Antiviral; Plasmodium; SARS-COV-2; Malaria; TLRs; HIV-1

Activated human monocyte-derived Langerhans-like cells (MoLC) have a reduced Th1 priming capacity and are less able to release IL-12p70 when exposed to 20 μM of chloroquine (CHQ). Chloroquine (20 μM) simultaneously stimulates the release of IL-17A from CD4+ T cells and improves the IL-1-induced IL-23 in MoLC [1]. In parental MDA-MB-231 cells, MMP-9 mRNA expression is inhibited by 25 μM of chloroquine under both normoxic and hypoxic conditions. The effects of chloroquine on MMP-2, MMP-9, and MMP-13 mRNA are depending on cell expression, dosage, and hypoxia [2]. Significantly less HuH7 cell proliferation was observed in vitro when TLR7 and TLR9 were inhibited with IRS-954 or chloroquine [3]. At low micromolar doses (EC50=1.13 μM), chloroquine (0.01–100 μM; 48 hours) inhibits SARS-CoV-2 infection and efficiently blocks virus infection in vero E6 cells. By raising the endosomal pH needed for virus/cell fusion and interfering with the glycosylation of SARS-CoV cell acquisition, chloroquine causes viral infection [4].

In an orthotopic mouse model, chloroquine (80 mg/kg, ip) does not stop triple-negative MDA-MB-231 cells from growing, regardless of how much TLR9 is expressed [2]. The growth of tumor xenograft models was considerably decreased by IRS-954 or chloroquine-induced TLR7 and TLR9 inhibition. Additionally, in the DEN/NMOR tumor model, chloroquine greatly reduces the formation of HCC [3].

Chloroquine suppressed matrix metalloproteinase (MMP)-2 and MMP-9 mRNA expression and protein activity, whereas MMP-13 mRNA expression and proteolytic activity were increased. Despite enhancing TLR9 mRNA expression, chloroquine suppressed TLR9 protein expression in vitro.[2]

In this study, we investigated the effect of CHQ on human monocyte-derived Langerhans-like cells (MoLC) and dendritic cells (MoDC) in response to IL-1β. The presence of CHQ reduced IL-12p70 release in both subsets, but surprisingly increased IL-6 production in MoDC and IL-23 in MoLC. Importantly, CHQ-treated MoLC promoted IL-17A secretion by CD4(+) T cells and elevated RORC mRNA levels, whereas IFN-γ release was reduced. The dysregulation of IL-12 family cytokines in MoLC and MoDC occurred at the transcriptional level. Similar effects were obtained with other late autophagy inhibitors, whereas PI3K inhibitor 3-methyladenine failed to increase IL-23 secretion. The modulated cytokine release was dependent on IL-1 cytokine activation and abrogated by a specific IL-1R antagonist. CHQ elevated expression of TNFR-associated factor 6, a common intermediate in IL-1R and TLR-dependent signaling. Accordingly, treatment with Pam3CSK4 and CHQ enhanced IL-23 release in MoLC and MoDC. CHQ inhibited autophagic flux, confirmed by increased LC3-II and p62 expression, and activated ERK, p38, and JNK MAPK, but only inhibition of p38 abrogated IL-23 release by MoLC. Thus, our findings indicate that CHQ modulates cytokine release in a p38-dependent manner, suggesting an essential role of Langerhans cells and dendritic cells in CHQ-provoked psoriasis, possibly by promoting Th17 immunity.[1]

Control and TLR9 siRNA MDA-MB-231 cells (5×105 cells in 100 μl) were inoculated into the mammary fat pads of four-week-old, immune-deficient mice (athymic nude/nu Foxn1; Harlan Sprague Dawley, Inc., Indianapolis, IN, USA). Treatments were started seven days after tumor cell inoculation. The mice were treated daily either with intraperitoneal (i.p.) chloroquine (80 mg/kg) or vehicle (PBS). The animals were monitored daily for clinical signs. Tumor measurements were performed twice a week and tumor volume was calculated according to the formula V = (π / 6) (d1 × d2)3/2, where d1 and d2 are perpendicular tumor diameters (9). The tumors were allowed to grow for 22 days, at which point the mice were sacrificed and the tumors were dissected for a final measurement. Throughout the experiments, the animals were maintained under controlled pathogen-free environmental conditions (20–21ºC, 30–60% relative humidity and a 12-h lighting cycle). The mice were fed with small-animal food pellets (Harlan Sprague Dawley) and supplied with sterile water ad libitum. The experimental procedures were reviewed and approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee.[2]

