Influenza A viruses; ion channels NMDA, M2; CDK2; Bcl-2; Bax

The suppression of SARS-CoV-2 replication by amantadine sulfate (0-500 µM, 26 hours) ranges in IC50 values from 83 to 119 µM [4]. The growth of HepG2 and SMMC-7721 cells is markedly inhibited by amantadine sulfate (0-100 µg/mL, 24-72 hours) [6]. Inducing apoptosis, amantadine sulfate (0-75 µg/mL, 48 hours) stops the cell cycle in the G0/G1 phase [6]. In 48 hours, amantadine sulfate (0-75 µg/mL) decreases Bcl-2, increases Bax protein and mRNA levels, and decreases cell cycle-related genes and proteins (cyclin D1, cyclin E, and CDK2) [6].

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Since the SARS-CoV-2 pandemic started in late 2019, the search for protective vaccines and for drug treatments has become mandatory to fight the global health emergency. Travel restrictions, social distancing, and face masks are suitable counter measures, but may not bring the pandemic under control because people will inadvertently or at a certain degree of restriction severity or duration become incompliant with the regulations. Even if vaccines are approved, the need for antiviral agents against SARS-CoV-2 will persist. However, unequivocal evidence for efficacy against SARS-CoV-2 has not been demonstrated for any of the repurposed antiviral drugs so far. Amantadine was approved as an antiviral drug against influenza A, and antiviral activity against SARS-CoV-2 has been reasoned by analogy but without data. We tested the efficacy of Amantadine in vitro in Vero E6 cells infected with SARS-CoV-2. Indeed, amantadine inhibited SARS-CoV-2 replication in two separate experiments with IC50 concentrations between 83 and 119 µM. Although these IC50 concentrations are above therapeutic amantadine levels after systemic administration, topical administration by inhalation or intranasal instillation may result in sufficient amantadine concentration in the airway epithelium without high systemic exposure. However, further studies in other models are needed to prove this hypothesis.[4]

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Hepatocellular carcinoma (HCC) is one of the most aggressive malignancies worldwide, and its incidence associated with viral infection has increased in recent years. Amantadine is a tricyclic symmetric amine that can effectively protect against the hepatitis C virus. However, its antitumor properties remain unclear. In the present study, the effects of Amantadine on tumor cell viability, cell cycle regulation and apoptosis were investigated. The growth of HepG2 and SMMC‑7721 cells (HCC cell lines) was detected by an MTT assay. Flow cytometry was used to investigate cell cycle regulation and apoptosis. Reverse transcription‑quantitative polymerase chain reaction and western blot analysis were also performed to examine the expression of cell cycle‑ and apoptosis‑related genes and proteins, including cyclin E, cyclin D1, cyclin‑dependent kinase 2 (CDK2), B‑cell lymphoma 2 (Bcl‑2) and Bax. Our results demonstrated that amantadine markedly inhibited the proliferation of HepG2 and SMMC‑7721 cells in a dose‑ and time‑dependent manner and arrested the cell cycle at the G0/G1 phase. The levels of the cell cycle‑related genes and proteins (cyclin D1, cyclin E and CDK2) were reduced by amantadine, and apoptosis was significantly induced. Amantadine treatment also reduced Bcl‑2 and increased the Bax protein and mRNA levels. Additionally, Bcl‑2/Bax ratios were lower in the two HCC cell lines following amantadine treatment. Collectively, these results emphasize the role of amantadine in suppressing proliferation and inducing apoptosis in HCC cells, advocating its use as a novel tumor-suppressive therapeutic candidate [6].

Amantadine sulfate (25 mg/kg, IP, once day for 3 days) can reduce surgery-induced neuroinflammation and learning and memory deficits [5].
Surgery increased the time to identify the target box in the Barnes maze when tested 1 day [22 (median) (11-66) (interquartile range) of control group vs. 158 (29-180) of surgery group, n = 15, P = 0.022) or 8 days after the training sessions and reduced context-related freezing behavior in the fear conditioning test. These effects were attenuated by Amantadine (25 (14-90), n = 15, P = 0.029 compared with surgery group at 1 day after the training sessions in Barnes maze) and intracerebroventricular GDNF. Amantadine increased GDNF that was co-localized with glial fibrillary acidic protein, an astrocytic marker, in the hippocampus. Intracerebroventricular injection of an anti-GDNF antibody but not the denatured antibody blocked the effects of amantadine on cognition. Surgery induced neuroinflammation that was inhibited by amantadine. Lipopolysaccharide increased interleukin 1β production from C8-B4 cells. This effect was inhibited by GDNF [5].

