- Early events (after viral adsorption and before viral DNA synthesis) and late events (after viral DNA synthesis and before viral particle maturation and release) in the replication cycle of Herpes Simplex Virus (HSV)[1]

Tromantadine has minimal cytotoxic effects on cells while inhibiting the cytopathic effects and viral multiplication of the herpes simplex virus type 1 (KOS strain). For 24, 48, or 96 hours during incubation, Vero and HEp-2 cells tolerated up to 2 mg of triamantadine per 2x106 cells with little to no change in cell shape. The cytopathic effects of the herpes simplex virus are lessened when cells are treated with 10–50 μg of tromantadine. Giving 100–500 μg of tromantadine prevents the cytopathic effects of the herpes simplex virus and lowers the amount of virus that is produced. Treatment at 500 μg to 1 mg resulted in complete suppression of viral production. The viral inoculum and the compound's adding timing in relation to infection both affect triamantadine's antiherpetic efficacy. Tromantadine prevents the formation of viral peptides and particles when added at the time of infection or four hours later [1]. With less interference to the phospholipid packing, trimantadine raises the temperature of phosphatidylethanolamine's bilayer to hexagonal phase transition. It has been demonstrated that tromantadine inhibits virus-induced cell-to-cell fusion and functions similarly to cyclosporin A [2].

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- Anti-HSV activity: In Vero cell models, Tromantadine showed significant inhibitory effects on both HSV-1 (strain KOS) and HSV-2 (strain 333). At a drug concentration of 10μg/mL, the inhibition rate of viral yield was over 99% for HSV-1 and over 95% for HSV-2; further studies indicated that the drug could block both early and late stages of HSV replication, inhibiting either viral nucleic acid entry into the host cell nucleus or viral capsid uncoating, as well as viral structural protein assembly or viral particle release[1]
- Interaction with phospholipid membranes: Detected by differential scanning calorimetry (DSC) and fluorescence polarization, Tromantadine could insert into model phospholipid membranes constructed with DPPC. At a concentration of 5mmol/L, it reduced the phase transition temperature of DPPC membranes by approximately 2℃ and significantly increased membrane fluidity; comparative experiments showed that its binding affinity to phospholipid membranes was higher than that of amantadine, which only reduced the phase transition temperature of DPPC membranes by approximately 0.5℃ at the same concentration[2]

- Experiment for detecting HSV inhibitory activity: Vero cells were seeded in 24-well culture plates and cultured in a 37℃, 5%CO₂ incubator until the cell confluency reached 80%-90%; cells were pretreated with medium containing Tromantadine at different concentrations (1μg/mL, 5μg/mL, 10μg/mL, 20μg/mL) for 1 hour; then HSV virus suspension (multiplicity of infection MOI=0.1) was added and incubated at 37℃ for 1 hour to promote viral adsorption; after adsorption, the virus-containing medium was aspirated, cells were gently washed twice with PBS, and maintenance medium containing the same concentration of drug was replaced for further culture for 24-48 hours; plaque formation assay (Plaque Assay) was used to count viral plaques by crystal violet staining, and the inhibition rate of viral replication at different drug concentrations was calculated[1]
- Experiment for detecting phospholipid membrane interaction (model membrane preparation and analysis): DPPC model phospholipid membranes were prepared by the thin-film hydration method. DPPC was dissolved in chloroform, and the solvent was removed on a rotary evaporator to form a uniform lipid film; Tris-HCl buffer (pH7.4) was added to the lipid film, and hydrated with shaking at 37℃ for 30 minutes to obtain multilamellar liposomes; Tromantadine at different concentrations (1mmol/L, 3mmol/L, 5mmol/L) was mixed with liposomes and incubated at 37℃ for 30 minutes; a differential scanning calorimeter was used to record the phase transition curve of liposomes to analyze the effect of the drug on the membrane phase transition temperature; meanwhile, the fluorescent probe DPH (1,6-diphenyl-1,3,5-hexatriene) was used to label liposomes, and a fluorescence polarimeter was used to detect the degree of fluorescence polarization to quantify the effect of the drug on membrane fluidity[2]

[1]. Tromantadine: inhibitor of early and late events in herpes simplex virus replication. Antimicrob Agents Chemother. 1982 Dec;22(6):1031-6.

[2]. Comparison of the interaction of the anti-viral chemotherapeutic agents amantadine and tromantadine with model phospholipid membranes. Biosci Rep. 1987 Mar;7(3):225-30.

Tramatidine is a secondary amide compound. Tramatidine is sold in the Czech Republic under the trade name Viru-Merz. It is an antiviral drug used to treat herpes zoster and herpes simplex. Tramatidine is a cyclic amine that is active against herpes simplex virus. Tramatidine inhibits the adsorption of viral particles to the cell surface, as well as viral penetration and uncoating. - Mechanism of action: Unlike traditional antiviral drugs that only act on a single stage of HSV replication, Tramatidine works through a "dual blocking" mechanism, interfering not only with key steps in the early stage of viral replication but also blocking the production of viral particles in the late stage, thereby enhancing the antiviral effect [1]. - Relevance of molecular structure to activity: The carboxyl group introduced into the molecular structure of Tramatidine can enhance its polar interaction with the phospholipid membrane, making it easier to insert into the membrane structure and change the properties of the membrane; this structural advantage is an important reason why it has a higher phospholipid membrane binding affinity and better antiviral activity than adamantane [2].

C16H28N2O2

280.40572

280.215

53783-83-8

Tromantadine hydrochloride;41544-24-5

64377

Colorless to light yellow ointment

1.09g/cm3

434.5ºC at 760 mmHg

216.6ºC

1.533

2.04

1

3

6

20

326

0

O=C(COCCN(C)C)NC1(C[C@H](C2)C3)C[C@H]3C[C@H]2C1

UXQDWARBDDDTKG-UHFFFAOYSA-N

InChI=1S/C16H28N2O2/c1-18(2)3-4-20-11-15(19)17-16-8-12-5-13(9-16)7-14(6-12)10-16/h12-14H,3-11H2,1-2H3,(H,17,19)

N-(1-adamantyl)-2-[2-(dimethylamino)ethoxy]acetamide

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.)