Toll like receptor 7 (TLR7); HSV-1

Iquimod hydrochloride increases NK cell activity, activates macrophage cytokines and NO, and causes B ischemia and mortality in animal models of innate immune response stimulation. Through the induction, synthesis, and release of cellular cytokines such as interferon-α (IFN-α), interleukin (IL)-6, and tumor factor (TNF)-α, imiquimod hydrochloride promotes 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
Good Skin Absorption (Cream Formulation) In adult patients with actinic keratosis, following topical application (75 mg three times a week for 16 weeks), 0.08–0.15% of the dose is excreted unchanged in the urine as its metabolites. In patients with HPV warts, following topical application, 0.11% and 2.41% of the dose, respectively, are excreted unchanged in the urine in men and women. Imiquimod is systemically absorbed after topical application. In adult patients with actinic keratosis treated with 5% topical imiquimod cream for 16 weeks, the mean peak serum concentration at the end of week 16 was approximately 0.1, 0.2, and 3.5 ng/mL in patients treated on the face (12.5 mg), scalp (25 mg), and hands/arms (75 mg), respectively. Systemic exposure appears to depend more on the surface area of the application site than on the total dose. In patients with genital and perianal human papillomavirus (HPV) warts treated with topical application of 5% imiquimod cream (mean dose 4.6 mg), the mean peak serum concentration was 0.4 ng/mL.

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Biological half-life
20 hours (topical application), 2 hours (subcutaneous application)
Studies with subcutaneous imiquimod showed an apparent half-life of 2 hours. Following topical application, imiquimod appears to persist for an extended period in the skin, as its half-life is approximately 10 times longer than that reported after subcutaneous administration.

Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation
There is currently no information regarding the use of imiquimod during lactation. However, due to the limited surface area of the skin on which this drug acts and poor absorption in the maternal circulation, it is unlikely to have any effect on the infant. If the mother needs to use imiquimod, she should not stop breastfeeding. Do not apply imiquimod to the breasts or nipples, and ensure that the infant's skin does not come into direct contact with the area where the drug has been applied.
◉ Effects on Breastfed Infants
As of the revision date, no relevant published information was found.
◉ Effects on Lactation and Breast Milk
As of the revision date, no relevant published information was found.

[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
Immunoadjuvant; Antineoplastic drug. Imiquimod is used topically to treat clinically typical, non-hyperkeratotic, non-hypertrophic actinic keratosis on the face or scalp of immunocompetent adults; to treat primary superficial basal cell carcinoma of immunocompetent adults confirmed by biopsy; and to treat external genital and perianal warts (condyloma acuminata) caused by human papillomavirus (HPV). /Included on US product label/ Topical application of imiquimod is effective in treating molluscum contagiosum in a small number of adults and children. /Not included on US product label/ 5% imiquimod cream has been used to treat external genital and perianal HPV warts in a small number of adults infected with human immunodeficiency virus (HIV); however, the treatment response rate in these patients appears to be lower than in HIV-free patients. /Not included on US product label/ For more complete data on the therapeutic uses of imiquimod (of 6 types), please visit the HSDB record page.

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Drug Warning
Adverse local reactions, including erythema, erosion, abrasion/scaling, and edema, are commonly reported at the application site and/or surrounding area of imiquimod. These reactions are usually mild to moderate; however, serious local reactions have been reported.
In controlled studies of adults with actinic keratosis, the most frequently reported local skin reactions in patients treated with imiquimod 5% cream (twice weekly for 16 weeks) were erythema (97%), desquamation/scaling/dryness (93%), crusting/scabs (79%), edema (49%), erosion/ulceration (48%), exudation/effusion (22%), and vesicles (9%).1In patients receiving topical imiquimod, 33% experienced application site reactions (e.g., bleeding, burning, induration, irritation, pain, itching, stinging, tenderness), compared to 14% in patients receiving placebo. In these studies, 16% of patients discontinued imiquimod treatment due to local or application site reactions, and 91% of these patients were able to resume treatment after a period of rest. Some patients receiving topical imiquimod treatment reported adverse skin reactions at sites distant from the application site. Distant site reactions included bleeding, burning sensation, edema, erosion, erythema, abrasions/desquamation, induration, pain, itching, tenderness, tinea cruris, and ulceration. In a controlled study of patients with genital and perianal HPV warts, after using 5% imiquimod cream (3 times a week for up to 16 weeks), 58-65% of patients receiving this treatment developed erythema, 30-31% developed erosion, 18-26% developed abrasions/desquamation, 12-18% developed edema, 4-13% developed crusting, 5-7% developed induration, 4-8% developed ulceration, and 2-3% developed vesicles. ¹In addition, patients receiving this drug experienced application site reactions, including itching (22-32%), burning sensation (9-26%), pain (2-8%), and soreness (0-3%). Furthermore, the incidence of fungal infection in patients receiving this drug ranged from 2% to 11%. Overall, 1.2% of patients in these studies discontinued treatment due to local or application site reactions.
For more complete data on drug warnings for imiquimod (25 of them), please visit the HSDB record page.