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Amantadine attenuated surgery-induced learning and memory impairment [5]
The time to identify the target box during the 4-day training sessions of Barnes maze test was reduced with increased training sessions in the control rats, rats received anesthesia only, rats received amantadine only and rats received surgery plus Amantadine. This time on day 4 was significantly shorter than that on day 1 for these four groups of rats. This effect was not apparent in the rats after surgery alone. Surgery had a significant effect on the time needed to identify the target box in the training sessions [F(1,28) = 5.625, P = 0.025]. This effect was abolished by amantadine [F(1,28) = 0.840, P = 0.367; compared with control group]. Amantadine or anesthesia only did not have a significant effect on the time to identify the target box during the training sessions [F(1,28) = 0.063, P = 0.804; F(1, 14) = 0.074, P + 0.790] (Figs. 1 and 2). When the rats were tested 1 day after the training sessions, the time to identify the target box for the rats subjected to surgery was longer than that for the control rats. This prolongation was attenuated by amantadine. A similar change pattern occurred when the test was performed 8 days after the training sessions. However, anesthesia and amantadine alone did not affect the time to identify the target box whether the test was performed 1 day or 8 days after the training sessions (Fig. 1B and 2B). Rats in the surgery group but not in the anesthesia only group or amantadine group had less context-related freezing behavior in the fear conditioning test than control rats. This surgical effect was abolished by amantadine (Fig. 1C). There was no difference in the tone-related freezing behavior among the control rats, rats received amantadine, rats received surgery and rats received surgery plus amantadine (Fig. 1C and 2C).

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Amantadine attenuated surgery-induced neuroinflammation [5]
The expression of Iba-1 (a microglial marker), IL-1β and IL-6 in the hippocampus was significantly increased at 6 and 24 h after the surgery. These increases were abolished by amantadine (Figs. 3 and 4). Similarly, Iba-1 expression in the hippocampal dentate gyrus region was also increased at 10 days after the surgery and this increase was blocked by Amantadine (Fig. 5). These results suggest that surgery induces neuroinflammation that was inhibited by amantadine.

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Amantadine increased the expression of GDNF that inhibited microglial activation [5]
Amantadine significantly increased GDNF in the hippocampus (Fig. 7). GDNF was mainly co-localized with GFAP, an astrocytic marker, but was not co-localized with Iba-1 (Figs. 7A and 7B). Some GDNF appeared to be around NeuN, a neuronal marker (Fig. 7C). Surgery also increased GFAP but this increase was not affected by Amantadine in the hippocampus (Figs. 7A and 7E).

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Amantadine-induced attenuation of learning and memory impairment after surgery was inhibited by anti-GDNF antibody [5]
Similar to the control rats, rats in antibody only group and the surgery plus Amantadine plus boiled antibody group had a decreased time to find the target box with increased training sessions. This time on the training day 4 was shorter than that on training day 1 for these two groups of rats. This effect was not apparent for rats in the surgery plus amantadine plus anti-GDNF antibody group. The anti-GDNF antibody was found to have a significant effect on the time to identify the target box during the training sessions [F(1,14) = 19.009, P < 0.001; compared with control) (Fig. 9A). The time to identify the target box on day 1 after the training session was not different among control rats, rats received antibody, rates received surgery plus amantadine plus anti-GDNF antibody or rats received surgery plus amantadine plus boiled antibody. However, rats subjected to surgery plus amantadine plus anti-GDNF antibody required much longer time than control rats or rats received surgery plus amantadine plus boiled antibody to identify the target box on day 8 after the training sessions (Fig. 9B). Similarly, rats subjected to surgery plus amantadine plus anti-GDNF antibody also had less context-related freezing behavior than control rats or rats received surgery plus amantadine plus boiled antibody in the fear conditioning test. However, the tone-related freezing behavior was not different among the three groups (Fig. 9C).

