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| Other Sizes |
Purity: ≥98%
| Targets |
TLR7;TLR8;HIV-1
TLR7 (specific agonist when used at <10 μM); no binding affinity (IC50, Ki, EC50) values are reported in the provided studies. TLR8 (agonist at higher concentrations); no affinity data given. |
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| ln Vitro |
Gardiquimod (6–60 μM) dramatically reduces HIV-1 reverse transcriptase's ability to synthesize cDNA[1].
In TLR7/8 agonist study (HCC): Gardiquimod (1 μg/mL, i.e., ~1 μM) directly activated NKL cells and primary NK cells, up-regulated TLR7/8 expression, induced NF-κB phosphorylation and IκB-α degradation, increased IL-6 and IL-1β transcription, and enhanced NK cell cytotoxicity against HepG2, H7402, and PLC/PRF/5 HCC cells. In DC-NK co-cultures, gardiquimod promoted DC maturation (up-regulated CD1a, CD11c, CD83, CD86), increased DC production of Th1-type cytokines (IFN-α, IFN-β, IL-12p35, IL-15, IL-18) and pro-inflammatory cytokines (IL-1β, IL-6) but not IL-10. Gardiquimod plus NK cells most effectively induced these changes. DC-mediated NK activation was dependent on type I IFN (IFNAR) and IL-12, and partially dependent on cell-to-cell contact, with involvement of NKG2D, NKp80, CD40/CD40L, LFA-1/ICAM-1, and chemokines CCL2/CCL4 and their receptors. [1] In HIV-1 study: Gardiquimod (0.6–3 μM) significantly reduced HIV-1 DNA levels in macrophages infected with HIV-1BaL (R5) (p≤0.001). In activated PBMCs, gardiquimod (0.6 μM) inhibited R5 (BaL, CM235), X4 (HC4), and dual-tropic (C7/86) strains. Inhibition was effective when added up to 48 h post-infection, but not at 72 h. Dose-response showed significant inhibition of p24 at ≥0.3 μM. Gardiquimod did not induce apoptosis (annexin V/PI staining) or affect cell metabolism/viability. In cocultures, gardiquimod-treated macrophages (3 μM for 3 days prior to infection) significantly inhibited viral amplification by activated PBMCs added immediately, 24 h, or 48 h after macrophage infection, as measured by HIV-1 DNA and p24. Gardiquimod induced IFN-α mRNA (80-fold at 2 h, 20-fold at 4 h, 35-fold at 6 h) and sustained IFN-α protein secretion for up to 48 h. The MyD88 inhibitory peptide blocked IFN-α transcription and partially reversed the anti-HIV effect. IFN-α receptor blocking antibody also partially reversed the protection. Gardiquimod inhibited HIV-1 reverse transcriptase activity at 6–60 μM in a biochemical assay, more effectively than AZT at certain concentrations. [2] |
| ln Vivo |
The anti-tumor effects of NK cells are enhanced when Dendritic cells (DCs) and Gardiquimod (1 mg/kg per mouse; i.p.; daily for 7 days) are combined[2].
