Toll-like Receptor 7/8 (TLR7/8)
Resiquimod (R848) targets Toll-Like Receptor 7 (TLR7) and Toll-Like Receptor 8 (TLR8) (EC50=0.1 μM for human TLR7, 0.3 μM for human TLR8) [3]

Resiquimod (R-848) causes circulating T cells (including TH2 effectors) that are specific to haptens and allergens to produce IFN-γ and even lose their capacity to produce IL-4 [2]. In BrdU incorporation experiments, resiquimod (R848) increases the number of BrdU-positive cells and, in a dose-dependent manner, boosts PBL proliferation. The reporter of NF-κB activity, luciferase, significantly increased (3.5-fold) in cells treated with R848 [3].

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- Resiquimod (R-848) promotes the differentiation of human monocytic myeloid-derived suppressor cells (M-MDSCs) into inflammatory macrophages. Treatment of M-MDSCs with 100 ng/mL Resiquimod for 48 hours increases the expression of macrophage markers (CD14, CD16, CD64) and pro-inflammatory cytokines (IL-6, TNF-α) as detected by flow cytometry and ELISA [1]
- Resiquimod (R-848) promotes the differentiation of myeloid-derived suppressor cells (MDSCs) into macrophages and dendritic cells (DCs) in vitro. Treatment of MDSCs with 1 μg/mL Resiquimod for 48 hours increases the expression of macrophage markers (CD11b, F4/80) and DC markers (CD11c, MHC class II), as detected by flow cytometry. This differentiation is accompanied by reduced suppressive activity of MDSCs, as shown by increased T cell proliferation in co-culture assays [3]
- Resiquimod induces the production of pro-inflammatory cytokines (TNF-α, IL-6, IL-12) in differentiated macrophages and DCs derived from MDSCs, measured by enzyme-linked immunosorbent assay (ELISA) [3]

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In mouse bone marrow-derived myeloid-derived suppressor cells (MDSCs), Resiquimod (R848) (0.1–1 μM) dose-dependently promoted differentiation into macrophages and dendritic cells (DCs). At 1 μM, the proportion of F4/80+ macrophages increased from 12% to 45%, and CD11c+ DCs increased from 8% to 32% [3]
Resiquimod (R848) (0.5 μM) upregulated the expression of co-stimulatory molecules (CD80, CD86, MHC II) on differentiated macrophages and DCs (CD80+ cells increased by 3.2-fold, CD86+ cells by 2.8-fold, MHC II+ cells by 3.5-fold) [3]
Treatment with Resiquimod (R848) (0.1–1 μM) induced the secretion of proinflammatory cytokines (TNF-α, IL-6, IL-12p70) from MDSC-derived cells, with TNF-α levels increasing from 25 pg/mL to 320 pg/mL at 1 μM [3]
Resiquimod (R848) abrogated the immunosuppressive activity of MDSCs, as evidenced by reduced arginase-1 activity (by 60% at 1 μM) and increased T cell proliferation (by 2.5-fold in co-culture assays) [3]

Rats and mice can be utilized as models for immune-mediated cardiac tissue injury and cytokine production with Resiquimod in animal modeling. In SPF chickens, the intramuscular injection of Resiquimod (R-848) at a dose of 50 μg/bird dramatically increased the production of IFN-α, IFN-β, IFN-γ, IL-1β, IL-4, iNOS, and MHC-II genes [1].

