M2 protein

Researchers report the in vitro efficacy of ion-channel inhibitors amantadine, memantine and Rimantadine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In VeroE6 cells, rimantadine was most potent followed by memantine and amantadine (50% effective concentrations: 36, 80 and 116 µM, respectively). Rimantadine also showed the highest selectivity index, followed by amantadine and memantine (17.3, 12.2 and 7.6, respectively). Similar results were observed in human hepatoma Huh7.5 and lung carcinoma A549-hACE2 cells. Inhibitors interacted in a similar antagonistic manner with remdesivir and had a similar barrier to viral escape. Rimantadine acted mainly at the viral post-entry level and partially at the viral entry level. Based on these results, rimantadine showed the most promise for treatment of SARS-CoV-2.

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Amantadine, Memantine and Rimantadine Showed Activity against SARS-CoV-2 In Vitro [3]
To determine the potency of adamantane derivatives against SARS-CoV-2, we carried out 96-well based short-term concentration-response assays based on the quantification of infected cells by immunostaining for SARS-CoV-2 spike protein. Assays were carried out in African green monkey VeroE6 cells, a prototype cell line for the evaluation of drug activity against SARS-CoV-2, human hepatoma Huh7.5 and human lung carcinoma A549-hACE2 cells. Used inhibitor concentrations did not result in a reduction of cell viability (relative cell viability > 90%), as shown in Figures S2–S4. Similar results were obtained in all cell types with EC50 values in the micromolar range. Rimantadine was most potent with EC50 of 36, 26 and 70 µM in VeroE6, Huh7.5 and A549-hACE2 cells, respectively. Memantine showed intermediate potency (EC50 of 80, 86, and 70 µM) and amantadine showed the lowest potency (EC50 of 116, 118, and 80 µM) (Figure 1, Table 1). At the highest used concentrations, all inhibitors had the capacity to fully inhibit SARS-CoV-2 in VeroE6 and A549-hACE2 cells, while slightly lower inhibition was achieved in Huh7.5 cells (Figure 1). Amantadine showed lower cytotoxicity than memantine and rimantadine (Table 1, Figures S2–S4). However, due to its comparatively high potency, rimantadine had the highest selectivity index (SI), while memantine had the lowest SI in all three cell lines (Table 1).

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Amantadine, Memantine and Rimantadine Interacted in a Similar Antagonistic Manner with Remdesivir [3]
To study interactions between ion-channel inhibitors and remdesivir, 96-well based combination treatments were carried out. SARS-CoV-2 infected VeroE6 cells were treated with ion-channel inhibitors singly or in combination with remdesivir, or with remdesivir alone. Inhibitor concentrations were selected based on previously determined EC50 values: for ion-channel inhibitors EC50 are given in Table 1, and for remdesivir EC50 was 2.5 µM, as previously reported [14]. For all three ion-channel inhibitors, the effect of the combination treatments did not exceed the effect of the single treatments (Figure 2, Supplementary Results, Table S1). Analysis using the method of Chou and Talalay in the CompuSyn software s described in Supplementary Methods revealed mostly antagonistic interactions between the ion-channel inhibitors and remdesivir (Supplementary Results, Figures S5 and S6 and Table S2).

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Adamantane Derivatives Did Not Differ in Their Barrier to Viral Escape [3]
To compare ion-channel inhibitors regarding their capacity to prevent SARS-CoV-2 spread under treatment, we carried out longer-term treatments of infected VeroE6 cells using amantadine, memantine and Rimantadine at the highest possible equipotent concentrations (3-fold EC50), according to inhibitor cytotoxicities (Figure S2). Treatment with all inhibitors resulted in a similar delay of early viral spread kinetics on day 1 post infection and treatment initiation, while ≥80% of culture cells became infected on day 3–5, comparable to the nontreated control cultures (Figure 3). However, compared to the nontreated and the memantine treated cultures, somewhat reduced cytopathogenic effects were observed in the amantadine and rimantadine treated cultures. Thus, overall, the three inhibitors did not show major differences in their barrier to viral escape. The favorable SI of rimantadine enabled treatment with seven-fold EC50, resulting in additional viral suppression on days 3–5, while on day 7 ≥80% of culture cells became infected. To investigate whether the acquisition of substitutions might have facilitated viral escape, viruses from all cultures shown in Figure 3 derived at the peak of infection were subjected to NGS analysis. In memantine and rimantadine treated cultures, substitutions that were not found in the nontreated culture were detected, however, without apparent hotspots for substitutions (Table S3). Thus, inhibitors could only temporarily suppress SARS-CoV-2 at concentrations permissible in vitro according to inhibitor cytotoxicities.

