Dengue virus (DENV) replication complex [1]
The antiviral mechanism involves modulation of host innate immune response genes including RIG-I, IFNα, RNase L, and PKR [1]
Not specified in the context of testicular toxicity; the mechanism is proposed to involve generation of free radicals leading to oxidative stress [2]
Not specified in the context of skin carcinogenesis; proposed mechanisms include inhibition of DMBA metabolism, down-regulation of ROS production, COX-2 inhibition, topoisomerase inhibition, and induction of interferon-α [3]

In the human liver cancer HepG2 cell line, quinine sulfate hydrate (150 μM, 30 minutes) suppresses the growth and cytostatic effects of dengue virus [1]. In the human liver cancer HepG2 cell line, quinine sulfate hydrate (37.5-150 μM, 24 hours) dramatically lowers viral DENV RNA and protein levels in a dose-dependent manner [1].

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In HepG2 cells infected with DENV-2 (MOI 1), treatment with Quinine sulfate dihydrate at 150 μM resulted in virus production being reduced to 19% of the untreated control, as measured by focus-forming unit (FFU) assay. At this concentration, cell viability remained above 80%. The EC50 for quinine against DENV-2 was 102.7 μM, and the CC50 was 322.2 μM, resulting in a therapeutic index (TI) of 3.137 [1].
Quinine sulfate dihydrate did not affect the adsorption of DENV-2 to the HepG2 cell surface nor the virus's internalization/entry into the cells [1].
Quinine sulfate dihydrate treatment significantly reduced viral RNA levels in a dose-dependent manner (37.5, 75, and 150 μM) in DENV-2-infected HepG2 cells at 24 hours post-infection, as shown by real-time RT-PCR [1].
Quinine sulfate dihydrate significantly reduced viral protein synthesis (DENV NS1, E, PrM, and C proteins) in a dose-dependent manner (37.5, 75, and 150 μM) in DENV-2-infected HepG2 cells at 24 hours post-infection, as shown by Western blot analysis [1].
Flow cytometry analysis showed that the percentage of DENV E antigen-positive HepG2 cells decreased in a dose-dependent manner from 29.64% (infected control) to 23.28%, 17.21%, and 12.05% after treatment with 37.5, 75, and 150 μM of Quinine sulfate dihydrate, respectively, at 24 hours post-infection [1].
Quinine sulfate dihydrate treatment (150 μM for 24 hours) of DENV-infected HepG2 cells led to a significant increase in the mRNA expression of antiviral genes including RIG-I, IFNα, RNase L, and PKR compared to DENV-infected control cells [1].
In DENV-2-infected A549 and EA.hy926 cells, treatment with Quinine sulfate dihydrate (37.5, 75, and 150 μM) resulted in a concentration-dependent inhibition of virus production [1].
In the testicular toxicity model, oral administration of Quinine sulfate dihydrate (10 mg/kg/day for 8 weeks) to rats induced oxidative stress. In the testes and epididymal sperm, this treatment significantly increased malondialdehyde (MDA) levels (a marker of lipid peroxidation) (Testes MF: 64.5±16.3 vs. control 28.5±13.4 μmol/mg protein; Epididymal sperm: 50.7±8.2 vs. control 23.8±4.7 μmol/mg protein). It significantly decreased the activity of catalase (CAT) in the mitochondrial fraction (MF) and post-mitochondrial supernatant (PMS) of the testes (MF: 0.97±0.02 vs. control 1.15±0.01; PMS: 1.02±0.05 vs. control 1.35±0.01 μmol H2O2 consumed/min/mg protein) and in epididymal sperm (1.21±0.01 vs. control 1.43±0.02). Superoxide dismutase (SOD) activity was significantly decreased only in the MF of the testes (1.20±0.43 vs. control 2.29±0.01 Units/mg protein). Ascorbic acid (AA) levels were significantly reduced in the testes MF (0.17±0.03 vs. control 0.21±0.02 mg/g tissue), testes PMS (0.43±0.01 vs. control 0.71±0.02), and epididymal sperm (0.15±0.03 vs. control 0.22±0.02). Glutathione (GSH) levels were non-significantly decreased [2].
The same treatment (10 mg/kg/day for 8 weeks) significantly reduced sperm count (55.2±7.21% vs. control 85.6±4.61%), sperm motility (58.0±8.41% vs. control 84.0±5.52%), live-dead ratio (75.8±9.13% vs. control 92.0±2.71%), daily sperm production (DSP) (4.62±1.51 x 10^7/g testis vs. control 8.92±1.62), and testicular sperm number (TSN) (29.12±3.21 x 10^6/g testis vs. control 50.35±2.61), while increasing the percentage of total sperm abnormalities (28.13±1.32% vs. control 17.25±1.12%), specifically bent tail abnormalities (7.3±1.7% vs. control 4.3±0.04%) [2].
In the skin carcinogenesis model, a single topical application of DMBA (100 μg/100 μL acetone) followed by croton oil (1% v/v in acetone, three times a week for 14 weeks) induced skin tumors. Oral administration of Quinine sulfate dihydrate at 12 mg/kg body weight throughout the promotion period significantly reduced the cumulative number of tumors, tumor weight (0.39±0.002g vs. positive control 1.15±0.09g), tumor yield (2.33±0.48 vs. 5.0±0.44), tumor burden (2.8±0.15 vs. 6±0.42), and tumor incidence (83.33% vs. 100%). It also increased the average latent period (11.7 weeks vs. 9.4 weeks). The inhibition of tumor multiplicity was 53.33% [3].