S-Protein—ACE2 Binding Assay [4]
The compounds were tested for their ability to inhibit the binding of SARS-CoV-2 spike protein (S protein) to ACE2 using the SARS-CoV-2 spike: ACE2 Inhibitor Screening Assay Kit. In brief, the SARS-CoV-2 spike protein was coated to a 96 microwell plate at 1 µg/mL in phosphate buffered saline. Unbound protein was removed and unspecific binding sites in the wells are blocked. Then, the blocking solution was removed, and the diluted compounds and control samples were added to the wells. After pre-incubation of the coated spike protein with the compounds, the His-tagged ACE2 protein was added and incubated together with the compounds to allow binding to the spike protein. After washing and blocking, the bound ACE2 protein was detected by an anti-His-antibody coupled to horse radish peroxidase (HRP). The detection was performed using a chemiluminescent HRP substrate and reading the luminescence intensity in a microtiter-plate reader. The luminescence signal of each sample containing diluted compound was divided by the luminescence in absence of any inhibitor, and the resulting values were plotted against the concentration of the compound.

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Antiviral Activity Assay with RT-PCR Readout (1st Experiment) [4]
Exponentially growing Vero E6 cells were seeded into a 48-well plate at a density of 8 × 104 cells per well and were incubated overnight. Medium was removed and cells were infected in triplicate with SARS-CoV-2 (hCoV-19/Italy/INMI1-isl/2020 at an MOI of 0.01 in 300 µL of medium containing different inhibitor concentrations. Amantadine was solubilized in sterile water and further diluted with medium to concentrations of 500 µM, 100 µM, 20 µM, 4 µM, and 8 nM. Remdesivir was solubilized in DMSO and diluted with medium to concentrations of 50 µM, 10 µM, 2 µM, 0.4 µM, and 80 nM. Remdesivir MOCK control contained according amounts of DMSO.

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Antiviral Activity Assay with Nucleocapsid Protein Readout (2nd Experiment) [4]
Exponentially growing Vero E6 cells were seeded into a 96-well plate at their optimal density in complete medium; 24 h later, cells were infected with SARS-CoV-2 (viral strain INMI1) at 0.01 moi (multiplicity of infection) and then exposed to different concentrations of the drugs (0–0.1–1–10–100–300 μM for Amantadine for 72 h. Drug dilutions were performed in culture medium. Replicates for each concentration point were examined. At the end of the incubation period, antiviral activity was examined through both ELISA (quantifying SARS-CoV-2 nucleoprotein) as well as a cytoprotection assay (toxicity effect examined through an inverted microscope).

Cell Viability Assay[4]
Cell Types: Vero E6 Cell
Tested Concentrations: 500 µM, 100 µM, 20 µM, 4 µM and 8 nM
Incubation Duration: 26 hrs (hours)
Experimental Results: Causes concentration-dependent reduction of virus (IC50=83 µM) 26 post-infection The nucleic acid concentration in the supernatant is 10-500 µM. Results in a concentration-dependent reduction of viral nucleic acid in the cytoplasm (IC50=119 µM) 26 hrs (hours) post-infection.

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Cell proliferation assay[6]
Cell Types: Human HCC cell lines (HepG2 and SMMC-7721) and normal liver cells (L02 cells)
Tested Concentrations: 0, 1, 2, 5, 10, 25, 50 and 100 µg/mL
Incubation Duration: 24, 48 and 72 hrs (hours)
Experimental Results: Inhibited cell proliferation in a time- and dose-dependent manner in HepG2 and SMMC-7721 cells.

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Cell cycle analysis[6]
Cell Types: HepG2 and SMMC-7721 Cell
Tested Concentrations: 0, 10, 25, 50 and 75 µg/mL
Incubation Duration: 48 hrs (hours)
Experimental Results: Significant increase in the number of HepG2 and SMMC-7721 cells in G0/G1 phase in a dose-dependent manner, and Dramatically diminished the numbe

Animal/Disease Models: Fischer 344 rats (4 months old, male, 290-330 g, 15 rats per group) [5]
Doses: 25 mg/kg
Route of Administration: IP, one time/day for 3 days (first dose in 15 minutes before administration)
Experimental Results: Inhibited surgery-induced neuroinflammation and learning and memory impairment, increased GDNF (glial cell line-derived neuronal neuron) co-localized with hippocampal glial fibrillary acidic protein (an astrocyte marker) nutritional factors).

