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
Chloroquine oral solution has a bioavailability of 52-102% and oral tablets have a bioavailability of 67-114%. Intravenous chloroquine reaches a Cmax of 650-1300µg/L and oral chloroquine reaches a Cmax of 65-128µg/L with a Tmax of 0.5h.
Chloroquine is predominantly eliminated in the urine. 50% of a dose is recovered in the urine as unchanged chloroquine, with 10% of the dose recovered in the urine as desethylchloroquine.
The volume of distribution of chloroquine is 200-800L/kg.
Chloroquine has a total plasma clearance of 0.35-1L/h/kg.
Chloroquine is rapidly and almost completely absorbed from the GI tract following oral administration, and peak plasma concn of the drug are generally attained within 1-2 hr. Considerable interindividual variations in serum concn of chloroquine have been reported. Oral administration of 310 mg of chloroquine daily reportedly results in peak plasma concn of about 0.125 ug/mL. If 500 mg of chloroquine is administered once weekly, peak plasma concn of the drug reportedly range from 0.15-0.25 ug/mL and trough plasma concn reportedly range from 0.02-0.04 ug/mL. Results of one study indicate that chloroquine may exhibit nonlinear dose dependent pharmacokinetics. In this study, administration of a single 500 mg oral dose of chloroquine resulted in a peak serum concentration of 0.12 ug/mL, and administration of a single 1 g oral dose of the drug resulted in a peak serum concentration of 0.34 ug/mL.
Results of one cross-over study in healthy adults indicate that the bioavailability of chloroquine is greater when the drug is administered with food than when the drug is administered in the fasting state. In this study, the rate of absorption of chloroquine was unaffected by the presence of food in the GI tract however, peak plasma concn of chloroquine and areas under the plasma concentration-time curves were higher when 600 mg of the drug was administered with food than when the same dose was administered without food.
Chloroquine is widely distributed into body tissues. The drug has an apparent volume of distribution of 116-285 L/kg in healthy adults. Animal studies indicate that concn of chloroquine in liver, spleen, kidney, and lung are at least 200-700 times higher than those in plasma, and concentration of the drug in brain and spinal cord are at least 10-30 times higher than those in plasma. Chloroquine binds to melanin containing cells in the eyes and skin; skin concn of the drug are considerably higher than plasma concentration. Animal studies indicate that the drug is concentrated in the iris and choroid and, to a lesser extent, in the cornea, retina, and sclera and is found in these tissues in higher concentration than in other tissues.
Chloroquine is also concentrated in erythrocytes and binds to platelets and granulocytes. Serum concentrations of chloroquine are higher than those in plasma, presumably because the drug is released from platelets during coagulation, and plasma concentrations are 10 to 15% lower than whole blood concentration of the drug.
For more Absorption, Distribution and Excretion (Complete) data for CHLOROQUINE (16 total), please visit the HSDB record page.

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Metabolism / Metabolites
Chloroquine is N-dealkylated primarily by CYP2C8 and CYP3A4 to N-desethylchloroquine. It is N-dealkylated to a lesser extent by CYP3A5, CYP2D6, and to an ever lesser extent by CYP1A1. N-desethylchloroquine can be further N-dealkylated to N-bidesethylchloroquine, which is further N-dealkylated to 7-chloro-4-aminoquinoline.
Chloroquine is partially metabolized; the major metabolite is desethylchloroquine. Desethylchloroquine also has antiplasmodial activity, but is slightly less active than chloroquine. Bisdesethylchloroquine, which is a carboxylic acid derivative, and several other unidentified metabolites are also formed in small amounts.
Hepatic (partially), to active de-ethylated metabolites. Principal metabolite is desethylchloroquine
Completely absorbed from gastrointestinal tract. Chloroquine is partially metabolized; the major metabolite is desethylchloroquine. Desethylchloroquine also has antiplasmodial activity, but is slightly less active than chloroquine. Bisdesethylchloroquine, which is a carboxylic acid derivative, and several other unidentified metabolites are also formed in small amounts (A625).
Route of Elimination: Excretion of chloroquine is quite slow, but is increased by acidification of the urine.
Half Life: 1-2 months

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Biological Half-Life
The half life of chloroquine is 20-60 days.
The plasma half-life of chloroquine in healthy individuals is generally reported to be 72-120 hr. In one study, serum concentrations of chloroquine appeared to decline in a biphasic manner and the serum half-life of the terminal phase increased with higher dosage of the drug. In this study, the terminal half-life of chloroquine was 3.1 hr after a single 250 mg oral dose, 42.9 hr after a single 500 mg oral dose, and 312 hr after a single 1 g oral dose of the drug.
Terminal elimination half-life is 1 to 2 months.
... extremely slow elimination, with a terminal elimination half-life of 200 to 300 hours)