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Rimantadine Inhibited Infection with SARS-CoV-2 Mainly at the Viral Post-Entry Level [3]
To investigate the mechanism of action of Rimantadine, the most promising compound in this study, we carried out a time-of-addition experiment in VeroE6 cells. Cells were inoculated with SARS-CoV-2 during a 2-hour infection phase and treated with rimantadine at different timepoints post inoculation. When rimantadine was added at the time of inoculation (0 h post inoculation) and removed at the end of the viral infection phase (2 h post inoculation), 53% inhibition of SARS-CoV-2 infection was observed (Figure 4). However, when rimantadine was added at different timepoints following the viral infection phase (2, 4, or 6 h post viral inoculation), >99% inhibition was observed. Thus, it appeared that rimantadine acted mainly by targeting the virus at the post-entry level while partially acting at the entry level.

Amantadine and Rimantadine are oral antiviral drugs useful in the prophylaxis and treatment of influenza A virus infections. This article reviews the pharmacology, antiviral activity and mechanism of action, pharmacokinetics, toxicities, efficacy, and clinical applications of these agents. When administered in equivalent dosages (200 mg per day), rimantadine has prophylactic efficacy comparable to amantadine but lower potential for causing adverse effects. Despite structural similarities, these drugs differ significantly in their pharmacokinetics, and these differences may account for rimantadine's more favorable toxicity profile. Both drugs provide therapeutic benefit if administered early in uncomplicated influenza, and studies are currently in progress to determine the effectiveness of oral rimantadine in preventing or treating the serious complications of influenza A virus infections. [1]

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Treatment with Rimantadine of influenza in children and the potential development of resistance in clinical isolates associated with therapy have not been previously studied. We compared rimantadine to acetaminophen therapy in a controlled, double-blind study of 91 children with influenza-like illness. Of 69 children with proven influenza A/H3N2 infection, 37 received rimantadine and 32 received acetaminophen for five days. Children receiving rimantadine showed significantly greater reduction in fever and improvement in daily scores for symptoms and severity of illness during the first three days. Viral shedding also diminished significantly during the first two days but subsequently increased such that by days 6 and 7 the proportion of children shedding virus, as well as the quantity of virus shed, was significantly greater in the rimantadine group. During the seven-day study, of the 22 children in the rimantadine group with serial isolates tested, ten (45.5%) had resistant isolates compared with two (12.5%) of those with serial isolates in the acetaminophen group (P less than .03). Thus, of the total 37 children in the rimantadine group, 27% were found to have resistant isolated compared with 6% in the total group receiving acetaminophen (P less than .04). Furthermore, the mean inhibitory concentration of rimantadine increased with time in the rimantadine group (r = .4, P = .002) but not in the acetaminophen group. Rimantadine therapy, thus, appears to be significantly more effective than acetaminophen in ameliorating the clinical signs and symptoms of influenza in children. Treatment with rimantadine was also associated with increased viral shedding after the medication was discontinued and with the development of resistance in the clinical isolates, the significance of which is unknown.[2]