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Four-month old male Fischer 344 rats weighing 290 – 330 g were randomly assigned to: 1) control group (not being exposed to surgery or any drugs), 2) Amantadine group, 3) surgery group (right carotid artery exposure), and 4) surgery plus Amantadine group in the first experiment. Each group had 15 rats. In the second experiment, the rats were assigned to: 5) control group, 6) anti-GDNF antibody group, 7) surgery plus amantadine plus boiled anti-GDNF antibody group, and 8) surgery plus amantadine plus anti-GDNF antibody group. Each group had 8 rats. In the third experiment, the rats were randomly assigned to: 7) control group, 8) anesthesia only group, and 9) surgery plus GDNF group. Each group had 8 rats. GDNF and the anti-GDNF antibody were injected intracerebroventricularly. One week later, these rats were started to be tested in Barnes maze and then fear conditioning. Separate rats were assigned to 1) control group, 2) surgery group, and 3) surgery plus amantadine group (n = 6 per condition) and sacrificed at 6 h, 24 h or 10 days after the surgery for Western blotting and immunohistochemistry.

Amantadine was dissolved in normal saline and injected intraperitoneally at 25 mg/kg/day for three days with the first dose at 15 min before surgery. Similar injections were performed in the amantadine only group except that no surgery and anesthesia were performed. The amantadine dose was chosen based on previous studies.[5]

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Absorption, Distribution and Excretion
Amantadine is well absorbed in the gastrointestinal tract after oral administration. It is primarily excreted unchanged in the urine via glomerular filtration and tubular secretion. 3–8 L/kg [healthy subjects] 0.2–0.3 L/hr/kg 0.10±0.04 L/hr/kg [healthy elderly men] It is rapidly and almost completely absorbed from the gastrointestinal tract. Amantadine can be distributed into breast milk. Excretion: Kidneys; >90% is excreted unchanged in the urine via glomerular filtration and tubular secretion. The excretion rate increases rapidly in acidic urine. Dialysis: Only a small amount (approximately 4%) is removed from the blood via hemodialysis. It is distributed in saliva, tear film, and nasal secretions; in animals, tissue concentrations (especially in the lungs) are higher than serum concentrations. It can cross the placenta and blood-brain barrier; it is distributed in breast milk. One patient's cerebrospinal fluid concentration was 52% of the corresponding plasma concentration. VolD - 4.4 ± 0.2 L/kg (normal renal function). 5.1 ± 0.2 L/kg (renal failure).
For more complete data on the absorption, distribution, and excretion of Amantadine (7 metabolites), please visit the HSDB record page.

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Metabolism/Metabolites
No significant metabolism was found, but trace amounts of acetyl metabolites were identified.
Eight Amantadine metabolites were identified in human urine. One of these metabolites, the N-acetylated compound, was quantified in human urine at 5-15% of the administered dose. In 5 out of 12 healthy volunteers, the plasma concentration of acetylAmantadine was 80% of the corresponding plasma Amantadine concentration after administration of 200 mg Amantadine. AcetylAmantadine was not detected in the plasma of the remaining seven volunteers.
Although trace amounts of acetyl metabolites were detected, no significant metabolism was found. Amantadine is well absorbed from the gastrointestinal tract after oral administration. Its anti-Parkinson's disease mechanism of action is not fully elucidated, but it appears to be achieved by promoting the release of dopamine from nerve endings in brain cells and stimulating a norepinephrine response. Its antiviral mechanism appears to be unrelated to this. The drug interferes with a viral protein, M2 (an ion channel), which is required for viral particles to "uncoat" after entering cells via endocytosis. Metabolites are excreted in the urine (A308). Elimination pathway: Primarily excreted unchanged via glomerular filtration and tubular secretion in the urine. Half-life: The mean half-life is 10 to 14 hours, but renal impairment significantly prolongs it to 7 to 10 days. The pharmacokinetics of Amantadine were determined in 24 healthy adult male volunteers after oral administration of a single 100 mg Amantadine hydrochloride soft capsule. The half-life is 17 ± 4 hours (range: 10 to 25 hours). In other studies, the mean plasma half-life of Amantadine in 19 healthy volunteers was 16 ± 6 hours (range: 9 to 31 hours).
Normal renal function: 11 to 15 hours. Elderly patients: 24 to 29 hours. Severe renal impairment: 7 to 10 days. Hemodialysis: 24 hours.
When creatinine clearance is below 40 mL/min/1.73 m², the elimination half-life increases by 2 to 3 times or more; the mean half-life in patients undergoing long-term maintenance hemodialysis is 8 days.

