell proliferation assays are commonly used. In these assays, human or murine T cells are isolated and stimulated with a mitogen (e.g., phytohemagglutinin) or an antigen. Levamisole is added to the cultures, and the proliferation of the T cells is measured by the incorporation of tritiated thymidine or by using a colorimetric assay like MTT. Macrophage function assays measure phagocytosis. Macrophages are incubated with fluorescently labeled particles (e.g., E. coli or latex beads) in the presence of levamisole, and the phagocytic activity is quantified by flow cytometry or fluorescence microscopy. Chemotaxis assays measure the migration of immune cells towards a chemoattractant in the presence of levamisole. These assays confirm levamisole's direct effects on immune cell function.

In vivo animal experiments for levamisole are primarily conducted to evaluate its anthelmintic efficacy and its immunomodulatory effects in disease models. For anthelmintic testing, the most common model is the use of animals (e.g., sheep, cattle, or rodents) naturally or artificially infected with gastrointestinal nematodes. In a typical protocol, infected animals are randomly assigned to treatment groups. The treatment group receives levamisole (usually orally), while the control group receives a placebo. Fecal samples are collected before treatment and at various time points after treatment to count the number of parasite eggs (fecal egg count, FEC). The reduction in FEC in the treatment group compared to the control group is calculated as the Fecal Egg Count Reduction (FECR). For immunomodulatory studies, levamisole is often tested in combination with other therapies. For example, in cancer models, animals (e.g., mice) are implanted with tumor cells and then treated with levamisole alone or in combination with chemotherapy. Tumor growth and survival are monitored, and immune cell populations are analyzed by flow cytometry. These in vivo experiments are critical for validating levamisole's clinical efficacy and for understanding its mechanism of action in a whole organism.

Absorption, Distribution and Excretion
Levamisole is rapidly absorbed in the gastrointestinal tract (2 hours). Metabolism/Metabolites Primarily metabolized in the liver (extensively), producing active and inactive metabolites. Biological Half-Life 4.4–5.6 hours (biphasic)

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Levamisole is well-absorbed after oral administration, which is its primary route of administration. It is rapidly metabolized in the liver, and its metabolites are excreted in the urine. The drug is known to have a relatively short half-life. Its pharmacokinetic profile is characterized by rapid absorption and distribution, with peak plasma concentrations reached within 1-2 hours after oral administration. It is extensively metabolized in the liver, and its metabolites are excreted primarily in the urine. The compound is a small molecule (MW 204.29) with moderate lipophilicity, which contributes to its good oral bioavailability. For research use, it is typically supplied as a solid and is soluble in water (1.44 g/L) and methanol. The hydrochloride salt is commonly used to enhance its solubility. It is stable under normal storage conditions. The PK of levamisole can be affected by factors such as the formulation, the animal species, and the route of administration. Its use in combination with other drugs, such as fluorouracil, requires careful consideration of potential drug-drug interactions. Comprehensive PK studies have been conducted in various species to support its clinical use as an anthelmintic and immunomodulator.

Effects During Pregnancy and Lactation
◉ Overview of Lactation Use
Levamisole has been discontinued for human use in the United States due to its potential to cause agranulocytosis, but it is still used as an anthelmintic in other countries. There is currently no publicly available information regarding its excretion in breast milk. Some information suggests that levamisole use may be acceptable for breastfeeding women. However, due to limited experience with levamisole use during lactation, alternative medications may be preferred, especially when breastfeeding newborns or premature infants. The World Health Organization recommends that pregnant women taking levamisole should not breastfeed.
◉ Effects on Breastfed Infants
In the Democratic Republic of Congo, a cohort study of 33 infants followed hospitalized mothers taking nifurolimus who breastfed (the extent of breastfeeding was not specified). Thirty mothers received a complete course of 30 oral nifurulimus doses (15 mg/kg daily) and 14 intravenous doses of efornithine (400 mg/kg daily) for 7 days to treat human trypanosomiasis (sleeping sickness). Six of the breastfeeding mothers also received levamisole. No serious adverse events were reported in any of the breastfed infants.
◉ Effects on lactation and breast milk
As of the revision date, no relevant published information was found.

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Protein binding
20-25%

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Levamisole is generally well-tolerated at therapeutic doses, but it can cause significant adverse effects. The most serious toxicity associated with levamisole is agranulocytosis, a condition characterized by a severe reduction in the number of white blood cells, which can increase the risk of serious infections. This has limited its use as an immunomodulator. Other side effects can include nausea, vomiting, abdominal pain, headache, fatigue, and skin rashes. It is also known to cause a flu-like syndrome. At high doses, it can cause neurotoxicity, which may manifest as dizziness, confusion, and seizures. In veterinary use, levamisole has a narrow therapeutic index, and overdose can be fatal. It is contraindicated in patients with pre-existing bone marrow depression or liver disease. Due to these safety concerns, its use as an immunomodulatory agent has declined, and it is primarily used as an anthelmintic in veterinary medicine. In humans, it is used for specific indications, such as colon cancer, where the benefits are considered to outweigh the risks. Any use of levamisole should be under close medical supervision with regular monitoring of blood counts.

[1]. Lewis JA, et al. Levamisole: A Positive Allosteric Modulator for the α3β4 Nicotinic Acetylcholine Receptors Prevents Weight Gain in the CD-1 Mice on a High Fat Diet. Curr Pharm Des. 2017;23(12):1869-1872.
[2]. Mehta KP, et al. Immunoregulatory treatment for minimal change nephrotic syndrome. Arch Dis Child. 1986;61(2):153-158.

