Toll like receptor 7 (TLR7); HSV-1

In animal models, imiquimod promotes the innate immune response through NK cell activation, nitric oxide and cytokine secretion from activated macrophages, and B lymphocyte differentiation and proliferation. Through the induction, synthesis, and release of cytokines such as interferon-a (IFN-α), interleukin (IL)-6, and tumor necrosis factor (TNF)-α, imiquimod enhances the innate immune response[1].

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Imiquimod can be used to create animal models of psoriasis. Imiquimod incites the innate immune response in animal models through NK cell activation, nitric oxide and cytokine secretion from activated macrophages, and B lymphocyte differentiation and proliferation. By triggering, producing, and releasing cytokines such as interleukin (IL)-6, tumor necrosis factor (TNF)-alpha, and interferon-alpha (IFN-alpha), imiquimod activates the innate immune response [1].

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Topical application of imiquimod (IMQ), a TLR7/8 ligand and potent immune activator, can induce and exacerbate psoriasis, a chronic inflammatory skin disorder. Recently, a crucial role was proposed for the IL-23/IL-17 axis in psoriasis. We hypothesized that IMQ-induced dermatitis in mice can serve as a model for the analysis of pathogenic mechanisms in psoriasis-like dermatitis and assessed its IL-23/IL-17 axis dependency. Daily application of IMQ on mouse back skin induced inflamed scaly skin lesions resembling plaque type psoriasis. These lesions showed increased epidermal proliferation, abnormal differentiation, epidermal accumulation of neutrophils in microabcesses, neoangiogenesis, and infiltrates consisting of CD4(+) T cells, CD11c(+) dendritic cells, and plasmacytoid dendritic cells. IMQ induced epidermal expression of IL-23, IL-17A, and IL-17F, as well as an increase in splenic Th17 cells. IMQ-induced dermatitis was partially dependent on the presence of T cells, whereas disease development was almost completely blocked in mice deficient for IL-23 or the IL-17 receptor, demonstrating a pivotal role of the IL-23/IL-17 axis. In conclusion, the sole application of the innate TLR7/8 ligand IMQ rapidly induces a dermatitis closely resembling human psoriasis, critically dependent on the IL-23/IL-17 axis. This rapid and convenient model allows further elucidation of pathogenic mechanisms and evaluation of new therapies in psoriasis.[5]

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Imiquimod (IMQ) induced human-like psoriasis in mice has been shown to be effective in testing and development of novel treatments. The IMQ psoriasis model has become widely used animal model, however, it is not completely characterized in different rat strains. We aimed to evaluate IMQ and betamethasone treatment for induction and reversal of psoriatic lesions on macroscopic, histological, genetic as well as cytokines and chemokines activation levels. Wistar rats were treated topically with IMQ. Adopted Psoriasis Area Severity Index (PASI) was calculated at the baseline, after the IMQ-symptoms induction and after betamethasone-symptoms reversal. Systematic effects were studied on cytokines and chemokines levels in plasma. Skin biopsy was taken to assess histological symptoms and selected inflammatory cytokines and receptors genes expression levels. Reversal of skin lesions, after betamethasone treatment, was significant (p = 0.03). Histological differences between untreated and IMQ-treated skin were significant for some markers (p < 0.05) though not significantly decreased by betamethasone treatment. Fourteen genes were significantly up-regulated after the IMQ and four genes were down-regulated after skin lesions reversal by betamethasone. This work provides new insights on biological effects of imiquimod induced psoriasis and its reversal by betamethasone treatment in Wistar rats. It also contributes to general knowledge of the rat model usage for testing of novel anti-psoriasis drugs.[6]

siRNA-mediated suppression of gene expression. [3]
Small interfering RNA (siRNA) specific for human cystatin A, siRNA specific for human adenosine receptor A1 [adenosine A1-R siRNA(h)], and a control siRNA were used. The siRNA (0.5 μg) was transfected into cells using FUGENE 6 reagent according to the manufacturer's instructions. After 24 h of culture, some, but not all, of the cells were treated with 10 μg of imiquimod/ml or not for 24 h. The cells were then infected with HSV-1 VR3 at an MOI of 1. The virus titer in the culture supernatant was determined as described above.