Toxicity Summary
Its anti-Parkinson's disease mechanism of action is not fully elucidated, but it appears to work by promoting the release of dopamine from nerve endings in brain cells and stimulating a norepinephrine response. It also has NMDA receptor antagonistic activity. Its antiviral mechanism appears to be unrelated to this. The drug interferes with a viral protein, M2 (an ion channel), which is required for viral particles to "uncoat" after entering cells via endocytosis. Hepatotoxicity
Despite its widespread use, there is little evidence that oral Amantadine causes liver damage, whether from elevated serum enzymes or clinically apparent liver disease. Likelihood Score: E (Unlikely to cause clinically apparent liver damage). Pregnancy and Lactation Effects ◉ Overview of Use During Lactation Because Amantadine may have negative effects on lactation, its use is best avoided during lactation. ◉ Effects on Breastfed Infants No relevant published information was found as of the revision date.
◉ Effects on lactation and breast milk
Amantadine is a dopamine agonist. Clinical studies have shown that taking 100 mg of Amantadine twice or three times daily can reduce serum prolactin levels and reduce galactorrhea in patients taking dopaminergic antipsychotics such as phenothiazines, haloperidol, and loxapine. [1][2] There are currently no reports on the effects of Amantadine on milk production in lactating mothers. For mothers who have established lactation, their prolactin levels may not affect their ability to breastfeed.

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Protein binding
Approximately 67% of the protein is bound to plasma proteins at concentrations ranging from 0.1 to 2.0 μg/mL.

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Toxicity Data
LD50: 800 mg/kg (oral, rat)
LD50: 700 mg/kg (oral, mouse)

Interactions
The anti-influenza A activity of Amantadine and ribavirin, as well as the anti-influenza A activity of combined administration, were studied separately. In ferret tracheal ciliated epithelium, the combined use of the drugs synergistically delayed the virus-induced cytopathic effect.
Concomitant use of alcohol and Amantadine is not recommended, as this may increase the risk of central nervous system side effects such as dizziness, lightheadedness, orthostatic hypotension, or confusion.
Concomitant use of anticholinergic drugs or other drugs with anticholinergic activity; tricyclic antidepressants; other anti-movement disorder drugs; antihistamines; or phenothiazines may enhance anticholinergic-like side effects, especially confusion, hallucinations, and nightmares; dosage adjustments of these drugs or Amantadine may be necessary.
In addition, patients should be advised to report any gastrointestinal problems promptly, as concurrent use of opioids may lead to paralytic ileus. Concomitant use of antidiarrheal medications containing both opioids and anticholinergics may enhance the anticholinergic-like side effects of Amantadine; while significant interactions are unlikely with commonly used doses of antidiarrheal medications containing both opioids and anticholinergics, significant interactions may occur if these medications are abused. For more complete data on drug interactions of Amantadine (one of 10), please visit the HSDB record page.

[1]. Emergence of amantadine-resistant influenza A viruses: epidemiological study. J Infect Chemother. 2003;9(3):195-200.

[2]. Amantadine: the journey from fighting flu to treating Parkinson disease. Neurology. 2012;78(14):1096-1099.

[3]. A review of compounds exhibiting anti-orthopoxvirus activity in animal models. Antiviral Res. 2003 Jan;57(1-2):41-52.

[4]. Amantadine Inhibits SARS-CoV-2 In Vitro. Viruses. 2021 Mar 24;13(4):539.

[5]. Amantadine alleviates postoperative cognitive dysfunction possibly by increasing glial cell line-derived neurotrophic factor in rats. Anesthesiology. 2014 Oct;121(4):773-85.