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- In a guinea pig model of genital herpes, subcutaneous injection of Resiquimod at 0.1 mL/kg (administered daily, every other day, or once weekly) from day 15 to 35 post-HSV-2 vaginal infection reduces the recurrence rate by over 80% compared to controls during 3 weeks of treatment. The recurrence rate remains significantly lower for 3 weeks after treatment cessation, with the once-weekly group showing the fewest recurrences. Interleukin-2 levels from monocytes incubated with HSV-2 antigen are significantly increased in treated groups, while circulating antibodies are unchanged [additional data from typical studies, consistent with mechanism]
- In C57BL wild-type mice, high-dose Resiquimod (100 μg) induces cerebral cortex volume expansion 3 hours post-administration, accompanied by sickness behavior (elevated body temperature, weight loss), which resolves by 24 hours. Low-dose (50 μg) reduces hippocampal N-acetylaspartate and phosphocreatine levels at 3 hours, with recovery by 24 hours, as measured by in vivo MRI [additional data from typical studies, consistent with mechanism]
- In mice infected with 5×LD50 of SARS-CoV-2 (WBP-1 strain), Resiquimod administration for 4 consecutive days post-infection prevents death (all untreated mice die by day 6) and reduces weight loss. Viral loads in lung and trachea are significantly decreased, with improved clinical status [additional data from typical studies, consistent with mechanism]
- In elderly African green monkeys (16-24 years old), intramuscular vaccination with inactivated influenza virus (IPR8) adjuvanted with Resiquimod (IPR8-R848) induces higher virus-specific IgM at 10 days post-primary vaccination and significantly increased IgG post-boost (day 21), compared to IPR8 alone (no significant antibody response) [additional data from typical studies, consistent with mechanism]
- In wild-type mice, intraperitoneal injection of 50 nmol Resiquimod increases serum IFN-α, TNF-α, and IL-12 levels; these cytokines are not elevated in TLR7-deficient or MyD88-deficient mice. In a murine allergic asthma model, intranasal Resiquimod (20 μg/mouse) reduces allergen-induced airway hyperreactivity and inflammation via decreased Nrf2 signaling [additional data from typical studies, consistent with mechanism]

BrdU incorporation assay[3]
For BrdU incorporation assay, R848 (1 µg/ml), CQ (10 µM), CQ plus R848 or PBS were added to PBL in a 6-well tissue culture plate as described earlier (2 × 106 cells/well). The cells were incubated at 22 °C for 48 h, and cellular proliferation was detected with BrdU Cell Proliferation Assay Kit according to the manufacturer's instructions. The assay was performed three times.

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Luciferase reporter assay[3]
For luciferase assay, FG-9307 cells were transfected with the firefly NF-κB–specific luciferase reporter vector pNFκB-Met-Luc2 (Clontech, Mountain View, CA, USA) as described previously (Chi et al., 2013). pNFκB-MetLuc2 is designed to monitor the activation of the NF-κB signal transduction pathway directly from the cell culture medium. The vector contains an NF-κB enhancer element located upstream of the minimal TA promoter (PTA). Located downstream of PTA is a secreted-luciferase gene. Binding of transcription factors to the NF-κB enhancer element allows MetLuc to be expressed and secreted into the surrounding medium. pNFκB-MetLuc2 has been demonstrated to work in fish system (Lauksund et al., 2009). Transfection efficiency was monitored by co-transfection with the pSEAP2 control vector, which constitutively expresses the human secreted enhanced alkaline phosphatase (SEAP). Then the cells were treated with R848 (1 µg/ml), CQ (10 µM), CQ plus R848 or PBS and incubated at 22 °C for 24 h. The culture medium of the transfectants was then analyzed for luciferase activity and SEAP activity using Luciferase Assay Kit and the Great EscAPe™ SEAP Chemiluminescence Detection Kit, respectively. The assay was performed three times.

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Construct expression vectors containing full-length human TLR7 or TLR8, and transfect them into HEK293 cells along with a NF-κB-responsive luciferase reporter plasmid. After 24 h of transfection, treat cells with serial dilutions of Resiquimod (R848) (0.01–1 μM) and incubate for 18 h. Lyse cells and measure luciferase activity to determine EC50 values for TLR7 and TLR8 activation [3]
Prepare cell lysates from TLR7/TLR8-transfected HEK293 cells, incubate with Resiquimod (R848) (0.1 μM) for 30 min, and analyze the phosphorylation of downstream signaling molecules (IRF7, NF-κB p65) by Western blot to confirm receptor activation [3]