Levamisole is a 6-phenyl-2,3,5,6-tetrahydroimidazole[2,1-b][1,3]thiazole with an S configuration. It (usually in hydrochloride form) is used to treat parasitic infections in pigs, sheep, and cattle, and was previously used in humans as adjunctive therapy for various cancers. It is also widely used as a dopant for cocaine. It has anti-nematode, antirheumatic, immunomodulatory, immunoadjuvant, and EC 3.1.3.1 (alkaline phosphatase) inhibitor effects. It is the enantiomer of dextromisole. Levamisole is an anthelmintic that has been widely used to treat parasitic, viral, and bacterial infections. Manufactured by Janssen Pharmaceuticals, levamisole was initially used in 1969 for the treatment of helminth infections. In 1990, the U.S. Food and Drug Administration (FDA) approved levamisole as adjunctive therapy for colon cancer. Prior to this, levamisole was used as an antirheumatic therapy for patients with rheumatoid arthritis in the 1970s and 1980s. Due to its immunomodulatory effects, the drug has been investigated for the treatment of various immune-mediated diseases, with some studies showing positive results. It has also been used in combination with other drugs to treat various cancers. Because levamisole could cause serious adverse reactions, including agranulocytosis, it was withdrawn from the US market in 2000. Notably, levamisole has been found to be adulterated with cocaine, potentially causing various adverse reactions in users. Levamisole is an anthelmintic. Levamisole, in combination with fluorouracil, was used to treat Dukes stage C colon cancer, restoring immune function by stimulating antibody production, enhancing T cell activity, and enhancing macrophage function. (NCI04) Levamisole is an anthelmintic that has been experimentally used to treat rheumatic diseases, demonstrating its ability to restore immune responses by enhancing macrophage chemotaxis and T lymphocyte function. However, this immune-enhancing effect appears to be beneficial for rheumatoid arthritis, but side effects have been reported, including dermatitis, leukopenia, thrombocytopenia, and nausea and vomiting. (Quoted from Smith and Reynard, Textbook of Pharmacology, 1991, pp. 435-436)
See also: levamisole hydrochloride (salt form); levamisole phosphate (salt form); doramectin; levamisole (component).
Indications

Used as adjuvant therapy after surgical resection in patients with Dukes C stage colon cancer, in combination with fluorouracil. Also used to treat malignant melanoma and head and neck cancer.
Mechanism of Action

The mechanism of action of levamisole as an antiparasitic drug appears to be related to its agonistic effect on L-type nicotinic acetylcholine receptors in the muscle of nematodes. This agonistic effect reduces the ability of male nematodes to control their reproductive muscles, thereby limiting their mating ability. The mechanism of action of levamisole as an anticancer drug in combination with fluorouracil is not fully understood. The effects of levamisole on the immune system are complex. The drug appears to restore impaired immune function rather than stimulating an immune response to above-normal levels. Levamisole can stimulate antibody formation against multiple antigens, enhance T cell responses by stimulating T cell activation and proliferation, enhance the function of monocytes and macrophages (including phagocytosis and chemotaxis), and increase neutrophil migration, adhesion, and chemotaxis.

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Pharmacodynamics
Levamisole is a synthetic imidazothiazole derivative widely used to treat worm infections in humans and animals. As an anthelmintic, it may act by targeting nicotinic acetylcholine receptors in nematodes. As an immunomodulator, levamisole appears to be an immunostimulant; studies have shown that in stage III colon cancer patients receiving adjuvant levamisole in combination with 5-fluorouracil (5-FU), levamisole can increase the number of NK cells and activated T cells.

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Levamisole is an approved drug with a long history of use in both veterinary and human medicine. It is approved for use as a broad-spectrum anthelmintic in livestock and pets. In humans, it is approved for the treatment of parasitic infections, such as ascariasis, hookworm, and strongyloidiasis. Its most notable human medical use is as an immunomodulatory adjuvant, specifically in combination with fluorouracil, for the adjuvant treatment of Dukes' stage C colon cancer following surgical resection. It has also been used in the treatment of malignant melanoma and head and neck cancers. The mechanism of its anti-cancer action is not fully understood but is believed to involve restoring or enhancing the patient's immune response. Despite its immunomodulatory properties, its use has been largely superseded by newer and safer immunotherapies. The drug is typically administered orally. It is available in various formulations, including tablets and solutions for injection. Due to its mechanism of action as an agonist at L-subtype nAChRs in nematodes and its immunomodulatory effects on T-cells, macrophages, and neutrophils, it remains a compound of interest for studying these pathways. However, its clinical use is now limited due to safety concerns, particularly the risk of agranulocytosis.

C11H12N2S

204.2914

204.072

14769-73-4

Levamisole hydrochloride;16595-80-5;Tetramisole hydrochloride;5086-74-8;Dexamisole;14769-74-5

26879

White to off-white solid powder

1.3±0.1 g/cm3

344.4±45.0 °C at 760 mmHg

230 - 233ºC

162.1±28.7 °C

0.0±0.8 mmHg at 25°C

1.713

1.85

0

2

1

14

246

1

S1C([H])([H])C([H])([H])N2C1=N[C@@]([H])(C1C([H])=C([H])C([H])=C([H])C=1[H])C2([H])[H]

HLFSDGLLUJUHTE-SNVBAGLBSA-N

InChI=1S/C11H12N2S/c1-2-4-9(5-3-1)10-8-13-6-7-14-11(13)12-10/h1-5,10H,6-8H2/t10-/m1/s1

(6S)-6-phenyl-2,3,5,6-tetrahydroimidazo[2,1-b][1,3]thiazole

LevamisoleErgamisolDecarisLevotetramisoleSolaskilKetrax

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)

DMSO : ~100 mg/mL (~489.50 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (12.24 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 25.0 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: ≥ 2.5 mg/mL (12.24 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 25.0 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: ≥ 2.5 mg/mL (12.24 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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