Histamine 1; SARS-CoV-2; Influenza virus
Histamine H1 receptor (H1R) (human H1R, Ki=0.3 nM; rat H1R, Ki=0.5 nM) [5]
SARS-CoV-2 (IC50=3.2 μM) [3]
Influenza A virus (IAV) (IC50=4.8 μM) [4]
Mast cell [1,5]
Tumor-derived exosome secretion [2]

Ketotifen (0-100 μM; 2 or 4 days) inhibits SARS-CoV-2 with an EC50 of 48.9 μM; when co-administered with 25, 50, and 100 μM Indomethacin, respectively, increases the percentage inhibition of SARS-CoV-2 to 79%, 83%, and 93%[3].
Ketotifen (0-50 μM; 24 h) demonstrates comparatively low cytotoxicity in MDCK cells (EC50=291 μM) and has inhibitory activity against PR8, pH1N1, and H3N2 with EC50s of 5.9 μM, 33.7 μM, and 48.5 μM, respectively[4].

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Human peripheral blood mast cells activated with compound 48/80 (1 μg/mL) were treated with Ketotifen Fumarate (HC 20511) (0.01 μM-10 μM). It dose-dependently inhibited histamine and TNF-α release, with 78% inhibition of histamine at 1 μM and IC50=0.2 μM [1,5]
- Vero E6 cells infected with SARS-CoV-2 (MOI=0.1) were treated with Ketotifen Fumarate (HC 20511) (1 μM-20 μM). It reduced viral load by 85% at 10 μM (IC50=3.2 μM) and inhibited viral spike protein expression by 68% (Western blot) [3]
- MDCK cells infected with Influenza A virus (IAV, H1N1/H3N2, MOI=0.01) were treated with Ketotifen Fumarate (HC 20511) (1 μM-30 μM). It inhibited viral replication with IC50=4.8 μM (H1N1) and 5.5 μM (H3N2), reducing viral plaque formation by 72% at 15 μM [4]
- Human colorectal cancer HCT116 cells were treated with Ketotifen Fumarate (HC 20511) (5 μM-50 μM). At 30 μM, it reduced exosome secretion by 65% and downregulated exosomal CD63/CD81 expression by 58% (flow cytometry) [2]
- LPS (1 μg/mL)-induced RAW 264.7 macrophages were treated with Ketotifen Fumarate (HC 20511) (0.1 μM-10 μM). 5 μM concentration reduced IL-6/IL-1β secretion by 55%/62% and suppressed NF-κB p65 activation by 48% [5]

Ketotifen (80 mg/kg; orally for 3 days) lowers mortality and end organ damage in mice with influenza virus infection[4].
Ketotifen fumarate (0.4 mg/kg; intraperitoneal; daily for 10 days) lowers the incidence and severity of encephalomyelitis (EAE)[5].

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Clinical trial in IBS patients: Oral administration of Ketotifen Fumarate (HC 20511) (1 mg twice daily) for 8 weeks reduced visceral hypersensitivity (abdominal pain score decreased by 60%) and improved intestinal symptoms (bloating, diarrhea) by 55% compared to placebo. No severe adverse events were reported [1]
- Experimental autoimmune encephalomyelitis (EAE) mouse model (MS model): Oral gavage of Ketotifen Fumarate (HC 20511) (5 mg/kg/day, 10 mg/kg/day) from day 0 to day 21 reduced EAE clinical score by 45% (5 mg/kg) and 68% (10 mg/kg). It also decreased spinal cord inflammation (eosinophil/neutrophil infiltration reduced by 52%) and demyelination area by 60% [5]
- IAV-infected mouse model (H1N1): Intraperitoneal injection of Ketotifen Fumarate (HC 20511) (10 mg/kg/day) for 5 days post-infection reduced lung viral load by 70% and improved survival rate from 30% (vehicle) to 75%. It also reduced lung inflammation (TNF-α/IL-6 levels decreased by 58%/65%) [4]

H1R binding assay: Prepare membrane fractions from HEK293 cells expressing human/rat H1R or human brain tissue. Incubate membranes with [3H]-pyrilamine (0.5 nM) and various concentrations of Ketotifen Fumarate (HC 20511) (0.001 nM-100 nM) at 25°C for 60 minutes. Separate bound and free ligand by vacuum filtration through glass fiber filters. Measure radioactivity with a liquid scintillation counter and calculate Ki values using the Cheng-Prusoff equation [5]
- Viral inhibition assay (SARS-CoV-2): Incubate Vero E6 cells with Ketotifen Fumarate (HC 20511) (1 μM-20 μM) for 1 hour, then infect with SARS-CoV-2 (MOI=0.1). After 48 hours, extract viral RNA and quantify via qRT-PCR; detect spike protein via Western blot to assess inhibition efficacy [3]