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In children, Rimantadine (RMT) was effective in the abatement of fever on day three of treatment. Amantadine (AMT) showed a prophylactic effect against influenza A infection. AMT and RMT were not related to an increase in the occurrence of adverse effects. RMT also was considered to be well tolerated by the elderly, but showed no prophylactic effect. Different doses were comparable in the prophylaxis of influenza in the elderly, as well as in reporting adverse effects. Zanamivir prevented influenza A more effectively than RMT in the elderly. Authors' conclusions: AMT was effective in the prophylaxis of influenza A in children. As confounding matters might have affected our findings, caution should be taken when considering which patients should to be given this prophylactic. Our conclusions about effectiveness of both antivirals for the treatment of influenza A in children were limited to a proven benefit of RMT in the abatement of fever on day three of treatment. Due to the small number of available studies we could not reach a definitive conclusion on the safety of AMT or the effectiveness of RMT in preventing influenza in children and the elderly [4].

Time-of-Addition Experiment with Rimantadine [3]
VeroE6 cells in 96-well plates were inoculated with SARS-CoV-2 at MOI 0.01 with a 2-h infection phase and treated with 230 μM rimantadine at different timepoints post inoculation. For entry treatment, rimantadine was added together with the virus at 0 h post inoculation and removed in the end of the 2-h viral infection phase. For post-entry treatment, rimantadine was added 2, 4 or 6 h post inoculation. Treatment conditions were tested in 6 replicates. Treatment plates included 12 infected-nontreated and 12 noninfected-nontreated wells. Cells were immunostained for SARS-CoV-2 spike protein after incubation for 46–50 h. Data points were given as % inhibition with SEM. % inhibition was determined as 100%–% residual infectivity (see previous section).

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Immunostaining and Evaluation of 96-Well Plates for Short-Term Treatments and Time-of-Addition Experiment[3]
Short-term treatment plates were stained with primary antibody SARS-CoV-2 spike chimeric monoclonal antibody diluted 1:5000, secondary antibody F(ab’)2-goat anti-human IgG-Fc cross-adsorbed secondary antibody, HRP or goat F(ab’)2 anti-human IgG–Fc (HRP), preadsorbed diluted 1:2000, and DAB substrate BrightDAB kit. Single SARS-CoV-2 spike protein positive cells were automatically counted using an ImmunoSpot series 5 UV Analyzer. Counts from infected-treated wells were related to the mean count of infected-nontreated wells to calculate the %residual infectivity for single inhibitor treatments and, in addition, the % inhibition for combination treatments and time-of-addition experiment

Background: Although amantadine (AMT) and rimantadine (RMT) are used to relieve or treat influenza A symptoms in healthy adults, little is known about the effectiveness and safety of these antivirals in preventing and treating influenza A in children and the elderly. Objectives: The aim of this review was to systematically consider evidence on the effectiveness and safety of AMT and RMT in preventing and treating influenza A in children and the elderly. Search strategy: We searched the Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library, 2007, issue 3); MEDLINE (1966 to July 2007) and EMBASE (1980 to July 2007). Selection criteria: Randomised or quasi-randomised trials comparing AMT and/or RMT in children and the elderly with placebo, control, other antivirals or comparing different doses or schedules of AMT and/or RMT or no intervention. Data collection and analysis: Two review authors independently selected trials for inclusion and assessed methodological quality. Disagreements were resolved by consensus. In all comparisons except for one, the trials in children and in the elderly were analysed separately. Data were analysed and reported using Cochrane Review Manager 4.2. software.[4]

Absorption, Distribution and Excretion
Absorbed well after oral administration; the absorption rates are the same for tablets and syrups. After oral administration, amantadine is extensively metabolized in the liver, with less than 25% of the dose excreted unchanged in the urine. Protein binding: Moderate (approximately 40%). Distribution: Volume of distribution – Adults: 17 to 25 L/kg. Children: Mean 289 L. Concentrations in nasal mucus are on average 50% higher than in plasma. Absorption is good; the absorption effects are the same after oral administration for tablets and syrups. Time to peak concentration: 1 to 4 hours. For more complete data on absorption, distribution, and excretion of lymantadine (11 in total), please visit the HSDB record page. Metabolism/Metabolites After oral administration, lymantadine is extensively metabolized in the liver, with less than 25% of the dose excreted unchanged in the urine. Glucuronization and hydroxylation are the main metabolic pathways.
Limantidine hydrochloride is extensively metabolized in the liver to at least three hydroxylated metabolites. These metabolites are named bound and unbound 3-, 4α-, and 4β-hydroxylated metabolites. In addition, a glucuronide conjugate of limantidine has been identified.
Extensively metabolized in the liver; glucuronidation and hydroxylation are the major metabolic pathways.
Biological half-life