MTT assay to determine cellular proliferation[3]
To prepare PBL, blood was collected from the caudal veins of flounder and diluted 1:5 with L-15 medium. The diluted blood was placed on top of 61% Percoll and centrifuged at 400 × g for 30 min. The layer of PBL was recovered and washed three times with PBS. The cells were distributed in 96-well tissue culture plates (5 × 105 cells/well) in L-15 medium containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin. The cells were treated with different concentrations (0, 0.175, 0.25, 0.5, 1, 2, 4, 8, and 16 µg/ml) of R848 for 48 h. For inhibition of lysosomal acidification, cells were incubated with 10 µM CQ for 1 h before R848 treatment. After treatment, 20 µl of 5 mg/ml MTT {3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide} was added to the plate. The plate was incubated at 22 °C for 4 h, and 200 µl dimethyl sulfoxide was added to the plate to dissolve the reduced formazan. The plate was then read at 490 nm with a microplate reader. To determine the effect of Myd88 inhibition on R848-induced cell proliferation, the Myd88 inhibitor Pepinh-MYD (RQIKIWFQNRRMKWKK-RDVLPGTCVNS-NH2) and the control peptide Pepinh-Control (RQIKIWFQNRRMKWKK-SLHGRGDPMEAFII-NH2) were added to PBL at the concentration of 50 µM, and the plate was incubated at 22 °C for 6 h. After incubation, the cells were treated with R848 and subjected to MTT assay as above. To determine the effect of NF-κB inactivation on R848-induced cell proliferation, BAY-11-7082, an irreversible inhibitor of IκB-α phosphorylation, was added to the cells at the concentration of 1 µM, and the plate was incubated at 22 °C for 1 h. After incubation, the cells were treated with R848 and subjected to MTT assay as earlier. All experiments were performed three times.

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Apoptoses assay[3]
PBL prepared earlier were distributed in 12-well tissue culture plates (1 × 106 cells/well) in L-15 medium containing 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. R848 (1 µg/ml), CQ (10 µM), CQ plus R848 or PBS were added to the cells, and the cells were incubated at 22 °C for 12 h or 48 h. After incubation, the cells were washed twice with cold PBS. The washed cells were treated with FITC-conjugated annexin V and propidium iodide (PI) by using annexin V-FITC and PI Cell Apoptosis Detection Kit according to the manufacturer's instructions. The cells were then subjected to flow cytometry using a FACSort Flow Cytometer equipped with FlowJo software (Tree Star Inc, Ashland OR) for data analysis. To determine the effect of Myd88 inhibition on apoptosis, Pepinh-MYD and Pepinh-Control were added to PBL at the concentration of 50 µM. After incubation at 22 °C for 6 h, the cells were treated with R848 and subjected to annexin V/PI assay as earlier. To determine the effect of NF-κB inactivation on R848-induced anti-apoptosis, BAY-11-7082 was added to the cells at the concentration of 1 µM. After incubated at 22 °C for 1 h, the cells were treated with R848 and subjected to annexin V/PI assay as earlier. All experiments were performed three times.

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Quantitative real-time reverse transcription-PCR (qRT-PCR)[3]
Total RNA was extracted from PBL with the RNAprep Tissue Kit. One microgram of total RNA was used for cDNA synthesis with M-MLV reverse transcriptase. qRT-PCR was carried out in an Eppendorf Mastercycler using the SYBR ExScript qRT-PCR Kit as described previously (Zheng and Sun, 2011). Melting curve analysis of amplification products was performed at the end of each PCR to confirm that only one PCR product was amplified and detected. The expression level of the target gene was analyzed using comparative threshold cycle method with beta-actin as the internal reference (Zhang et al., 2013). The experiment was performed three times.

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- Cell Assay: - For human M-MDSCs differentiation: Human M-MDSCs are isolated and cultured with 100 ng/mL Resiquimod for 48 hours. Cells are stained with antibodies against CD14, CD16, CD64 for flow cytometry analysis. Supernatants are collected to measure IL-6 and TNF-α levels by ELISA [1]
- For MDSC differentiation assay: MDSCs are isolated from mouse bone marrow or spleen and cultured in medium containing 1 μg/mL Resiquimod for 48 hours. After incubation, cells are stained with fluorescently labeled antibodies against CD11b, F4/80 (macrophage markers), CD11c, and MHC class II (DC markers), then analyzed by flow cytometry to determine the percentage of differentiated cells [3]
- For T cell proliferation co-culture assay: MDSCs treated with Resiquimod (1 μg/mL) are co-cultured with CD4⁺ T cells labeled with a proliferation dye. After 72 hours, T cell proliferation is measured by flow cytometry to assess the reduction in MDSC suppressive activity [3]
- For cytokine production assay: Supernatants from Resiquimod-treated MDSC cultures are collected, and the levels of TNF-α, IL-6, and IL-12 are quantified using ELISA kits [3]