There is a certain inhibitory effect of quinine sulfate hydrate (oral gavage, 12 or 15 mg/kg, weekly, 16 weeks) on Swiss albino mice skin cancer [2]. The antioxidant defense system, which includes SOD, CAT, and GSH enzyme activities, will decline in the testicular tissue of male adult albino rats given quinine sulfate hydrate (oral gavage, 10 mg/kg, daily, for 8 weeks) [3].

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In a rat model of testicular toxicity, oral administration of Quinine sulfate dihydrate (10 mg/kg/day) for 8 weeks induced oxidative stress. This was evidenced by significantly decreased activities of antioxidant enzymes (SOD, CAT) and non-enzymatic antioxidants (ascorbic acid), and increased lipid peroxidation (MDA) in the testes and epididymal sperm. It also led to histopathological changes in the testes, including disruption of the testicular basement membrane of seminiferous tubules and loss of spermatozoa. These effects were associated with a significant reduction in sperm count, motility, viability, daily sperm production, and testicular sperm number, as well as an increase in sperm abnormalities (particularly bent tail). A slight, non-significant decrease in serum testosterone levels was also observed [2].
In a DMBA-induced and croton oil-promoted two-stage skin carcinogenesis model in Swiss albino mice, oral administration of Quinine sulfate dihydrate at a dose of 12 mg/kg body weight (starting from the first croton oil application until the end of the 16-week experiment) demonstrated chemopreventive effects. This treatment led to a significant reduction in tumor incidence (83.33% vs. 100% in positive control), tumor yield (2.33±0.48 vs. 5.0±0.44), tumor burden (2.8±0.15 vs. 6±0.42), tumor weight (0.39±0.002g vs. 1.15±0.09g), and cumulative number of tumors. The average latent period for tumor appearance was also prolonged (11.7 weeks vs. 9.4 weeks). However, a higher oral dose of 15 mg/kg body weight did not show a protective effect, with tumor parameters being much closer to the positive control group (e.g., tumor yield 4.33±0.41, tumor burden 5.2±0.27, tumor incidence 100%) [3].

Cell proliferation assay [1]
Cell Types: Human hepatoma cell line (HepG2)
Tested Concentrations: 150 μM
Incubation Duration: 30 minutes
Experimental Results: Compared with untreated, DENV virus replication was inhibited and the yield reached 19%. diminished DENV-positive cells from 23.28% to 12.05% in a dose-dependent manner.