Absorption, Distribution and Excretion
The bioavailability of oral chloroquine solution is 52-102%, and that of oral tablets is 67-114%. The peak plasma concentration (Cmax) of chloroquine after intravenous injection is 650-1300 µg/L, while the peak plasma concentration (Cmax) of oral chloroquine is 65-128 µg/L, with a time to peak concentration (Tmax) of 0.5 hours. Chloroquine is primarily excreted in the urine. 50% of the dose is excreted unchanged in the urine, and 10% is excreted as desethylchloroquine. The volume of distribution of chloroquine is 200-800 L/kg. The total plasma clearance of chloroquine is 0.35-1 L/h/kg. After oral administration, chloroquine is rapidly and almost completely absorbed from the gastrointestinal tract, typically reaching peak plasma concentrations within 1-2 hours. Significant individual variability in chloroquine serum concentrations has been reported. A daily oral dose of 310 mg of chloroquine resulted in a peak plasma concentration of approximately 0.125 μg/mL. A weekly dose of 500 mg of chloroquine was reported to have peak plasma concentrations ranging from 0.15 to 0.25 μg/mL, with trough concentrations ranging from 0.02 to 0.04 μg/mL. One study indicated that chloroquine may exhibit non-linear dose-dependent pharmacokinetic characteristics. In this study, a single oral dose of 500 mg chloroquine resulted in a peak serum concentration of 0.12 μg/mL, and a single oral dose of 1 g chloroquine resulted in a peak serum concentration of 0.34 μg/mL. A crossover study in healthy adults showed that chloroquine has higher bioavailability when taken with food compared to taking it on an empty stomach. This study found that the presence of food in the gastrointestinal tract did not affect the absorption rate of chloroquine; however, compared to taking the same dose on an empty stomach, taking 600 mg of chloroquine on an empty stomach resulted in higher peak plasma concentrations and a higher area under the plasma concentration-time curve. Chloroquine is widely distributed throughout the body. In healthy adults, the apparent volume of distribution of this drug is 116-285 L/kg. Animal studies have shown that chloroquine concentrations in the liver, spleen, kidneys, and lungs are at least 200-700 times higher than plasma concentrations, and in the brain and spinal cord at least 10-30 times higher than plasma concentrations. Chloroquine can bind to melanocytes in the eyes and skin; drug concentrations in the skin are much higher than in plasma. Animal studies have shown that the drug is mainly concentrated in the iris and choroid, with smaller concentrations in the cornea, retina, and sclera, and in these tissues the concentrations are higher than in other tissues. Chloroquine also concentrates in erythrocytes and binds to platelets and granulocytes. Serum concentrations of chloroquine are higher than in plasma, possibly because the drug is released from platelets during coagulation, and plasma concentrations are 10% to 15% lower than whole blood concentrations. For more complete data on absorption, distribution, and excretion of chloroquine (16 items in total), please visit the HSDB records page.

---

Metabolism/Metabolites
Chloroquine is primarily N-dealkylated via CYP2C8 and CYP3A4 to produce N-deethylchloroquine. Chloroquine is primarily N-dealkylated via CYP3A5 and CYP2D6, and secondarily via CYP1A1, the latter to a lesser extent. N-Deethylchloroquine can be further N-dealkylated to N-bis(deethylchloroquine), which can be further N-dealkylated to 7-chloro-4-aminoquinoline.
Chloroquine is partially metabolized; the main metabolite is deethylchloroquine. Deethylchloroquine also has antimalarial activity, but slightly lower than chloroquine. Small amounts of bis(deethylchloroquine) (a carboxylic acid derivative) and several other unidentified metabolites are also produced.
It is (partially) metabolized in the liver to an active deethylated metabolite. The main metabolite is deethylchloroquine.
It is completely absorbed from the gastrointestinal tract. Chloroquine is partially metabolized; the main metabolite is deethylchloroquine. Desethylchloroquine also has antimalarial activity, but slightly lower than chloroquine. Small amounts of didesethylchloroquine (a carboxylic acid derivative) and several other unidentified metabolites are also produced (A625). Elimination pathway: Chloroquine is excreted slowly, but acidification of urine can accelerate its excretion. Half-life: 1-2 months. The half-life of chloroquine is 20-60 days. It has been reported that the plasma half-life of chloroquine in healthy individuals is typically 72-120 hours. One study showed that serum concentrations of chloroquine decreased in a biphasic manner, and the terminal serum half-life increased with increasing drug dose. In this study, the terminal half-life was 3.1 hours after a single oral dose of 250 mg chloroquine; 42.9 hours after a single oral dose of 500 mg chloroquine; and 312 hours after a single oral dose of 1 g chloroquine. The terminal elimination half-life is 1 to 2 months. … (Drug elimination is extremely slow, with a terminal elimination half-life of 200 to 300 hours.)