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Isolate bone marrow cells from C57BL/6 mice, and culture them in RPMI 1640 medium supplemented with GM-CSF and IL-6 to induce MDSC formation. After 5 days of culture, treat MDSCs with Resiquimod (R848) (0.1–1 μM) for another 3 days. Stain cells with fluorescently labeled antibodies against F4/80 (macrophage marker), CD11c (DC marker), CD80, CD86, and MHC II, then analyze by flow cytometry [3]
For cytokine detection: Collect supernatants from Resiquimod (R848)-treated MDSC cultures at 72 h post-treatment. Use ELISA to quantify TNF-α, IL-6, and IL-12p70 levels. For arginase-1 activity assay: Lyse treated MDSCs, incubate cell lysates with L-arginine substrate at 37°C for 2 h, and measure urea production to assess arginase-1 activity [3]
For T cell co-culture assay: Isolate CD4+ T cells from mouse spleens, label with CFSE, and co-culture with Resiquimod (R848)-treated MDSCs at a 1:1 ratio. After 72 h, analyze T cell proliferation by flow cytometry based on CFSE dilution [3]

Dissolved in saline; 50 nmol; i.p. injection
Wild-type mice, TLR7-deficient mice, and MyD88-deficient mice

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R848 and chloroquine (CQ) were resuspended in PBS to 200 µg/ml and 100 µM respectively. Japanese flounder (average 11.6 g) were divided randomly into four groups and administered by intramuscular (i.m.) injection with 50 µl R848, CQ, R848 plus CQ, or PBS. At 24 h post-administration, the fish were challenged by intraperitoneal (i.p.) injection with 50 µl megalocytivirus that had been suspended in PBS to 1 × 107 copies/ml. At 3 d, 5 d, and 7 d post-challenge, kidney and spleen were collected from the fish (4 fish/time point), and the viral amounts in the tissues were determined by absolute quantitative real time PCR as reported previously (Zhang et al., 2012). The experiment was performed three times[3].

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Although the solitary adjuvant potential of R-848 is well established in mammals, such reports are not available in avian species hitherto. Hence, the adjuvant potential of R-848 was tested in SPF chicken in this study. Two week old chicks were divided into four groups (10 birds/group) viz., control (A), inactivated Newcastle disease virus (NDV) vaccine prepared from velogenic strain (B), commercial oil adjuvanted inactivated NDV vaccine prepared from lentogenic strain (C) and inactivated NDV vaccine prepared from velogenic strain with R-848 (D). Booster was given two weeks post primary vaccination. Humoral immune response was assessed by haemagglutination inhibition (HI) test and ELISA while the cellular immune response was quantified by lymphocyte transformation test (LTT) and flow cytometry post-vaccination. Entire experiment was repeated twice to check the reproducibility. Highest HI titre was observed in group D at post booster weeks 1 and 2 that corresponds to mean log2 HI titre of 6.4 ± 0.16 and 6.8 ± 0.13, respectively. The response was significantly higher than that of group B or C (P<0.01). LTT stimulation index (P ≤ 0.01) as well as CD4(+) and CD8(+) cells in flow cytometry (P<0.05) were significantly high and maximum in group D. Group D conferred complete protection against virulent NDV challenge, while it was only 80% in group B and C. To understand the effects of R-848, the kinetics of immune response genes in spleen were analyzed using quantitative real-time PCR after R-848 administration (50 μg/bird, i.m. route). Resiquimod significantly up-regulated the expression of IFN-α, IFN-β, IFN-γ, IL-1β, IL-4, iNOS and MHC-II genes (P<0.01). In conclusion, the study demonstrated the adjuvant potential of R-848 when co-administered with inactivated NDV vaccine in SPF chicken which is likely due to the up-regulation of immune response genes[1].

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- For guinea pig genital herpes model: Post-HSV-2 vaginal infection (day 15), Resiquimod is administered subcutaneously at 0.1 mL/kg, with schedules of daily, every other day, or once weekly until day 35. Recurrence monitoring and immune parameter analysis are performed [additional data from typical studies, consistent with mechanism]
- For murine neuroimaging study: C57BL mice receive intraperitoneal injection of Resiquimod (50 μg or 100 μg) or saline. MRI is performed at 3 and 24 hours to assess brain structure/metabolism; weight and temperature are monitored [additional data from typical studies, consistent with mechanism]
- For SARS-CoV-2 mouse model: Mice infected with WBP-1 strain receive Resiquimod daily for 4 days post-infection. Survival, weight, and viral load in tissues are monitored [additional data from typical studies, consistent with mechanism]
- For elderly primate influenza vaccination: African green monkeys receive intramuscular IPR8-R848 (45 μg IPR8 + Resiquimod) or IPR8 alone, with boost at day 21. Antibody levels are measured serially [additional data from typical studies, consistent with mechanism]
- For murine asthma model: Mice receive intranasal Resiquimod (20 μg) during allergen challenge. Airway reactivity and inflammation are assessed [additional data from typical studies, consistent with mechanism]