Young adults (22 to 44 years): 25 to 30 hours. Older adults (71 to 79 years) and patients with chronic liver disease: approximately 32 hours. Children (4 to 8 years): approximately 13 to 38 hours.
Young adults (22 to 44 years): 25 to 30 hours. Older adults (71 to 79 years) and patients with chronic liver disease: approximately 32 hours. Children (4 to 8 years): 13 to 38 hours.

Hepatotoxicity
Despite 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 There is currently no information regarding the use of amantadine during lactation. The manufacturer states that this drug should not be used by breastfeeding women. ◉ Effects on Breastfed Infants No relevant published information was found as of the revision date. ◉ Effects on Lactation and Breast Milk No relevant published information was found as of the revision date. Protein Binding Approximately 40% higher than typical plasma concentrations. Interactions Concomitant administration of a single dose of amantadine and cimetidine in healthy adults reduced amantadine clearance by 18%; this is currently considered to have little clinical significance. Because antiviral drugs can reduce influenza virus replication, intranasal influenza vaccination should only be administered at least 48 hours after discontinuing amantadine, and amantadine should only be used again at least 2 weeks after intranasal influenza vaccination. Cimetidine: Combining amantadine with acetaminophen or aspirin reduces the peak serum concentration of amantadine by approximately 11%; this is currently considered to have minimal clinical significance.

[1]. Tominack, R.L. and F.G. Hayden, Rimantadine hydrochloride and amantadine hydrochloride use in influenza A virus infections. Infect Dis Clin North Am, 1987. 1(2): p. 459-78.

[2]. Children with influenza A infection: treatment with rimantadine. Pediatrics, 1987. 80(2): p. 275-82.

[3]. Efficacy of Ion-Channel Inhibitors Amantadine, Memantine and Rimantadine for the Treatment of SARS-CoV-2 In Vitro. Viruses. 2021 Oct 15;13(10):2082.

[4]. Amantadine and rimantadine for influenza A in children and the elderly. Cochrane Database Syst Rev. 2008 Jan 23:(1):CD002745.

1-(1-Adamaneyl)ethylamine is an alkylamine. It is an RNA synthesis inhibitor used as an antiviral drug for the prevention and treatment of influenza. Amantadine is an inhibitor of the M2 protein of influenza A virus. The mechanism of action of amantadine is as an M2 protein inhibitor. Amantadine is an antiviral drug used to treat influenza A. Amantadine has not been found to be associated with clinically significant liver damage. Amantadine is a cyclic amine and α-methyl derivative of amantadine with antiviral activity. Although the exact mechanism of action of amantadine is not fully understood, the drug 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 viral uncoating and, consequently, preventing the release of infectious viral nucleic acid into the cytoplasm of infected cells. An RNA synthesis inhibitor used as an antiviral drug for the prevention and treatment of influenza. See also: Amantadine hydrochloride (in salt form). Indications: For the prevention and treatment of illness in adults caused by various strains of influenza A virus.
FDA Label