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Binding to adenosine receptor determined by competition assay. [3]
3H-labeled DPCPX, which is a selective antagonist of adenosine A1 receptor, was purchased from Perkin-Elmer. CHO-A1 or CHO-K1 cells (17) were seeded into a 96-well plate at a density of 2 × 103 cells per well, followed by incubation overnight. The cells were incubated with [3H]DPCPX (550 Bq/well) and competition chemicals (imiquimod and resiquimod [each at 100 μM] or DPCPX [1 μM]) in the IMDM containing 10% FBS and penicillin-streptomycin for 20 min in a CO2 incubator. After incubation, the cells were washed four times with PBS (−) and lysed with lysis buffer (1% NP-40, 5% glycerol, 5 mM EDTA, 100 mM NaCl, 50 mM HEPES [pH 7.4]). The counts per minute (cpm) of [3H]DPCPX in the lysate were measured using a LS6500 liquid scintillation counter. Selective binding amounts of [3H]DPCPX on adenosine receptor A1 of CHO-A1 cells were calculated as the cpm value of CHO-A1 subtracted from the cpm value of CHO-K1 cells as a control. The cpm value without competitors was set to 100%.

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Imiquimod, a small-molecule immune response modifier of the imidazoquinoline family, has shown profound antitumoral and antiviral efficacy both in vitro and in clinical applications in vivo. It has been demonstrated that this activity is mediated through the Toll-like receptor (TLR)7- and TLR8-signaling cascade resulting in the secretion of proinflammatory cytokines and, consecutively, induction of a tumor-directed cellular immune response. In addition, imiquimod exerts a direct proapoptotic activity in tumor cells. We demonstrate here that imiquimod induces activation of the transcription factor NF-kappaB and the downstream production of proinflammatory cytokines in the absence of TLR7 and TLR8. In Chinese hamster ovary cells stably transfected with the human adenosine receptor subtypes, we then show in radioligand-binding competition experiments that imiquimod binds to adenosine receptors at concentrations relevant in clinical settings, with highest affinities to the A(1) and A(2A) subtypes. The effect on the receptor-mediated activation of adenylyl cyclase was also studied, and these experiments revealed that imiquimod acts as an adenosine receptor antagonist. In addition, imiquimod had an inhibitory effect on adenylyl cyclase activity downstream from the receptor. Finally, using transformed human keratinocytes, we provide experimental evidence that imiquimod and A(2A) adenosine receptor-specific compounds similarly induce proinflammatory cytokines in the absence of immune cells. Thus, imiquimod appears to suppress an important feedback mechanism of inflammation by antagonism of adenosine receptor-dependent increase of cAMP and a concomitant receptor-independent inhibition of cAMP production. These novel mechanisms presumably act synergistic with the positive induction of proinflammatory cytokines and can, at least in part, explain the profound inflammation observed in some patients in vivo.

Infection experiments and cell viability assay. [3]
FL cells were seeded at 105 cells/ml in a 6-cm dish and cultured for 24 h. For analyses of the effects of imiquimod on the cell viability and virus replication, the cells were cultured in a medium containing imiquimod for 12 or 24 h before virus infection. The cells were then inoculated with HSV-1 at a multiplicity of infection (MOI) of 0.1 or 1, incubated for 1 h for absorption, and then cultured for a further 24 h. Virus titers in the supernatants were determined by plaque assay using Vero cells as described previously. Cell viability was determined with a modified 3-(4,5-dimethylthoazol-2-yl)-2,5-diphenyltetrazolium bromide assay using Cell Counting Kit 8.

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Microarray analysis. [3]
Total cellular RNA was prepared from FL cells that were cultured for 24 h with or without imiquimod at 10 μg/ml. Microarray analysis was performed using the 3D-Gene Human OligoChip 25K.