Hepatotoxicity
Despite use for more than 50 years, chloroquine has rarely been linked to serum aminotransferase elevations or to clinically apparent acute liver injury. In patients with acute porphyria and porphyria cutanea tarda, chloroquine can trigger an acute attack with fever and serum aminotransferase elevations, sometimes resulting in jaundice. Hydroxychloroquine does not cause this reaction and appears to have partial beneficial effects in porphyria. In clinical trials of chloroquine for COVID-19 prevention and treatment, there were no reports of hepatotoxicity, and rates of serum enzyme elevations during chloroquine treatment were low and similar to those in patients receiving placebo or standard of care.
Likelihood score: D (possible rare cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Very small amounts of chloroquine are excreted in breast milk; when given once weekly, the amount of drug is not sufficient to harm the infant nor is the quantity sufficient to protect the child from malaria. United Kingdom malaria treatment guidelines recommend that weekly chloroquine 500 mg be given until breastfeeding is completed and primaquine can be given. Breastfeeding infants should receive the recommended dosages of chloroquine for malaria prophylaxis.In HIV-infected women, elevated viral HIV loads in milk were decreased after treatment with chloroquine to a greater extent than other women who were treated with the combination of sulfadoxine and pyrimethamine. Because no information is available on the daily use of chloroquine during breastfeeding, hydroxychloroquine or another agent may be preferred in this situation, especially while nursing a newborn or preterm infant.
◉ Effects in Breastfed Infants
Several authors have pointed out that malaria prophylaxis in nursing mothers with chloroquine is common in endemic areas. As of the revision date, no reports of adverse reactions in breastfed infants have been published.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
Chloroquine is 46-74% bound to plasma proteins. (-)-chloroquine binds more strongly to alpha-1-acid glycoprotein and (+)-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 that is quinoline which is substituted at position 4 by a [5-(diethylamino)pentan-2-yl]amino group at at position 7 by chlorine. It is used for the treatment of malaria, hepatic amoebiasis, lupus erythematosus, light-sensitive skin eruptions, and rheumatoid arthritis. It has a role as an antimalarial, an antirheumatic drug, a dermatologic drug, an autophagy inhibitor and an anticoronaviral agent. It is an aminoquinoline, a secondary amino compound, a tertiary amino compound and an organochlorine compound. It is a conjugate base of a chloroquine(2+).
Chloroquine is an aminoquinolone derivative first developed in the 1940s for the treatment of malaria. It was the drug of choice to treat malaria until the development of newer antimalarials such as [pyrimethamine], [artemisinin], and [mefloquine]. Chloroquine and its derivative [hydroxychloroquine] have since been repurposed for the treatment of a number of other conditions including HIV, systemic lupus erythematosus, and rheumatoid arthritis. **The FDA emergency use authorization for [hydroxychloroquine] and chloroquine in the treatment of COVID-19 was revoked on 15 June 2020.** Chloroquine was granted FDA Approval on 31 October 1949.
Chloroquine is an Antimalarial.
Chloroquine is an aminoquinoline used for the prevention and therapy of malaria. It is also effective in extraintestinal amebiasis and as an antiinflammatory agent for therapy of rheumatoid arthritis and lupus erythematosus. Chloroquine is not associated with serum enzyme elevations and is an extremely rare cause of clinically apparent acute liver injury.
Chloroquine has been reported in Cocos nucifera, Cinchona calisaya, and other organisms with data available.
Chloroquine is a 4-aminoquinoline with antimalarial, anti-inflammatory, and potential chemosensitization and radiosensitization activities. Although the mechanism is not well understood, chloroquine is shown to inhibit the parasitic enzyme heme polymerase that converts the toxic heme into non-toxic hemazoin, thereby resulting in the accumulation of toxic heme within the parasite. This agent may also interfere with the biosynthesis of nucleic acids. Chloroquine's potential chemosensitizing and radiosensitizing activities in cancer may be related to its inhibition of autophagy, a cellular mechanism involving lysosomal degradation that minimizes the production of reactive oxygen species (ROS) related to tumor reoxygenation and tumor exposure to chemotherapeutic agents and radiation.
Chloroquine is only found in individuals that have used or taken this drug. It is a prototypical antimalarial agent with a mechanism that is not well understood. It has also been used to treat rheumatoid arthritis, systemic lupus erythematosus, and in the systemic therapy of amebic liver abscesses. [PubChem]The mechanism of plasmodicidal action of chloroquine is not completely certain. Like other quinoline derivatives, it is thought to inhibit heme polymerase activity. This results in accumulation of free heme, which is toxic to the parasites. nside red blood cells, the malarial parasite must degrade hemoglobin to acquire essential amino acids, which the parasite requires to construct its own protein and for energy metabolism. Digestion is carried out in a vacuole of the parasite cell.During this process, the parasite produces the toxic and soluble molecule heme. The heme moiety consists of a porphyrin ring called Fe(II)-protoporphyrin IX (FP). To avoid destruction by this molecule, the parasite biocrystallizes heme to form hemozoin, a non-toxic molecule. Hemozoin collects in the digestive vacuole as insoluble crystals.Chloroquine enters the red blood cell, inhabiting parasite cell, and digestive vacuole by simple diffusion. Chloroquine then becomes protonated (to CQ2+), as the digestive vacuole is known to be acidic (pH 4.7); chloroquine then cannot leave by diffusion. Chloroquine caps hemozoin molecules to prevent further biocrystallization of heme, thus leading to heme buildup. Chloroquine binds to heme (or FP) to form what is known as the FP-Chloroquine complex; this complex is highly toxic to the cell and disrupts membrane function. Action of the toxic FP-Chloroquine and FP results in cell lysis and ultimately parasite cell autodigestion. In essence, the parasite cell drowns in its own metabolic products.
The prototypical antimalarial agent with a mechanism that is not well understood. It has also been used to treat rheumatoid arthritis, systemic lupus erythematosus, and in the systemic therapy of amebic liver abscesses.
See also: Chloroquine Phosphate (has salt form); Chloroquine Sulfate (has salt form); Chloroquine Hydrochloride (has salt form) ... View More ...