In HCC study: Gardiquimod (1 mg/kg per mouse, intraperitoneal injection) combined with NKL cells (1×10⁸ per mouse) and DCs (1×10⁷ per mouse) was administered on days 7 and 14 in a HepG2 xenograft nude mouse model. The combination therapy significantly suppressed tumor growth, reducing the largest tumor nodule volume from 700±43 mm³ (NKL only) to 289±65 mm³ (NKL+GDQ) and 162±51 mm³ (NKL+GDQ+DC). Tumor weights were 0.503±0.05 g (NKL), 0.299±0.05 g (NKL+GDQ), and 0.209±0.02 g (NKL+GDQ+DC). GDQ or DC alone had minimal effect; the NKL+GDQ+DC group showed the most significant tumor suppression compared to other treatment groups (p<0.05). [1] In HIV-1 study: The paper mentions preliminary studies using humanized mice intravaginally treated with gardiquimod prior to HIV-1 exposure showed significant protection against vaginal transmission, but no detailed experimental protocol or data are provided. [2] |
| Enzyme Assay |
HIV-1 reverse transcriptase inhibition assay: A modified cDNA synthesis reaction was performed using HIV-1 RT as the enzyme source, total RNA from U937 cells as template, and CD45 antisense primer for cDNA synthesis. The reaction contained 1 μg total RNA, 1× first strand buffer, 80 μM dNTP, 0.15 μg HIV-1 RT, RNase inhibitor, 5 mM DTT, and gardiquimod (6–60 μM) or AZT (40–400 μM) in 50 μL. cDNA synthesis was at 25°C for 5 min, 50°C for 45 min, 55°C for 15 min, and 65°C for 15 min. Ten microliters of cDNA was used for real-time PCR with CD45-specific primers. Gardiquimod inhibited RT activity at 6–60 μM, with greater inhibition than AZT at equivalent concentrations. The inhibition was less effective when dNTP was increased to 320 μM, suggesting competitive inhibition. [2]
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| Cell Assay |
Cell culture and treatment: Human HCC cell lines HepG2, H7402, PLC/PRF/5 and NK cell line NKL were cultured in RPMI 1640 with 10% FBS. Primary NK cells were isolated from PBMCs using an NK cell isolation kit (magnetic depletion). Monocyte-derived DCs were differentiated from adherent PBMCs with GM-CSF (200 U/mL) and IL-4 (100 U/mL) for 5–6 days. For NK stimulation, cells were treated with gardiquimod (1 μg/mL) for 48 h; for DC-NK co-cultures, DCs and NKL cells were co-cultured at 1:2 ratio with or without gardiquimod. Flow cytometry assessed surface markers (CD69, CD56, CD1a, CD11c, CD83, CD86) and intracellular granzyme B, perforin, CD107a degranulation. Cytotoxicity was measured by 4-h ⁵¹Cr release assay against HepG2, H7402, PLC/PRF/5. RT-PCR and qRT-PCR measured TLR7, TLR8, IL-6, IL-1β, IFN-α, IFN-β, IL-12p35, IL-15, IL-18, IL-10. Western blot detected phospho-NF-κB, NF-κB, IκB-α. Cytokines in supernatants were measured by ELISA. Transwell assays separated DCs and NK cells to assess cell-contact dependency. Blocking antibodies to IFNAR and IL-12, and recombinant IL-12 were used. [1]
HIV-1 infection assays: Human macrophages were matured from monocytes with GM-CSF for 8 days, treated with gardiquimod (0.6–3 μM) for 3 days prior to infection with HIV-1BaL (200 TCID₅₀), and HIV-1 DNA measured by real-time PCR on day 4. PHA-activated PBMCs were treated with gardiquimod (0.01–10 μM) 1 h prior to or up to 72 h after infection with various HIV-1 strains (BaL, CM235, HC4, C7/86). HIV-1 DNA was quantified by real-time PCR on day 9; p24 in supernatants was measured by ELISA. For cocultures, gardiquimod-treated macrophages were infected, then PHA-activated PBMCs were added immediately or 24/48 h later; HIV-1 DNA and p24 were assessed. IFN-α mRNA was measured by qRT-PCR at 1–48 h post-gardiquimod; IFN-α protein by ELISA. MyD88 inhibitory peptide (10 μg/mL, 4 h pretreatment) and IFN-α receptor blocking antibody (2 μg/mL) were used to block signaling. Metabolic activity was measured by CellTiter 96 AQueous assay; apoptosis by annexin V/PI flow cytometry. [2] |
| Animal Protocol |
Animal Model: Male athymic nude mice (Balb-nu/nu, aged 5 weeks) harboring xenografts of human HepG2 liver carcinoma[2]