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Pharmacodynamics
Imiquimod is an immunomodulator 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 directly combat the virus that causes warts, but it helps to alleviate and control the formation of warts. It is not intended to treat warts in the vagina, penis, or rectum. Imiquimod is also used to treat a facial and scalp skin condition called actinic keratosis. Imiquimod can also be used to treat certain types of skin cancer, such as superficial basal cell carcinoma. Imiquimod is particularly useful for areas where surgery or other treatments may be difficult, complex, or unsuitable, especially the face and lower legs. Imiquimod is an imidazoquinoline fused [4,5-c] derivative with isobutyl and amino substituents at the N-1 and C-4 positions, respectively. As a prescription drug, it is used as an immunomodulator to treat genital warts, superficial basal cell carcinoma, and actinic keratosis. It has antitumor activity and can induce interferon production. Imiquimod is a prescription drug approved by the U.S. Food and Drug Administration (FDA) for topical use only. Imiquimod has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of certain skin conditions, including:
Acute keratosis (a skin condition that can develop into skin cancer)
Gentle warts (warts on the outside of the genitals) and perianal warts (warts around the anus).
Gentle warts and perianal warts are caused by human papillomavirus (HPV). HPV may be an opportunistic infection (OI) of HIV.
Imiquimod is an immunomodulator that acts as a Toll-like receptor 7 agonist. Imiquimod is typically applied 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 directly combat the virus that causes warts, but it helps to alleviate and control the growth of warts. Imiquimod is also used to treat a skin condition of the face and scalp called actinic keratosis, and certain types of skin cancer called superficial basal cell carcinoma.
Imiquimod's mechanism of action is as an interferon inducer. Imiquimod's physiological effects are achieved by increasing cytokine activity and production. Imiquimod is a synthetic drug with immunomodulatory activity. As an immunomodulator (IRM), imiquimod can stimulate the production of cytokines (especially interferon) and exhibits antitumor activity, particularly against skin cancer. Imiquimod's pro-apoptotic activity appears to be related to the overexpression of Bcl-2 in susceptible tumor cells. (NCI04) Imiquimod is a small molecule drug, with its highest clinical trial stage being Phase IV (covering all indications). It was first approved in 1997 and currently has 6 approved indications and 47 investigational indications. This drug carries a black box warning from the U.S. Food and Drug Administration (FDA). Imiquimod is a topically applied aminoquinoline immunomodulator that induces interferon production. It is used to treat genital warts and perianal warts, superficial basal cell carcinoma, and actinic keratosis.

C14H17CLN4

276.7646

276.114

99011-78-6

Imiquimod;99011-02-6;Imiquimod maleate;896106-16-4

13982876

White to off-white solid powder

4.205

2

3

2

19

294

0

RGKLRAHQVIHCCH-UHFFFAOYSA-N

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

1-(2-methylpropyl)imidazo[4,5-c]quinolin-4-amine;hydrochloride

Imiquimod hydrochloride; 99011-78-6; Imiquimod (hydrochloride); 1-(2-methylpropyl)imidazo[4,5-c]quinolin-4-amine;hydrochloride; 1-Isobutyl-1H-imidazo[4,5-c]quinolin-4-amine hydrochloride; 1H-Imidazo[4,5-c]quinolin-4-amine, 1-(2-methylpropyl)-,monohydrochloride; Imiquimod HCl; R 837 hydrochloride;

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)

MEthanol : ~24 mg/mL (~86.72 mM)
DMSO : ~8 mg/mL (~28.91 mM )
H2O : ~4.8 mg/mL (~17.34 mM)
Ethanol : ~3.85 mg/mL (~13.91 mM)

Solubility in Formulation 1: ≥ 0.8 mg/mL (2.89 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 8.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 0.8 mg/mL (2.89 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 8.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 0.8 mg/mL (2.89 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 8.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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