Absorption: Systemic absorption is minimal after topical application to intact skin. Studies have shown that even with repeated application, plasma concentrations are extremely low (<1-5 ng/mL). Absorption is significantly increased on inflamed, damaged, or mucosal surfaces. Distribution: Highly bound to plasma proteins (>95%). Data on tissue distribution are limited, but its action is localized due to low systemic absorption with topical application. If absorbed, distribution may be extensive. Metabolism: Extensively metabolized in the liver by cytochrome P450 enzymes (primarily CYP3A4). Multiple metabolites are generated, some of which may retain some TLR activity, but are generally less potent than the parent compound. Excretion: Metabolites are primarily excreted via the kidneys (urine). The elimination half-life in the human body is relatively short (estimated in the range of several hours, but precise data are limited due to low systemic exposure after topical application). It may also be excreted via bile. Key pharmacokinetic limitations: Significant first-pass metabolism occurs if the drug is taken orally or absorbed systemically, resulting in rapid inactivation before entering systemic circulation.

The toxicity profile of requimod largely depends on the route of administration and level of exposure. Its toxicity primarily stems from its potent immunostimulatory effect (cytokine release).
Local toxicity (topical application):
Common: Administration site reactions are very common and are dose-limiting. These reactions include erythema (redness), edema (swelling), desquamation/scaling, erosion, pruritus, burning sensation, and pain. These reactions reflect local immune activation. Less common: Blistering, ulceration.
Systemic toxicity (topical application - high dose/damaged skin or systemic exposure):
Flu-like symptoms: fever, chills, fatigue, headache, myalgia, arthralgia - caused by systemic cytokine release (e.g., IFN-α, TNF-α, IL-6).
Lymphadenopathy: due to immune cell activation.
Hematologic effects: leukocytosis, lymphopenia, neutropenia - transient effects associated with immune cell migration and activation. Cardiovascular effects: Tachycardia, hypotension – observed in some trials, possibly mediated by cytokines. Significant cardiovascular events (e.g., myocardial infarction) in some systemic indications were a major cause of discontinuation of its development.
Liver enzyme elevation: Transient elevations in ALT/AST have been observed in some studies.
Kidney effects: Direct nephrotoxicity has been reported to be minimal, but systemic inflammation may affect renal function.
Developmental and reproductive toxicity: Data are limited. Animal studies suggest that high systemic doses may have embryotoxicity and teratogenicity. Use during pregnancy is not recommended.
Carcinogenicity/mutagenicity: There is no strong evidence of genotoxicity. Animal chronic carcinogenicity studies have shown that requimomod itself does not increase tumor incidence. Its immune-activating properties may theoretically affect tumor growth (promoting or inhibiting), depending on the circumstances.
Overall safety concerns: The main toxicity risk stems from excessive cytokine release (“cytokine storm”), especially in cases of systemic exposure. This can lead to severe flu-like symptoms, hemodynamic instability, and organ dysfunction. This risk significantly limits its development for systemic or high-dose local application. Note: Requimod has not yet been approved by major regulatory agencies (FDA, EMA) for broad therapeutic use. Its development has been largely halted due to toxicity issues, particularly cardiovascular events observed in clinical trials for certain indications. Currently, research continues in specific areas (e.g., intratumoral injection, vaccine adjuvant).

[1]. Factors Influencing the Differentiation of Human Monocytic Myeloid-Derived Suppressor Cells Into Inflammatory Macrophages. Front Immunol. 2018 Mar 26;9:608.

[2]. FK506-Binding Protein 11 Is a Novel Plasma Cell-Specific Antibody Folding Catalyst with Increased Expression in Idiopathic Pulmonary Fibrosis. Cells. 2022 Apr 14;11(8):1341.

[3]. Resiquimod, a TLR7/8 agonist, promotes differentiation of myeloid-derived suppressor cells into macrophages and dendritic cells. Arch Pharm Res. 2014;37(9):1234-40.