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Cells (uninfected or DENV-infected) were incubated with serially diluted drugs for 24 hours. Cell viability was assessed using a PrestoBlue Cell Viability Assay by adding the reagent to cultures, incubating at 37°C for 30 minutes, and measuring absorbance at 570/600 nm. Results were reported as percent cell viability compared to untreated controls.
Focus-Forming Unit (FFU) Assay: Culture supernatants were collected 24 hours after drug treatment, serially diluted 10-fold, and inoculated into Vero cells in a 96-well plate. After 24 hours, an overlay medium containing 1.5% carboxymethyl cellulose was added. The plate was incubated for 3 days at 37°C. Cells were then washed, fixed with 3.7% formaldehyde/PBS, and permeabilized with 1% Triton X-100. An anti-DENV E antibody (4G2 clone) was added as the primary antibody for 1 hour, followed by an HRP-conjugated secondary antibody for 30 minutes. Substrate (3-3’-diaminobenzidine) was used to stain the foci, which were then counted under a light microscope.
Virus Adsorption Assay: HepG2 cells were collected as a cell suspension in a tube and inoculated with the drug and DENV (MOI 1) for 30 minutes at 4°C. Cells were washed with PBS, blocked with 5% BSA/PBS, and stained for surface DENV E antigen using an anti-DENV E antibody (3H5 clone) for 1 hour on ice, followed by an HRP-conjugated secondary antibody for 30 minutes on ice. Surface expression was analyzed by flow cytometry.
Virus Internalization Assay: HepG2 cells were infected with DENV (MOI 1) for 30 minutes at 4°C. Cells were washed with PBS to remove unbound virus, the drug was added, and cells were incubated at 37°C for 2 hours. Cells were then collected, and total RNA was extracted. RNA was reverse-transcribed into cDNA, and real-time RT-PCR was performed using primers for DENV E mRNA to detect internalized virus.
Real-time RT-PCR for mRNA Expression: HepG2 cells were infected with DENV at MOI 1 for 24 hours, then unbound virus was removed, and the drug was added for another 24 hours. Total RNA was extracted, reverse-transcribed into cDNA, and real-time RT-PCR was performed using specific primers for genes of interest (RIG-I, IFNα, IFNAR1, OAS-3, RNase L, PKR). GAPDH was used as a housekeeping gene. The relative expression (2-ΔΔCt) was analyzed.
Western Blot Analysis for DENV Protein Synthesis: HepG2 cells infected with DENV and treated with the drug were collected at 24 hours and lysed with RIPA buffer. Equal concentrations of protein were separated by SDS-PAGE and blotted onto a nitrocellulose membrane. The membrane was blocked with 5% non-fat dairy milk in PBST. DENV proteins were detected using specific primary antibodies (anti-DENV NS1, E, PrM, C) incubated overnight at 4°C, followed by HRP-conjugated secondary antibodies for 1 hour at room temperature. Chemiluminescent substrate was used for detection, and band intensity was quantified using ImageJ software.

Animal/Disease Models: Swiss albino mice 7-8 weeks (weight 24 grams) [2]
Doses: 12 mg/kg, 15 mg/kg
Route of Administration: po (oral gavage); weekly; 16-week
Experimental Results: 12 mg/kg dose Tumor size and weight were Dramatically diminished at 15 mg/kg, with little effect at higher doses of 15 mg/kg.

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Male adult albino rats (Wistar strain, weighing 220-240g) were used. Quinine sulfate dihydrate was administered orally at a dose of 10 mg/kg body weight/day for 8 weeks. The drug was given via the oral route. Rats in the control group received normal saline (0.7 mL/kg). Rats were sacrificed 24 hours after the last treatment by cervical dislocation.

In vitro, the CC50 (50% cytotoxic concentration) of Quinine sulfate dihydrate was 322.2 μM in HepG2 cells, and the therapeutic index (TI) was found to be 3.137 [1].
In a rat model, chronic administration of a therapeutic dose of Quinine sulfate dihydrate (10 mg/kg/day for 8 weeks) induced testicular toxicity. This was manifested as oxidative stress (decreased antioxidant enzymes and increased lipid peroxidation), histopathological changes in the testes (disruption of seminiferous tubule basement membrane, loss of spermatozoa), and adverse effects on spermiogram (reduced sperm count, motility, viability, daily production, and increased abnormalities) [2].
In a mouse skin carcinogenesis model, an oral dose of 15 mg/kg body weight of Quinine sulfate dihydrate did not show protective effects and resulted in tumor parameters (e.g., tumor yield 4.33±0.41, tumor burden 5.2±0.27, 100% tumor incidence) that were much closer to the positive control group. The study suggests this dose rate may be toxic or mitogenic [3].

[1]. Drug repurposing of quinine as antiviral against dengue virus infection. Virus Res. 2018 Aug 15;255:171-178. doi: 10.1016/j.virusres.2018.07.018. Epub 2018 Jul 25.