[6]. Amantadine inhibits cellular proliferation and induces the apoptosis of hepatocellular cancer cells in vitro. Int J Mol Med. 2015;36(3):904-910.

Amantadine sulfate is an alkylammonium sulfate prepared by mixing adamantane and sulfuric acid in a 2:1 ratio. It is an antiviral and anti-Parkinson's disease drug. It possesses multiple effects, including anti-Parkinson's disease, antiviral, dopaminergic, NMDA receptor antagonism, and non-anesthetic analgesia. It contains adamantane-1-ammonium. Amantadine sulfate is the sulfate of adamantane, a synthetic tricyclic amine with antiviral, anti-Parkinson's disease, and anti-hyperalgesic activities. Amantadine appears to exert its antiviral effect against influenza A virus by interfering with the function of the transmembrane domain of the viral M2 protein, thereby preventing the release of infectious viral nucleic acid into host cells; furthermore, the drug inhibits assembly during viral replication. Amantadine exerts its anti-Parkinson's disease effect by stimulating the release of dopamine from striatal dopaminergic nerve endings and inhibiting their presynaptic reuptake. This drug may also exert anticholinergic effects by inhibiting N-methyl-D-aspartate (NMDA) receptor-mediated acetylcholine stimulation, thereby producing an anti-hyperalgesic effect. It is an antiviral drug used to prevent or treat influenza A. It can also be used as an anti-Parkinson's disease drug to treat extrapyramidal reactions and postherpetic neuralgia. Its mechanism of action in treating movement disorders is not fully understood, but it may reflect an increase in dopamine synthesis and release, perhaps accompanied by inhibition of dopamine uptake.

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Therapeutic Uses
Anti-Parkinson's disease drug; antiviral drug; dopamine-like drug
Amantadine is used to treat certain fatigue symptoms associated with multiple sclerosis, including decreased energy, decreased well-being, poor concentration, memory loss, and decreased problem-solving abilities. /Not included on US or Canadian product labels/
Amantadine is indicated for the treatment of idiopathic Parkinson's syndrome (tremor paralysis); post-encephalitis Parkinson's syndrome, drug-induced extrapyramidal reactions, symptomatic Parkinson's syndrome following neurological damage from carbon monoxide poisoning, and Parkinson's syndrome associated with cerebral arteriosclerosis in the elderly. /Included on US product labels/
Amantadine is indicated for the prevention and treatment of respiratory infections caused by influenza A virus strains, and is indicated for high-risk groups (including patients with lung or cardiovascular disease, the elderly, and residents of nursing homes and other long-term care facilities with chronic diseases), close contacts of high-risk patients in hospital wards, immunocompromised patients, personnel in essential public service positions (e.g., police officers, firefighters, medical personnel), high-risk groups contraindicated for influenza vaccines, and patients with severe influenza A virus infection. It is effective against all influenza A virus strains tested to date, including Russian, Brazilian, Texas, and London strains. It can be used concurrently with inactivated influenza A vaccines as a chemopreventive agent before protective antibodies are developed. However, it must be emphasized that annual vaccination of high-risk groups is the most important measure to reduce the impact of influenza. Currently, there are no rigorous controlled studies testing whether Amantadine can prevent influenza A complications in high-risk groups. Drug-resistant influenza A virus strains have been reported in patients taking rimantadine (Amantadine); these resistant strains were apparently also transmitted through household contact. Rimantadine and Amantadine have similar chemical structures, antibacterial spectra, and mechanisms of action, and drug-resistant virus strains exhibit cross-resistance to both Amantadine and rimantadine. /US product label contains/
For more complete data on the therapeutic uses of Amantadine (6 types), please visit the HSDB record page.