Tissue ELISA revealed that imiquimod specifically reduced IL-1β and IL-6 secretion in the treated mouse paws (Panels F-G), whereas neutrophil infiltration (visualized by MPO quantification, Panel H) and TNF-α production (panel I) remained unaffected. RTqPCR and western blot analysis further documented the negative effect of imiquimod on the transcription of the Il-1β and Il-6 genes (Panels J-K) and IL-1β pro-form expression (Panel L).

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Imiquimod-induced psoriasis-like skin inflammation in mice [5]
Mice at 8 to 11 wk of age received a daily topical dose of 62.5 mg of commercially available imiquimod/IMQ cream (5%) on the shaved back and the right ear for 5 or 6 consecutive days, translating in a daily dose of 3.125 mg of the active compound. This dose was empirically determined to cause most optimal and reproducible skin inflammation in mice (data not shown). Control mice were treated similarly with a control vehicle cream. CD3+ cells were depleted by injection of the mice with 400 μg/mouse rat-anti-mouse CD3 mAb 17A2 on days −3, 0, and 3, relative to the start of IMQ application.

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Skin inflammation induction and symptom reversal evaluation [6]
Following the treatment with imiquimod, animals were divided into two groups: IMQ Group—for the histological evaluation of imiquimod effects on Day 7, and BTM Group—for evaluation of imiquimod/IMQ induced changes reversal determined by calculation of PASI prior and after the treatment with betamethasone ointment (0.5 mg/g). Experimental design is provided in Fig. 1.

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PASI evaluation of skin inflammation [6]
The evaluation of rat skin lesions was performed by adopted Psoriasis Area Severity Index (PASI) calculation on day 0 (before imiquimod application), day 7 (after period of imiquimod application) and day 11 in BTM group. PASI evaluation was scored individually for three indicators (erythema, scaling, and thickening) using psoriasis scoring tables. As the size of experimental area did not differ, the area score was not taken into account. Scores were assigned as follows: 0—none; 1—slight; 2—moderate; 3—marked and 4—very marked. The cumulative score served as a measure of the inflammation severity (maximum score of 12).

Absorption, Distribution and Excretion
Well absorbed through skin (as a cream)
Following topical application to the skin in adults with actinic keratosis (75-mg doses 3 times weekly for 16 weeks), 0.08-0.15% of the dose is eliminated in urine as unchanged drug and metabolites. Following topical application in patients with HPV warts, 0.11 or 2.41% of the dose is eliminated in urine as unchanged drug and metabolites in men or women, respectively.
Imiquimod is absorbed systemically following topical application to skin. In adults with actinic keratosis who received topical imiquimod 5% cream 3 times weekly for 16 weeks, mean peak serum concentrations at the end of week 16 were approximately 0.1, 0.2, or 3.5 ng/mL in those treated on the face (12.5-mg doses), scalp (25-mg doses), or hands/arms (75-mg doses), respectively. Systemic exposure appeared to depend more on the surface area of the application site than on the total applied dose. In patients with external genital and perianal human papillomavirus (HPV) warts who received topical imiquimod 5% cream (average dose 4.6 mg), mean peak serum concentrations were 0.4 ng/mL.

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Biological Half-Life
20 hours (topical dose), 2 hours (subcutaneous dose)
Studies using subcutaneous imiquimod indicate the drug has an apparent half-life of 2 hours. Following topical application, imiquimod appears to be retained in the skin for prolonged periods since the half-life is approximately 10 times greater than that reported following subcutaneous administration.

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the use of imiquimod during breastfeeding. However, because the drug is used on a limited surface area and poorly absorbed into the maternal circulation, effects on the infant are unlikely. If imiquimod is required by the mother, it is not a reason to discontinue breastfeeding. Do not apply imiquimod to the breast or nipple and ensure that the infant's skin does not come into direct contact with the areas of skin that have been treated.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

[1]. Imiquimod - A toll like receptor 7 agonist - Is an ideal option for management of COVID 19. Environ Res. 2020 Sep; 188: 109858.