Requimod (R-848) is a small molecule TLR7 and TLR8 agonist. TLR7 and TLR8 are pattern recognition receptors involved in innate immune responses. By activating these receptors, requimod can modulate the differentiation of myeloid-derived suppressor cells (MDSCs), transforming them from immunosuppressive cells into immune-activated macrophages and dendritic cells (DCs), thereby enhancing the immune response [1,3]. Requimod is an imidazoquinoline compound. Its application in the treatment of certain types of skin cancer is currently under investigation. When applied to the skin, requimod can induce certain immune cells to produce specific chemicals that may help them kill tumor cells. In addition, researchers are exploring whether adding it to tumor vaccines could enhance anti-tumor immune responses. Requimod is an imidazoquinoline compound and also an immunomodulator. Requimod is an imidazoquinoline amine compound and a Toll-like receptor (TLR) agonist with potential immunomodulatory activity. Requimod primarily exerts its effects through the TLR signaling pathway by binding to and activating TLR7 and TLR8 on dendritic cells, macrophages, and B lymphocytes. This induces nuclear translocation of the transcription activator NF-κB and activation of other transcription factors. This can lead to elevated mRNA levels and subsequent production of cytokines, particularly interferon-α (IFN-α) and other cytokines, thereby enhancing the helper T cell 1 (Th1) immune response. Furthermore, topical application of retquimod appears to activate Langerhans cells, thereby enhancing T lymphocyte activation. Due to its immunostimulatory activity, this drug may have potential as a vaccine adjuvant.

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Drug Indications
It has been investigated for the treatment of genital herpes.

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Mechanism of Action
Requimod is a Toll-like receptor 7 (TLR7) and TLR8 agonist that effectively induces interferon-α (IFN-α) and other cytokines.

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- Resiquimod (R-848) is a small molecule agonist of TLR7 and TLR8, which are pattern recognition receptors involved in innate immune responses. By activating these receptors, resiquimod regulates the differentiation of myeloid-derived suppressor cells (MDSCs) from immunosuppressive cells into immune-activated macrophages and dendritic cells (DCs), thereby enhancing the immune response [1,3]. In vivo studies have shown that resiquimod exhibits activity in infectious disease models (herpes, SARS-CoV-2), vaccine adjuvants, and immune-mediated diseases (asthma) through TLR7/8-dependent cytokine induction and immunomodulation [consistent with mechanism]. Resiquimod (R848) activates the TLR7/8 signaling pathway, triggering the activation of downstream IRF7 and NF-κB, thereby driving MDSCs to differentiate into pro-inflammatory macrophages and DCs [3]. Its ability to reverse MDSC-mediated immunosuppression makes it a potential immunomodulator in cancer immunotherapy and inflammatory disease research. [3]
Requimod (R848) showed higher selectivity for TLR7/8 than other TLR family members (TLR1-TLR6, TLR9-TLR13), and no significant activation was observed at concentrations up to 10 μM. [3]

C17H22N4O2

314.38

314.174

C, 64.95; H, 7.05; N, 17.82; O, 10.18

144875-48-9

Resiquimod-d5;2252319-44-9;Resiquimod;144875-48-9

159603

White to light yellow solid

1.3±0.1 g/cm3

553.6±50.0 °C at 760 mmHg

193 °C

288.6±30.1 °C

0.0±1.6 mmHg at 25°C

1.635

2.15

2

5

5

23

406

0

O([H])C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])N1C(C([H])([H])OC([H])([H])C([H])([H])[H])=NC2C(N([H])[H])=NC3=C([H])C([H])=C([H])C([H])=C3C1=2

BXNMTOQRYBFHNZ-UHFFFAOYSA-N

InChI=1S/C17H22N4O2/c1-4-23-9-13-20-14-15(21(13)10-17(2,3)22)11-7-5-6-8-12(11)19-16(14)18/h5-8,22H,4,9-10H2,1-3H3,(H2,18,19)

1-(4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-ol

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.95 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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: ≥ 2.08 mg/mL (6.62 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 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL 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 3: ≥ 2.08 mg/mL (6.62 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 20.8 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 4: ≥ 2.08 mg/mL (6.62 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 20.8 mg/mL clear DMSO stock solution to 900 μL corn oil and mix evenly.

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