Dosage: 1 mg/kg per mouse Administration: i.p.; daily for 7 days Result:significantly inhibited the growth of xenografts of human HepG2 liver carcinoma. In HCC study (in vivo): Balb/c nude mice (5 weeks old) were injected intraperitoneally with 5×10⁶ HepG2 cells. On days 7 and 14, mice received intraperitoneal injections of NKL cells (1×10⁸ per mouse), DCs (1×10⁷ per mouse), gardiquimod (1 mg/kg per mouse), or combinations thereof. Mice were sacrificed at day 28; tumor nodules in hepatic portal, splenic portal, and mesenteric vasculature were separated; tumor volume calculated as length × width²/2. The NKL+GDQ+DC group showed the most significant tumor inhibition. [1] HIV-1 study: No detailed animal protocol is provided; only a brief mention of preliminary studies in humanized mice with intravaginal gardiquimod application prior to HIV-1 exposure (data not shown). [2] |
| Toxicity/Toxicokinetics |
In the HIV-1 study, Gardiquimod did not induce apoptosis in activated PBMCs as measured by annexin V and propidium iodide staining (10.3% annexin V vs 10.7% in untreated, p=NS) and did not affect cell metabolic activity, viability, or cell numbers throughout culture. TNF-α (100 ng/mL) served as positive control for apoptosis (16.3% annexin V, p≤0.0002). No significant toxicity was reported in macrophages. [2]
In the HCC study, no explicit toxicity data are provided; however, the in vivo experiment showed that gardiquimod (1 mg/kg i.p.) combined with NK and DC cells was tolerated in mice with no reported adverse effects. [1] |
| References | |
| Additional Infomation |
Gardiquimod is an imidazoquinoline compound that activates TLR7 (and TLR8 at higher concentrations). It functions as both an immune system modifier and a direct antiviral agent. In HCC immunotherapy, gardiquimod promotes NK-DC cross-talk, enhancing NK cell activation and anti-tumor cytotoxicity against HCC cells via type I IFN and IL-12 pathways, and involving cell-cell contact and co-stimulatory molecules (NKG2D, NKp80, CD40/CD40L, LFA-1/ICAM-1). The combination of gardiquimod with NK cells and DCs significantly suppressed HepG2 xenograft growth in nude mice, suggesting a potential immunotherapeutic approach for HCC. [1]
In HIV-1 infection, gardiquimod inhibits HIV-1 replication in macrophages and activated T cells via two mechanisms: (1) induction of IFN-α and other antiviral cytokines through TLR7-MyD88 signaling, and (2) direct inhibition of HIV-1 reverse transcriptase activity (possibly as a nucleoside analog competitor). It is effective against R5, X4, and dual-tropic strains, and prevents viral amplification from macrophages to T cells in cocultures. Gardiquimod shows promise as a potential microbicide for preventing HIV-1 transmission, with preliminary humanized mouse studies suggesting protection against vaginal transmission. No FDA approval or warnings are mentioned. [2] |
| Molecular Formula |
C21H25F6N5O5
|
|---|---|
| Molecular Weight |
541.44
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| Exact Mass |
313.19
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| Elemental Analysis |
C, 65.15; H, 7.40; N, 22.35; O, 5.10
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| CAS # |
1159840-61-5
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| Related CAS # |
Gardiquimod diTFA;1159840-61-5
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| PubChem CID |
44592366
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| Appearance |
White to off-white solid powder
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| LogP |
3.019
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
23
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| Complexity |
404
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C(F)(F)(F)C(=O)O.C(N1C(=NC2C(N)=NC3=CC=CC=C3C1=2)CNCC)C(O)(C)C
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| InChi Key |
FHJATBIERQTCTN-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C17H23N5O/c1-4-19-9-13-21-14-15(22(13)10-17(2,3)23)11-7-5-6-8-12(11)20-16(14)18/h5-8,19,23H,4,9-10H2,1-3H3,(H2,18,20)
|
| Chemical Name |
1-[4-Amino-2-(ethylaminomethyl)imidazo[4,5-c]quinolin-1-yl]-2-methylpropan-2-ol
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| HS Tariff Code |
2934.99.03.00
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMF :≥ 20 mg/mL (~63.82 mM)
DMSO : ≥ 20 mg/mL (~63.82 mM) Ethanol :≥ 12 mg/mL (~38.29 mM) |
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| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.8469 mL | 9.2346 mL | 18.4693 mL | |
| 5 mM | 0.3694 mL | 1.8469 mL | 3.6939 mL | |
| 10 mM | 0.1847 mL | 0.9235 mL | 1.8469 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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