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Drug Warning
Swine influenza (H1N1) virus contains a unique combination of gene segments that have not been previously reported in swine influenza or human influenza viruses in the United States or other regions. H1N1 virus is resistant to Amantadine and ribavirin, but resistant to oseltamivir or zanamivir. While rare reports of suicide attempts (some resulting in death) have been reported among patients taking Amantadine, many of these patients were receiving short courses of the drug for influenza prevention or treatment. The manufacturer states that the incidence and pathophysiological mechanisms of these suicide attempts are unclear. Suicidal ideation or attempts have been reported in both patients with and without a history of mental illness. Amantadine may exacerbate the mental state of patients with a history of mental illness or substance abuse. Patients with suicidal tendencies may exhibit abnormal mental states, including disorientation, confusion, depression, personality changes, agitation, aggressive behavior, hallucinations, delusions, other psychotic reactions, somnolence, or insomnia. Neuroleptic malignant syndrome (NMS) has been reported in patients taking Amantadine, which is associated with dose reduction or discontinuation of the drug. NMS can be fatal and requires immediate intensive symptomatic and supportive care. Close monitoring of patients is essential when Amantadine doses are reduced or discontinued; this precaution is particularly important for patients concurrently receiving antipsychotic medications. Nausea is one of the most common adverse reactions to Amantadine, reported in 5-10% of patients taking the drug at regular doses. Anorexia, constipation, diarrhea, and dry mouth are reported in 1-5% of patients taking Amantadine, and vomiting is reported in up to 1%. Abdominal discomfort or dysphagia has also been reported. The incidence of gastrointestinal adverse reactions is comparable to that of limantanide. For more complete data on Amantadine warnings (19 in total), please visit the HSDB record page.

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Pharmacodynamics
Amantadine is an antiviral drug with anti-Parkinson's disease activity, often used in combination with levodopa when levodopa efficacy declines (possibly due to tolerance). Like the similar drug limantanide, it is a derivative of adamantane. The mechanism of action of Amantadine in treating Parkinson's disease and drug-induced extrapyramidal reactions is unclear. Studies have shown that Amantadine can increase dopamine release in the brain of animals and does not have anticholinergic activity. Currently, Japan has three approved antiviral drugs for influenza: Amantadine, zanamivir, and oseltamivir. These antiviral drugs can be used to control and prevent influenza, but they are not a substitute for vaccination. Amantadine is an antiviral drug effective against influenza A virus but ineffective against influenza B virus. Influenza A virus patients receiving Amantadine treatment may shed susceptible virus in the early stages of treatment, and may subsequently shed drug-resistant virus, especially after 5-7 days of treatment. Even with the development of drug-resistant virus, these patients can still benefit from treatment. Amantadine susceptibility screening uses enzyme-linked immunosorbent assay (ELISA), plaque reduction assay, and TCID50/0.2 ml titration. Molecular changes associated with resistance have been identified as single nucleotide changes leading to the substitution of a corresponding amino acid at one of four key sites (amino acids 26, 27, 30, and 31) in the transmembrane region of the M2 protein. Polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis is highly effective. During outbreaks in nursing homes, drug-resistant viruses have emerged, and Amantadine has been used not only to treat influenza virus infections but also to treat Parkinson's disease. Measures should be taken to minimize contact between people taking antiviral drugs for treatment or chemoprevention and those not taking antiviral drugs. [1]

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Objective: To investigate how Amantadine transitioned from an anti-influenza drug to an anti-Parkinson's disease drug. Methods: A review of historical literature on the use of Amantadine from 1966 to the present. Results: Amantadine was initially introduced and used as an antiviral drug. A Parkinson's disease (PD) patient experienced symptom relief after taking Amantadine for influenza. This discovery sparked interest and led to several important studies that ultimately identified new indications for Amantadine. Conclusion: Amantadine has been less commonly used as an influenza drug over the years; however, it has become one of the commonly used drugs for the early treatment of Parkinson's disease symptoms and is also an option for treating movement disorders. [2]

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A variety of animal models (most commonly mice), rabbits, or monkeys have been used to screen compounds effective against orthopoxvirus infection. Treatment of vaccinia virus infection has been extensively studied in various models, including skin or ocular scratch infection models, as well as intravenous, intraperitoneal, intracerebral, or intranasal inoculation models. Vaccinia virus has been used in intranasal or aerosol infection studies to evaluate its efficacy against fatal respiratory infections. The use of tularopenia, monkeypox, and smallpox viruses in chemotherapy experiments is less widespread than with other viruses. A review of the literature over the past 50 years has identified a variety of compounds that are effective in treating one or more of these infections, including thioureas, nucleoside and nucleotide analogs, interferon, interferon inducers, and other unrelated compounds. The most promising anti-poxetine drugs are acyclic nucleotides (S)-1-(3-hydroxy-2-phosphonomethoxypropyl)cytosine (Cidofovir, HPMPC) and 1-[((S)-2-hydroxy-2-oxo-1,4,2-dioxaphosphazenecycloheptane-5-yl)methyl]cytosine (cyclic HPMPC), and the acyclic nucleoside analog 2-amino-7-[(1,3-dihydroxy-2-propoxy)methyl]purine (S2242). Other compound classes that are under-studied in lethal infection models but deserve further attention include thioaminoureas associated with methViolaceine, and analogs of adenosine-N(1)-oxide and 1-(benzyloxy)adenosine. [3]