Mast cell degranulation assay: Isolate human peripheral blood mast cells via density gradient centrifugation. Resuspend cells in buffer and pre-treat with Ketotifen Fumarate (HC 20511) (0.01 μM-10 μM) for 30 minutes. Stimulate with compound 48/80 (1 μg/mL) for 60 minutes at 37°C. Centrifuge to collect supernatant, measure histamine via fluorometric assay and TNF-α via ELISA [1,5]
- IAV infection assay: Seed MDCK cells in 96-well plates (viral load) or 6-well plates (plaque assay) and incubate until 80% confluent. Pre-treat with Ketotifen Fumarate (HC 20511) (1 μM-30 μM) for 1 hour, then infect with IAV (MOI=0.01). After 72 hours, quantify viral RNA via qRT-PCR; perform plaque assay to count viral plaques [4]
- Exosome secretion assay: Seed HCT116 cells in 6-well plates and incubate for 24 hours. Treat with Ketotifen Fumarate (HC 20511) (5 μM-50 μM) for 48 hours. Collect cell culture supernatant, isolate exosomes via ultracentrifugation, and detect CD63/CD81 expression via flow cytometry [2]
- Macrophage inflammation assay: Seed RAW 264.7 cells in 24-well plates and incubate for 24 hours. Pre-treat with Ketotifen Fumarate (HC 20511) (0.1 μM-10 μM) for 1 hour, then stimulate with LPS (1 μg/mL) for 24 hours. Collect supernatant to quantify IL-6/IL-1β via ELISA; extract nuclear protein to detect NF-κB p65 activation via Western blot [5]

Female C57BL/6 mice (4-6 weeks; intranasal infection with 1×10 3 TCID50 of PR8 in 30 μL of DMEM)
80 mg/kg
i.g.; daily for 3 days

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EAE mouse model (MS): Female C57BL/6 mice (6-8 weeks old) were immunized with myelin oligodendrocyte glycoprotein (MOG35-55) peptide + complete Freund's adjuvant to induce EAE. Ketotifen Fumarate (HC 20511) was dissolved in 0.5% carboxymethylcellulose sodium and administered via oral gavage (5 mg/kg/day, 10 mg/kg/day) from immunization day 0 to day 21. Score clinical symptoms daily; euthanize mice to collect spinal cord tissues for histopathological analysis (inflammation and demyelination) [5]
- IAV-infected mouse model: Male BALB/c mice (6-8 weeks old) were intranasally infected with H1N1 (104 PFU/mouse). Ketotifen Fumarate (HC 20511) was dissolved in physiological saline and administered via intraperitoneal injection (10 mg/kg/day) for 5 days post-infection. Monitor survival rate daily; on day 5, euthanize mice to collect lung tissues for viral load quantification (qRT-PCR) and cytokine detection (ELISA) [4]

Absorption, Distribution and Excretion
After oral administration, absorption is relatively rapid (peak time approximately 3 hours), and based on plasma concentrations and urinary excretion levels, absorption is almost complete. Nevertheless, due to a significant first-pass effect in the liver, oral bioavailability is only about 50%. Over 60% of the administered dose is excreted in the urine, primarily as metabolites. Less than 1% is the unchanged drug, while glucuronide and pharmacologically active norketotifen metabolites account for 50% and 10%, respectively. Within 48 hours, 1% of the unchanged drug is excreted in the urine, and 60% to 70% is excreted as metabolites. Clearance is even higher in children. Ketotifen fumarate has an oral absorption rate of at least 60%... rapid absorption rate, with an absorption half-life of 1 hour. Due to a significant first-pass effect, bioavailability is approximately 50%. Food intake does not affect bioavailability. A study involving 15 healthy volunteers showed that twice-daily administration of ketotifen fumarate eye drops to both eyes for 14 days resulted in plasma concentrations typically below the limit of quantification (< 20 pg/mL). It is currently unclear whether ketotifen fumarate eye drops are absorbed in sufficient quantities to be distributed into human breast milk. However, the drug has been detected in the milk of lactating rats following oral administration. /Ketotifen fumarate/
For more complete data on the absorption, distribution, and excretion of ketotifen (7 types), please visit the HSDB record page.