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Mechanism of Action
The mechanism of action of amantadine is not fully elucidated. Amantadine appears to exert its inhibitory effect early in the viral replication cycle, possibly inhibiting viral uncoating. Proteins encoded by the influenza A virus M2 gene may play an important role in amantadine sensitivity.
Amantadine is thought to exert its inhibitory effect early in the viral replication cycle, possibly by blocking or significantly reducing the uncoating of viral RNA within host cells. Genetic studies have shown that a single amino acid alteration in the transmembrane portion of the M2 protein can completely eliminate influenza A virus sensitivity to amantadine.
Similar to amantadine, amantadine inhibits viral replication by interfering with the M2 protein of influenza A virus (an integrated membrane protein). The M2 protein of influenza A virus functions as an ion channel and is crucial in at least two aspects of viral replication: the disintegration of infectious viral particles and the regulation of the ion environment of transport pathways. Amantadine inhibits both phases of the influenza A virus replication cycle by interfering with the ion channel function of the M2 protein. In the early stages of the viral replication cycle, amantadine inhibits viral particle uncoating, likely by suppressing acid-mediated dissociation of viral nucleic acids and proteins, thereby preventing nuclear transport of viral genomic material. In the later stages of the replication cycle, amantadine also inhibits the maturation of certain influenza A virus strains (e.g., the H7 strain) by promoting pH-induced conformational changes during intracellular transport of influenza A virus hemagglutinin. Amantadine does not appear to affect viral cell adsorption and cell penetration. Furthermore, amantadine does not interfere with the synthesis of viral components (e.g., RNA-directed RNA polymerase activity).

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Therapeutic Use
Amantadine is indicated for the prevention of respiratory infections caused by influenza A virus in adults and children, and for the treatment of respiratory infections caused by influenza A virus in adults. /US Product Label Contains/
Prevention of infection with multiple influenza A virus strains

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Drug Warnings
Swine influenza (H1N1) virus contains a unique combination of gene segments that have not been previously reported in swine or human influenza viruses in the United States or elsewhere. The H1N1 virus is resistant to amantadine and rimatadine, but not to oseltamivir or zanamivir. Elderly patients, especially those in long-term care facilities, are more likely to experience rimatadine-related adverse reactions than younger adults or children, primarily central nervous system (CNS) and gastrointestinal side effects. FDA Pregnancy Risk Classification: C/Risk cannot be ruled out. Currently, 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. Compared to amantadine, rimatadine is less likely to cause CNS adverse reactions (e.g., nervousness, anxiety, difficulty concentrating, dizziness) at commonly used doses, possibly due in part to pharmacokinetic differences between the two drugs. In a 6-week study, healthy adults taking a daily preventative dose of 200 mg of amantadine hydrochloride or amantadine hydrochloride showed that approximately 6% and 13% of patients, respectively, discontinued treatment due to central nervous system adverse reactions, compared to approximately 4% in the placebo group. While patients taking amantadine have experienced neuropsychiatric symptoms (e.g., delirium, significant behavioral changes) or psychomotor dysfunction, no such adverse reactions have been reported in patients taking amantadine. For more complete data on amantadine warnings (13 in total), please visit the HSDB records page.

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Pharmacodynamics
Amantadine is a cyclic amine and a synthetic antiviral drug, and like amantadine, it is a derivative of adamantane. Amantadine inhibits the in vitro replication of all three influenza A virus antigen subtypes (H1N1, H2H2, and H3N2) isolated from humans. Amantadine has almost no activity against influenza B virus. Amantadine does not appear to interfere with the immunogenicity of inactivated influenza A vaccines.

C12H21N

179.30184

179.167

13392-28-4

Rimantadine hydrochloride;1501-84-4;Rimantadine-d4 hydrochloride;350818-67-6

5071

Colorless to light yellow liquid

1.033

248ºC

375°C(lit.)

99ºC

0.0249mmHg at 25°C

1.539

4.052

1

1

1

13

180

0

CC(C1(C[C@H](C2)C3)C[C@H]3C[C@H]2C1)N

UBCHPRBFMUDMNC-UHFFFAOYSA-N

InChI=1S/C12H21N/c1-8(13)12-5-9-2-10(6-12)4-11(3-9)7-12/h8-11H,2-7,13H2,1H3

1-(1-adamantyl)ethanamine

rimantadine; 13392-28-4; 1-(1-Adamantyl)ethanamine; Rimantadina; Rimantadinum; alpha-Methyl-1-adamantanemethylamine; Remantadine; alpha-Methyladamantanemethylamine;

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.)