[2]. Imiquimod: a review. J Cutan Med Surg. Nov-Dec 2002;6(6):554-60.

[3]. Imiquimod suppresses propagation of herpes simplex virus 1 by upregulation of cystatin A via the adenosine receptor A1 pathway. J Virol. 2012 Oct;86(19):10338-46.

[4]. The small antitumoral immune response modifier imiquimod interacts with adenosine receptor signaling in a TLR7- and TLR8-independent fashion. J Invest Dermatol. 2006 Jun;126(6):1338-47.

[5]. Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis. J Immunol. 2009, 182, 9.

[6]. Molecular and histopathological profiling of imiquimod induced dermatosis in Swiss Wistar rats: contribution to the rat model for novel anti-psoriasis treatments. Mol Biol Rep. 2021, 48, 5.

[7]. Development and Optimization of Imiquimod-Loaded Nanostructured Lipid Carriers Using a Hybrid Design of Experiments Approach. Int J Nanomedicine. 2023.

Therapeutic Uses
Adjuvants, Immunologic; Antineoplastic Agents
Imiquimod is used topically for the treatment of clinically typical, nonhyperkeratotic, nonhypertrophic actinic keratosis on the face or scalp in immunocompetent adults; treatment of biopsy-confirmed, primary superficial basal cell carcinoma in immunocompetent adults; and treatment of external genital and perianal exophytic warts (condylomata acuminata) caused by human papillomavirus (HPV). /Included in US product label/
Topical imiquimod has been effective when used in a limited number of adults and children for the treatment of molluscum contagiosum. /NOT included in US product label/
Imiquimod 5% cream has been used for the topical treatment of external genital and perianal HPV warts in a limited number of adults with human immunodeficiency virus (HIV) infection; however, the response rate appears to be lower in these individuals than in those who are not HIV infected. /NOT included in US product label/
For more Therapeutic Uses (Complete) data for Imiquimod (6 total), please visit the HSDB record page.

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Drug Warnings
Adverse local reactions, including erythema, erosion, excoriation/flaking, and edema, commonly occur at the site of application of imiquimod and/or surrounding areas. These reactions usually are mild to moderate in severity; however, severe local reactions have been reported.
In controlled studies in adults with actinic keratosis, the most frequently reported local skin reactions in those receiving imiquimod 5% cream (twice weekly for 16 weeks) were erythema (97%), flaking/scaling/dryness (93%), scabbing/crusting (79%), edema (49%), erosion/ulceration (48%), weeping/exudate (22%), and vesicles (9%).1 Application site reactions (e.g., bleeding, burning, induration, irritation, pain, pruritus, stinging, tenderness) occurred in 33% of those receiving topical imiquimod compared with 14% of those receiving placebo. In these studies, 16% of patients discontinued imiquimod treatment because of local or application site reactions and 91% of these were able to resume treatment after a rest period.
Adverse dermatologic reactions at sites away from the site of application have been reported in some patients receiving topical imiquimod. Remote site reactions have included bleeding, burning, edema, erosion, erythema, excoriation/flaking, induration, pain, pruritus, tenderness, tinea cruris, and ulceration.
When imiquimod 5% cream was used in controlled studies in patients with genital and perianal HPV warts (3 times weekly for up to 16 weeks), erythema occurred in 58-65%, erosion in 30-31%, excoriation/flaking in 18-26%, edema in 12-18%, scabbing in 4-13%, induration in 5-7%, ulceration in 4-8%, and vesicles in 2-3% of those receiving the drug.1 In addition, application site reactions in those receiving the drug included pruritus (22-32%), burning (9-26%), pain (2-8%), and soreness (0-3%). In addition, fungal infections occurred in 2-11% of patients receiving the drug. Overall, 1.2% of patients in these studies discontinued treatment because of local or application site reactions.
For more Drug Warnings (Complete) data for Imiquimod (25 total), please visit the HSDB record page.