Hepatotoxicity
Although chloroquine has been used for over 50 years, it is rarely associated with elevated serum transaminases or clinically significant acute liver injury. In patients with acute porphyria and porphyria cutanea latitudinalis, chloroquine can induce acute exacerbations with fever and elevated serum transaminases, sometimes leading to jaundice. Hydroxychloroquine does not cause this reaction and appears to have partial efficacy for porphyria. In clinical trials of chloroquine for the prevention and treatment of COVID-19, no hepatotoxicity was reported, and the incidence of elevated serum enzymes during chloroquine treatment was low, similar to patients receiving placebo or standard treatment. Probability score: D (likely to cause clinically significant liver injury, albeit rarely).

---

Effects During Pregnancy and Lactation
◉ Overview of use during lactation: Very small amounts of chloroquine are excreted into breast milk; once a week, the dose is insufficient to harm the infant or protect them from malaria infection. The UK malaria treatment guidelines recommend 500 mg of chloroquine weekly until breastfeeding is discontinued and primaquine can be taken. Breastfeeding infants should receive chloroquine at the recommended dose for malaria prevention. In HIV-infected women, chloroquine treatment resulted in a significantly lower HIV viral load in breast milk compared to women taking a combination of sulfadoxine and pyrimethamine. Since there is currently no information on daily chloroquine use during breastfeeding, hydroxychloroquine or other medications may be a better option in such cases, especially for newborns or premature infants.
◉ Effects on breastfed infants
Some authors have noted that chloroquine use for malaria prevention is common among breastfeeding women in malaria-endemic areas. As of the revision date, no reports of adverse reactions in breastfed infants have been published.
◉ Effects on breastfeeding and breast milk
As of the revision date, no relevant published information was found.
Protein binding

Chloroquine binds to plasma proteins at a rate of 46-74%. (-)-Chloroquine binds more strongly to α1-acid glycoprotein, while (+)-chloroquine binds more strongly to serum albumin.

[1]. Said A, et al. Chloroquine promotes IL-17 production by CD4+ T cells via p38-dependent IL-23 release by monocyte-derived Langerhans-like cells. J Immunol. 2014 Dec 15;193(12):6135-43.
[2]. Tuomela J, et al. Chloroquine has tumor-inhibitory and tumor-promoting effects in triple-negative breast cancer. Oncol Lett. 2013 Dec;6(6):1665-1672.
[3]. Mohamed FE, et al. Effect of toll-like receptor 7 and 9 targeted therapy to prevent the development of hepatocellular carcinoma. Liver Int. 2014 Jul 2. doi: 10.1111/liv.12626.
[4]. Colson P, et al. Chloroquine and hydroxychloroquine as available weapons to fight COVID-19. Int J Antimicrob Agents. 2020;55(4):105932.
[5]. Savarino A, et al. The anti-HIV-1 activity of chloroquine. J Clin Virol. 2001;20(3):131-135.