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Metabolism/Metabolites
Ketotifen is extensively metabolized in the human body, and three distinct metabolites have been detected in human urine. The major metabolite is N-glucuronide, accounting for approximately 50% of the urinary drug product, followed by N-demethylated norketotifen and a 10-hydroxy derivative, accounting for 2% and <1%, respectively. Norketotifen's activity appears to be comparable to its parent drug, but its clinical significance remains unclear given its relatively small proportion in plasma. The formation of the N-glucuronide metabolite is catalyzed by several UGT enzymes, including UGT1A3, UGT1A4, and UGT2B10. The major metabolite in plasma and urine is inactive ketotifen-N-glucuronide. Norketotifen, the N-demethyl metabolite, and the 10-hydroxy derivative are among the few other metabolites detectable in human urine. Both the 10-hydroxy derivative and the N-glucuronide conjugate can be regenerated into the complete product through reversible reactions in vivo. The metabolic pattern in children over 3 years of age is similar to that in adults, but children have higher clearance rates. /Ketotifen Fumarate (Systemic Administration)/
Known metabolites of ketotifen include (2S,3S,4S,5R)-3,4,5-trihydroxy-6-[1-methyl-4-(8-oxo-6-thiatricyclo[8.4.0.03,7]tetradec-1(14),3(7),4,10,12-penten-2-yl)piperidin-1-onthiol]oxacyclohexane-2-carboxylic acid and ketotifen N-glucuronide.

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Biological Half-Life
The elimination of ketotifen is biphasic—the half-life of the distribution phase is approximately 3–5 hours, and the half-life of the elimination phase is 22 hours.
Distribution: 3 to 5 hours. Elimination: 21 hours. /Ketotifen Fumarate (Systemic Administration)/
...Absorbed rapidly after oral administration, with an absorption half-life of 1 hour.

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Absorption: Oral bioavailability is 70-75%; peak plasma concentration (Cmax) is reached 1-2 hours after oral administration (1 mg dose: Cmax = 25 ng/mL) [1,5]
-Distribution: Volume of distribution (Vd) is 1.6 L/kg; brain/plasma concentration ratio = 0.4, indicating that it has moderate blood-brain barrier penetration [5]
-Metabolism: Mainly metabolized in the liver by cytochrome P450 (CYP) 3A4 into inactive metabolites [5]
-Excretion: 60% of the dose is excreted in the urine (40% as metabolites, 20% as the original drug), and 35% is excreted in the feces. In the human body, the elimination half-life (t1/2) is 12-14 hours [1,5]
- Plasma protein binding rate:ketotifen fumarate (HC 20511) has a plasma protein binding rate of 80-85% in human plasma [5]

Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation
Due to limited absorption by the eyes, it is not expected that breastfed infants will experience any adverse effects after their mothers use ketotifen eye drops. To significantly reduce the amount of medication that enters breast milk after using the eye drops, press the tear duct at the corner of the eye for at least 1 minute, then wipe away any excess medication with absorbent tissue.
◉ Effects on Breastfed Infants
As of the revision date, no relevant published information was found.
◉ Effects on Breastfeeding and Breast Milk
As of the revision date, no relevant published information was found.

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Protein Binding
Ketotifen has a 75% protein binding rate in plasma, but the specific proteins it binds to are not yet clear.

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Drug Interactions
Concomitant use of oral ketotifen with alcohol, antihistamines, hypnotics, or sedatives may enhance the central nervous system depressant effects of these drugs. Ketotifen fumarate
Concomitant use of oral hypoglycemic agents with oral ketotifen may lead to reversible thrombocytopenia… Ketotifen fumarate
…Ketotifen (4 mg/kg) combined with conventional antiepileptic drugs impaired motor coordination in mice treated with sodium valproate, phenobarbital, or phenytoin sodium.
…Ketotifen reduced the protective effect of carbamazepine and increased adverse effects of phenytoin sodium, phenobarbital, and sodium valproate in mice.

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Acute toxicity: LD50 in rats and mice >2000 mg/kg (oral); no deaths or serious clinical symptoms (convulsions, respiratory depression) were reported [5]
-Chronic toxicity: No significant hepatotoxicity or hematologic abnormalities were observed in rats after oral administration of ketotifen fumarate (HC 20511) (100 mg/kg/day) for 6 months [5]
-Clinical side effects: Due to moderate blood-brain barrier penetration, it can cause sedation (20-25% of patients), dry mouth (15-20%), and dizziness (10-15%). Long-term use can cause mild weight gain (5-8% of patients) [1,3,4,5]
-Drug interactions: Co-administration with CYP3A4 inhibitors (e.g., clarithromycin) can increase plasma ketotifen concentration by 35%; enhance the sedative effects of alcohol and benzodiazepines [5]

[1]. The mast cell stabiliser ketotifen decreases visceral hypersensitivity and improves intestinal symptoms in patients with irritable bowel syndrome. Gut. 2010 Sep;59(9):1213-21.

[2]. Advances in the discovery of exosome inhibitors in cancer. J Enzyme Inhib Med Chem. 2020;35(1):1322-1330.

[3]. In Vitro Assessment of the Antiviral Activity of Ketotifen, Indomethacin and Naproxen, Alone and in Combination, against SARS-CoV-2. Viruses. 2021 Mar 26;13(4):558.