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Pharmacodynamics
Imiquimod is an immune response modifier that acts as a toll-like receptor 7 agonist. Imiquimod is commonly used topically to treat warts on the skin of the genital and anal areas. Imiquimod does not cure warts, and new warts may appear during treatment. Imiquimod does not fight the viruses that cause warts directly, however, it does help to relieve and control wart production. It is not used on warts inside the vagina, penis, or rectum. Imiquimod is also used to treat a skin condition of the face and scalp called actinic keratoses. Imiquimod can also be used to treat certain types of skin cancer called superficial basal cell carcinoma. Imiquimod is particularly useful on areas where surgery or other treatments may be difficult, complicated or otherwise undesirable, especially the face and lower legs.

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Imiquimod is an imidazoquinoline fused [4,5-c] carrying isobutyl and amino substituents at N-1 and C-4 respectively. A prescription medication, it acts as an immune response modifier and is used to treat genital warts, superficial basal cell carcinoma, and actinic keratosis. It has a role as an antineoplastic agent and an interferon inducer.

Imiquimod is a prescription medicine approved by the U.S. Food and Drug Administration (FDA). It is a cream for topical use only. Imiquimod is FDA-approved for the treatment of certain skin conditions, including:

Actinic keratosis (a skin condition that may develop into skin cancer)
External genital warts (warts on the outside of the genitals) and perianal warts (warts around the outside of the anus)
External genital and perianal warts are caused by the human papillomavirus (HPV). HPV can be an opportunistic infection (OI) of HIV.

Imiquimod is an immune response modifier that acts as a toll-like receptor 7 agonist. Imiquimod is commonly used topically to treat warts on the skin of the genital and anal areas. Imiquimod does not cure warts, and new warts may appear during treatment. Imiquimod does not fight the viruses that cause warts directly, however, it does help to relieve and control wart production. Miquimod is also used to treat a skin condition of the face and scalp called actinic keratoses and certain types of skin cancer called superficial basal cell carcinoma.

The mechanism of action of imiquimod is as an Interferon Inducer. The physiologic effect of imiquimod is by means of Increased Cytokine Activity, and Increased Cytokine Production.

Imiquimod is a synthetic agent with immune response modifying activity. As an immune response modifier (IRM), imiquimod stimulates cytokine production, especially interferon production, and exhibits antitumor activity, particularly against cutaneous cancers. Imiquimod's proapoptotic activity appears to be related to Bcl-2 overexpression in susceptible tumor cells. (NCI04)

IMIQUIMOD is a small molecule drug with a maximum clinical trial phase of IV (across all indications) that was first approved in 1997 and has 6 approved and 47 investigational indications. This drug has a black box warning from the FDA.

A topically-applied aminoquinoline immune modulator that induces interferon production. It is used in the treatment of external genital and perianal warts, superficial CARCINOMA, BASAL CELL; and ACTINIC KERATOSIS.

C18H20N4O4

356.375803947449

356.148

896106-16-4

Imiquimod;99011-02-6;Imiquimod hydrochloride;99011-78-6

11595577

White to off-white solid powder

3

7

4

26

413

0

CC(C)CN1C=NC2=C1C3=CC=CC=C3N=C2N.C(=C\C(=O)O)\C(=O)O

KYQFLNAOJRNEDV-BTJKTKAUSA-N

InChI=1S/C14H16N4.C4H4O4/c1-9(2)7-18-8-16-12-13(18)10-5-3-4-6-11(10)17-14(12)15;5-3(6)1-2-4(7)8/h3-6,8-9H,7H2,1-2H3,(H2,15,17);1-2H,(H,5,6)(H,7,8)/b;2-1-

(Z)-but-2-enedioic acid;1-(2-methylpropyl)imidazo[4,5-c]quinolin-4-amine

Imiquimod maleate; 896106-16-4; Imiquimod (maleate); (Z)-but-2-enedioic acid;1-(2-methylpropyl)imidazo[4,5-c]quinolin-4-amine; R 837 maleate; Imiquimodmaleate; SCHEMBL3188539;

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)

DMSO : ~25 mg/mL (~70.15 mM)

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.)