[2]. Chemoprevention of DMBA induced skin carcinogenesis in swiss albino mice by quinine sulfate.(2016): 2636-2640.

[3]. Quercetin protects against testicular toxicity induced by chronic administration of therapeutic dose of quinine sulfate in rats. J Basic Clin Physiol Pharmacol. 2012 Feb 27;23(1):39-44.

Quinine sulfate is the sulfate form of quinine, an alkaloid isolate of quinidine. Quinine has multiple mechanisms of action, including reducing oxygen uptake and carbohydrate metabolism; interfering with DNA replication and transcription through DNA insertion; and reducing muscle fiber excitability by altering calcium distribution. This drug can also inhibit the drug efflux pump P-glycoprotein, which is overexpressed in multidrug-resistant tumors and may therefore enhance the efficacy of certain antitumor drugs. (NCI04)
Quinine is an alkaloid extracted from the bark of the cinchona tree. It is used as an antimalarial drug and is the active ingredient in cinchona extracts, which have been used for malaria treatment since before 1633. Quinine is also a mild antipyretic and analgesic, and has been used to treat the common cold. It was once widely used as a bittering agent and flavoring agent and is still used to treat babesiosis. Quinine is also effective against certain muscle disorders, particularly nocturnal leg cramps and congenital myotonia, because it acts directly on muscle cell membranes and sodium channels. The mechanism of its antimalarial effect is not fully understood. See also: Quinine sulfate (note moved to).

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Quinine sulfate dihydrate is a natural compound extracted from the Cinchona tree and is widely used to treat chloroquine-resistant falciparum malaria [1].
It has been shown to decrease virus replication in vitro against herpes simplex virus (HSV) and influenza [1].
In the context of dengue virus infection, Quinine sulfate dihydrate is proposed to exert its antiviral activity by restricting viral RNA and protein synthesis and exocytosis, and also by facilitating the upregulation of antiviral genes (RIG-I, IFNα, RNase L, PKR), thereby boosting the host innate immune response [1].
Quinine is known to have a low therapeutic index in malaria treatment. The prescribed oral dosage for adult malarial patients is 600 mg/day, and its plasma concentration should be maintained at 10-15 mg/L. The therapeutic dosage should be thoroughly monitored if repurposed [1].
The testicular toxicity of quinine sulfate is proposed to be, in part, due to impairment of testicular antioxidant defense, disruption of spermatogenesis, and enhancement of lipid peroxidation [2].
Quinine sulfate contains a quinoline scaffold, which is present in many biologically-active compounds. Other quinoline derivatives have shown antineoplastic activity, and some (like imiquimod) are used as antitumor agents. The chemopreventive efficacy of quinine sulfate in the mouse skin cancer model suggests its potential role as an anticancer agent, possibly through mechanisms such as COX-2 inhibition, topoisomerase inhibition, or induction of interferon-α [3].

C20H28N2O7S

440.51

782.356

6119-70-6

Quinine hydrochloride dihydrate;6119-47-7;Quinine;130-95-0;Quinine hemisulfate;804-63-7;Quinine hydrobromide;549-49-5

16211610

White to off-white solid powder

495.9ºC at 760 mmHg

233-235ºC

253.7ºC

6.521

6

14

8

55

538

8

COC1=CC2=C(C=CN=C2C=C1)[C@H]([C@@H]3C[C@@H]4CCN3C[C@@H]4C=C)O.COC1=CC2=C(C=CN=C2C=C1)[C@H]([C@@H]3C[C@@H]4CCN3C[C@@H]4C=C)O.O.O.OS(=O)(=O)O

ZHNFLHYOFXQIOW-LPYZJUEESA-N

InChI=1S/2C20H24N2O2.H2O4S.2H2O/c2*1-3-13-12-22-9-7-14(13)10-19(22)20(23)16-6-8-21-18-5-4-15(24-2)11-17(16)18;1-5(2,3)4;;/h2*3-6,8,11,13-14,19-20,23H,1,7,9-10,12H2,2H3;(H2,1,2,3,4);2*1H2/t2*13-,14-,19-,20+;;;/m00.../s1

(1R)-(6-methoxyquinolin-4-yl)((2S,4S,5R)-5-vinylquinuclidin-2-yl)methanol hemisulfate hydrate

Chinini sulfas Quinine hemisulfate salt monohydrate Quinine sulfate dihydrate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), 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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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