Chloroquine is an aminoquinoline compound with a quinoline structure, where the 4-position is replaced by a [5-(diethylamino)pentan-2-yl]amino group and the 7-position by a chlorine atom. It is used to treat malaria, hepatic amebiasis, lupus erythematosus, photosensitive rashes, and rheumatoid arthritis. It possesses multiple effects, including antimalarial, antirheumatic, skin disease treatment, autophagy inhibition, and anticoronavirus activity. Chloroquine is an aminoquinoline compound belonging to the secondary, tertiary, and organochlorine compounds. It is the conjugate base of chloroquine(2+). Chloroquine is an aminoquinoline derivative initially developed in the 1940s for the treatment of malaria. Before the advent of newer antimalarial drugs such as pyrimethamine, artemisinin, and mefloquine, chloroquine was the drug of choice for treating malaria. Subsequently, chloroquine and its derivatives (hydroxychloroquine) have been used to treat a variety of other diseases, including AIDS, systemic lupus erythematosus, and rheumatoid arthritis. On June 15, 2020, the U.S. Food and Drug Administration (FDA) revoked the Emergency Use Authorization for hydroxychloroquine and chloroquine for the treatment of COVID-19. Chloroquine was approved by the FDA on October 31, 1949. Chloroquine is an antimalarial drug. It is an aminoquinoline drug used for the prevention and treatment of malaria. Chloroquine is also effective against extraintestinal amebiasis and can be used as an anti-inflammatory drug to treat rheumatoid arthritis and lupus. Chloroquine does not cause elevated serum enzymes and rarely causes clinically significant acute liver injury. It has been reported to be found in coconuts (Cocos nucifera), cinchona trees (Cinchona calisaya), and other organisms with relevant data. Chloroquine is a 4-aminoquinoline compound with antimalarial, anti-inflammatory, and potential chemosensitizing and radiosensitizing effects. Although its mechanism of action is not fully understood, studies have shown that chloroquine inhibits heme polymerase, an enzyme that converts toxic heme into non-toxic heme in parasites, leading to the accumulation of toxic heme within the parasite. Furthermore, chloroquine may interfere with nucleic acid biosynthesis. The potential chemosensitizing and radiosensitizing effects of chloroquine in cancer may be related to its inhibition of autophagy. Autophagy is a cellular mechanism involving lysosomal degradation that minimizes the production of reactive oxygen species (ROS) associated with tumor reoxygenation and tumor exposure to chemotherapeutic drugs and radiation. Chloroquine is only present in individuals who have used or taken the drug. It is a typical antimalarial drug with an incompletely understood mechanism of action. Chloroquine has also been used to treat rheumatoid arthritis, systemic lupus erythematosus, and amoebic liver abscess systemically. [PubChem] The mechanism of action of chloroquine in killing malaria parasites is not fully understood. Like other quinoline derivatives, it is thought to inhibit the activity of heme polymerase. This leads to the accumulation of free heme, which is toxic to the parasite. Within red blood cells, Plasmodium must degrade hemoglobin to obtain essential amino acids, which are necessary for the parasite to build its own proteins and perform energy metabolism. The digestive process takes place in the vacuoles of the Plasmodium cells. During this process, the parasite produces a toxic and soluble molecular heme. The heme portion consists of a porphyrin ring called Fe(II)-protoporphyrin IX (FP). To avoid destruction by heme, the parasite biocrystallizes the heme into non-toxic molecular heme crystals. These heme crystals accumulate in the digestive vacuoles as insoluble crystals. Chloroquine enters the red blood cells, Plasmodium cells, and digestive vacuoles through simple diffusion. Because the digestive vacuoles are acidic (pH 4.7), chloroquine is subsequently protonated (generating CQ2+), preventing it from leaving the digestive vacuoles by diffusion. Chloroquine binds to the heme crystals, preventing further biocrystallization of heme, thus leading to heme accumulation. Chloroquine binds to heme (or phosphatidylserine, FP) to form the so-called FP-chloroquine complex; this complex is highly toxic to cells and disrupts cell membrane function. The toxicity of the FP-chloroquine complex and the action of FP lead to cell lysis, ultimately resulting in autophagy of the parasite cells. Essentially, the parasite cells are overwhelmed by their own metabolic products. Chloroquine is a typical antimalarial drug, but its mechanism of action is not fully understood. It has also been used to treat rheumatoid arthritis, systemic lupus erythematosus, and amoebic liver abscesses systemically. See also: Chloroquine phosphate (in salt form); Chloroquine sulfate (in salt form); Chloroquine hydrochloride (in salt form)...see more...

---

Drug Indications
Chloroquine is indicated for the treatment of Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, and Plasmodium falciparum infections sensitive to chloroquine. It is also used to treat extraintestinal amebiasis. Chloroquine has also been used off-label for the treatment of rheumatic diseases, as well as for the treatment and prevention of Zika virus. Chloroquine is currently undergoing clinical trials for the treatment of COVID-19.
FDA Label