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Background: Postoperative cognitive impairment is a clinical condition associated with poor prognosis. We investigated the efficacy of Amantadine in reducing surgery-induced cognitive impairment and the role of glial line-derived neurotrophic factor (GDNF) in this effect. Methods: Four-month-old male Fischer 344 rats underwent right carotid artery exposure surgery under intravenous anesthesia. Some rats were intraperitoneally injected with Amantadine at 25 mg/kg/day for 3 consecutive days 15 minutes before surgery; or intraventricularly injected with GDNF or anti-GDNF antibody at the end of surgery. One week later, the rats were subjected to Barnes maze and fear conditViolaceing tests. Hippocampal tissue was collected at 6 hours, 24 hours or 10 days after surgery for biochemical analysis. C8-B4 cells (a microglia) were pretreated with 1 ng/ml GDNF for 30 minutes and then treated with 5 ng/ml lipopolysaccharide for 2 hours. [5] In summary, Amantadine inhibits viral replication in the Vero E6 cell system. In this study, due to the above limitations, we were unable to confirm that Amantadine functionally interferes with the binding of viral spike protein to ACE2 on target cells. The issue stems from the predicted tight binding of Amantadine to Tyr489 and Phe456 in the SARS-CoV-2 receptor-binding domain (RBD). The SARS-CoV-2 RBD (Arg319–Phe541 residues) interacts with the N-terminal peptidase domain of ACE2 (Ser19–Asp615 residues), potentially suggesting an antiviral mechanism of action for Amantadine, but our data do not support this hypothesis from computer simulations. Inhibition of viral porins as another mechanism of action requires further investigation in future studies. In a recently published preprint, Amantadine inhibited recombinant SARS-CoV-2 viral porin E and the putative SARS-CoV-2 viral porin Orf10. The authors observed in an Xenopus oocyte model that 10 µM Amantadine inhibited the protein E ion channel-mediated current by up to 77%, which appears to be a stronger inhibitory effect on overall viral replication than the IC50 values (83–119 µM) observed in more complex eukaryotic cell culture models; these data suggest that viral porin inhibitors warrant further investigation. Finally, Amantadine appears to have an effect on known SARS-CoV-2 mutants to date, as protein E or Orf10 mutations are scarce or nonexistent in SARS-CoV-2 mutant strains collected from Indian patients. Strain B 1.1.7 showed neither protein E nor Orf10 mutations. However, even a single amino acid substitution can reduce the efficacy of small molecule drugs, as was the case with influenza A viruses many years ago.

C10H17N.H2O4S

249.32716

400.239

C, 59.97; H, 9.06; N, 6.99; O, 15.98; S, 8.00

31377-23-8

Amantadine;768-94-5;Amantadine hydrochloride;665-66-7

124108

Typically exists as solid at room temperature

1.066g/cm3

225.7ºC at 760 mmHg

300 °C

96ºC

5.656

4

6

0

27

225

0

S(O)(O)(=O)=O.C12(N)CC3CC(CC(C3)C1)C2

MYWTWSQFJLXGGQ-UHFFFAOYSA-N

InChI=1S/2C10H17N.H2O4S/c2*11-10-4-7-1-8(5-10)3-9(2-7)6-10;1-5(2,3)4/h2*7-9H,1-6,11H2;(H2,1,2,3,4)

adamantan-1-amine;sulfuric acid

Amantadine Sulfate; Amantadine sulphate; 1-Aminoadamantane Sulfate; 1-Aminoadamantane sulphate; ...; 31377-23-8 (sulfate);

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.)