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Drug Indication
Chloroquine is indicated to treat infections of _P. vivax_, _P. malariae_, _P. ovale_, and susceptible strains of _P. falciparum_. It is also used to treat extraintestinal amebiasis. Chloroquine is also used off label for the treatment of rheumatic diseases, as well as treatment and prophylaxis of Zika virus. Chloroquine is currently undergoing clinical trials for the treatment of COVID-19.
FDA Label

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Mechanism of Action
Chloroquine inhibits the action of heme polymerase in malarial trophozoites, preventing the conversion of heme to hemazoin. _Plasmodium_ species continue to accumulate toxic heme, killing the parasite. Chloroquine passively diffuses through cell membranes and into endosomes, lysosomes, and Golgi vesicles; where it becomes protonated, trapping the chloroquine in the organelle and raising the surrounding pH. The raised pH in endosomes, prevent virus particles from utilizing their activity for fusion and entry into the cell. Chloroquine does not affect the level of ACE2 expression on cell surfaces, but inhibits terminal glycosylation of ACE2, the receptor that SARS-CoV and SARS-CoV-2 target for cell entry. ACE2 that is not in the glycosylated state may less efficiently interact with the SARS-CoV-2 spike protein, further inhibiting viral entry.
The exact mechanism of antimalarial activity of chloroquine has not been determined. The 4-aminoquinoline derivatives appear to bind to nucleoproteins and interfere with protein synthesis in susceptible organisms; the drugs intercalate readily into double-stranded DNA and inhibit both DNA and RNA polymerase. In addition, studies using chloroquine indicate that the drug apparently concentrates in parasite digestive vacuoles, increases the pH of the vacuoles, and interferes with the parasite's ability to metabolize and utilize erythrocyte hemoglobin. Plasmodial forms that do not have digestive vacuoles and do not utilize hemoglobin, such as exoerythrocytic forms, are not affected by chloroquine.
The 4-aminoquinoline derivatives, including chloroquine, also have anti-inflammatory activity; however, the mechanism(s) of action of the drugs in the treatment of rheumatoid arthritis and lupus erythematosus has not been determined. Chloroquine reportedly antagonizes histamine in vitro, has antiserotonin effects, and inhibits prostaglandin effects in mammalian cells presumably by inhibiting conversion of arachidonic acid to prostaglandin F2. In vitro studies indicate that chloroquine also inhibits chemotaxis of polymorphonuclear leukocytes, macrophages, and eosinophils.
Antiprotozoal-Malaria: /Mechanism of action/ may be based on ability of chloroquine to bind and alter the properties of DNA. Chloroquine also is taken up into the acidic food vacuoles of the parasite in the erythrocyte. It increases the pH of the acid vesicles, interfering with vesicle functions and possibly inhibiting phospholipid metabolism. In suppressive treatment, chloroquine inhibits the erythrocytic stage of development of plasmodia. In acute attacks of malaria, chloroquine interrupts erythrocytic schizogony of the parasite. its ability to concentrate in parasitized erythrocytes may account for its selective toxicity against the erythrocytic stages of plasmodial infection.
Antirheumatic-Chloroquine is though to act as a mild immunosuppressant, inhibiting the production of rheumatoid factor and acute phase reactants. It also accumulates in white blood cells, stabilizing lysosomal membranes and inhibiting the activity of many enzymes, including collagenase and the proteases that cause cartilage breakdown.

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.

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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.

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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.

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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.

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