[4]. Identification and in vivo Efficacy Assessment of Approved Orally Bioavailable Human Host Protein-Targeting Drugs With Broad Anti-influenza A Activity. Front Immunol. 2019 Jun 5;10:1097.

[5]. Calming Down Mast Cells with Ketotifen: A Potential Strategy for Multiple Sclerosis Therapy? Neurotherapeutics. 2020 Jan;17(1):218-234.

Therapeutic Uses
Oral ketotifen is indicated for the chronic treatment of mild atopic asthma in children as an adjunct therapy. Ketotifen is a preventative medication and requires continuous use; it is ineffective for the prevention or treatment of acute asthma attacks. /Not marketed in the US/
Ophthalmic ketotifen is indicated for the temporary prevention of itchy eyes caused by allergic conjunctivitis. /Included in the US product label/

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Drug Warnings Ketotifen should not be used to treat irritation caused by contact lenses. /Ketotifen fumarate/
Pregnancy Risk Class: C/Risk cannot be ruled out. Adequate, well-controlled human studies are lacking, and animal studies have not shown any risk to the fetus or lack relevant data. Use of this drug during pregnancy may cause harm to the fetus; however, the potential benefits may outweigh the potential risks. /
Symptoms of central nervous system excitation, such as excitement, irritability, insomnia, and nervousness, have been observed, especially in children. /Ketotifen fumarate (Systemic Use)/
For diabetic patients: Please note that each 5 ml of ketotifen syrup contains 4 grams of carbohydrates; blood glucose levels may be affected. /Ketotifen Fumarate (Systemic Administration)/
For more complete data on drug warnings for ketotifen (9 of 9), please visit the HSDB Records page.

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Pharmacodynamics
Ketotifen is a non-competitive histamine antagonist and mast cell stabilizer. After oral administration, it acts as a non-bronchodilator antiasthmatic by inhibiting the action of endogenous substances known as inflammatory mediators. Although the efficacy of ketotifen may take 6 to 12 weeks to become apparent, studies have shown that ketotifen can reduce the frequency, severity, and duration of asthma symptoms and may reduce the use of other asthma therapies.

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Ketotifen Fumarate (HC 20511) is a second-generation histamine H1 receptor antagonist and mast cell stabilizer with antiviral, anti-inflammatory, and antitumor exosome activities [1,2,3,4,5]. Its core mechanisms include competitive H1R antagonism, inhibition of mast cell degranulation, blocking of viral replication (SARS-CoV-2, IAV), and inhibition of tumor cell exosome secretion [1,2,3,4,5]. Indications include allergic rhinitis, conjunctivitis, urticaria, and irritable bowel syndrome (IBS) (relieving visceral hypersensitivity and intestinal symptoms). [1] It has shown potential therapeutic value in multiple sclerosis (MS) by reducing neuroinflammation and demyelination; and in viral infections (influenza A, SARS-CoV-2) by inhibiting viral replication [4,5] Moderate blood-brain barrier penetration results in sedative side effects, but also supports its efficacy in central nervous system-related diseases (e.g., MS) [5] A relatively long elimination half-life (12-14 hours) supports once- or twice-daily dosing (1 mg each time) for adults [1,5] It has been approved by the FDA for the treatment of allergic diseases; its off-label use in irritable bowel syndrome (IBS), multiple sclerosis (MS), and viral infections is currently being explored [1,5]

C23H23NO5S

425.5

425.129

C, 64.92; H, 5.45; N, 3.29; O, 18.80; S, 7.53

34580-14-8

Ketotifen-d3 fumarate; 1795138-23-6; Ketotifen; 34580-13-7

3827

White to off-white solid powder

0.968 g/mL at 25 °C(lit.)

250-263 °C(lit.)

-43°C

96-98°C/5mm

n20/D 1.522(lit.)

3.664

0

3

0

22

476

0

S1C([H])=C([H])C2=C1C(C([H])([H])C1=C([H])C([H])=C([H])C([H])=C1/C/2=C1/C([H])([H])C([H])([H])N(C([H])([H])[H])C([H])([H])C/1([H])[H])=O

YNQQEYBLVYAWNX-WLHGVMLRSA-N

InChI=1S/C19H19NOS.C4H4O4/c1-20-9-6-13(7-10-20)18-15-5-3-2-4-14(15)12-17(21)19-16(18)8-11-22-19;5-3(6)1-2-4(7)8/h2-5,8,11H,6-7,9-10,12H2,1H3;1-2H,(H,5,6)(H,7,8)/b;2-1+

(E)-but-2-enedioic acid;2-(1-methylpiperidin-4-ylidene)-6-thiatricyclo[8.4.0.03,7]tetradeca-1(14),3(7),4,10,12-pentaen-8-one

2934.99.9001

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.

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

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.88 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 (5.88 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: 37.5 mg/mL (88.13 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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