---

Mechanism of Action
Chloroquine inhibits the activity of heme polymerase in the trophozoite of Plasmodium, preventing the conversion of heme into heme. Plasmodium continuously accumulates toxic heme, eventually leading to the death of the parasite. Chloroquine passively diffuses across the cell membrane into endosomes, lysosomes, and Golgi vesicles; in these organelles, chloroquine is protonated, trapped within the organelles, and raises the surrounding pH. The elevated pH in the endosomes prevents viral particles from using their activity to fuse and enter the cell. Chloroquine does not affect the expression level of ACE2 on the cell surface, but it inhibits the terminal glycosylation of ACE2, which is the target receptor for SARS-CoV and SARS-CoV-2 entry into cells. The interaction efficiency of unglycosylated ACE2 with the SARS-CoV-2 spike protein may be lower, further inhibiting viral entry into cells.
The exact mechanism of chloroquine's antimalarial activity is not yet clear. 4-Aminoquinoline derivatives appear to bind to nucleoproteins, interfering with protein synthesis in susceptible organisms; these drugs readily intercalate into double-stranded DNA and inhibit DNA and RNA polymerases. Furthermore, studies using chloroquine have shown that the drug appears to accumulate in the digestive vacuoles of the parasite, increasing vacuolar pH and interfering with the parasite's metabolism and ability to utilize hemoglobin from erythrocytes. Plasmodium morphologies lacking digestive vacuoles and hemoglobin utilization, such as the erythrocyte morphology, are unaffected by chloroquine. 4-Aminoquinoline derivatives, including chloroquine, also possess anti-inflammatory activity; however, the mechanisms of action of these drugs in treating rheumatoid arthritis and lupus erythematosus remain unclear. Chloroquine has been reported to antagonize histamine in vitro, exhibit antiserotonin activity, and inhibit prostaglandin activity in mammalian cells, possibly by inhibiting the conversion of arachidonic acid to prostaglandin F2. In vitro studies have also shown that chloroquine inhibits chemotaxis of polymorphonuclear leukocytes, macrophages, and eosinophils. Antiprotozoan-malaria: /Mechanism of action/ is likely based on chloroquine's ability to bind to and alter DNA properties. Chloroquine can also be absorbed by the acidic food vesicles of intraerythrocyte parasites. It increases the pH of these acidic vesicles, interfering with vesicle function and potentially inhibiting phospholipid metabolism. In suppressive therapy, chloroquine inhibits the developmental stage of Plasmodium within erythrocytes. During acute malaria attacks, chloroquine blocks the erythrocyte cleavage of Plasmodium. Its ability to accumulate in infected erythrocytes may explain its selective toxicity to the erythrocyte phase of Plasmodium infection. The antirheumatic drug chloroquine is believed to have mild immunosuppressive effects, inhibiting the production of rheumatoid factor and acute-phase reactants. It can also accumulate in leukocytes, stabilize lysosomal membranes, and inhibit the activity of various enzymes, including collagenase and proteases that cause cartilage degradation.

C18H26CLN3

319.87

319.181

C, 67.59; H, 8.19; Cl, 11.08; N, 13.14

54-05-7

Chloroquine phosphate;50-63-5;Chloroquine-d5;1854126-41-2;Chloroquine dihydrochloride;3545-67-3;Chloroquine-d5 diphosphate; 132-73-0 (sulfate); 1854126-42-3; 54-05-7 ;151-69-9 (acetate) ; 1446-17-9 (phosphate); 3545-67-3 (HCl) ; 50-63-5 (diphosphate) ;

2719

WHITE TO SLIGHTLY YELLOW, CRYSTALLINE POWDER
Colorless crystals

1.1±0.1 g/cm3

460.6±40.0 °C at 760 mmHg

87ºC

232.3±27.3 °C

0.0±1.1 mmHg at 25°C

1.592

4.69

1

3

8

22

309

0

ClC1C([H])=C([H])C2C(C=1[H])=NC([H])=C([H])C=2N([H])C([H])(C([H])([H])[H])C([H])([H])C([H])([H])C([H])([H])N(C([H])([H])C([H])([H])[H])C([H])([H])C([H])([H])[H]

WHTVZRBIWZFKQO-UHFFFAOYSA-N

InChI=1S/C18H26ClN3/c1-4-22(5-2)12-6-7-14(3)21-17-10-11-20-18-13-15(19)8-9-16(17)18/h8-11,13-14H,4-7,12H2,1-3H3,(H,20,21)

N4-(7-chloroquinolin-4-yl)-N1,N1-diethylpentane-1,4-diamine

RP 3377; RP-3377; RP3377;Imagon; NSC 187208; NSC-187208; NSC187208;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Ethanol : ~100 mg/mL (~312.63 mM)
DMSO : ~50 mg/mL (~156.31 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.82 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 25.0 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.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (7.82 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 25.0 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.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (7.82 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

Solubility in Formulation 4: 10 mg/mL (31.26 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; Need ultrasonic and warming and heat to 44°C.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

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