Metabolism and Interactions of Chloroquine and Hydroxychloroquine with Human Cytochrome P450 Enzymes and Drug Transporters

Abstract

Background:

In clinical practice chloroquine and hydroxychloroquine are often co-administered with other drugs in the treatment of malaria, chronic inflammatory diseases, and COVID-19. Therefore, their metabolic properties and the effects on activity of cytochrome P450 (P450, CYP) enzymes and drug transporters should be considered into when developing the most efficient treatments for patients.

Methods:

Scientific literature on the interactions of chloroquine and hydroxychloroquine, with human P450 enzymes and drug transporters was searched using PUBMED.Gov (https://pubmed.ncbi.nlm.nih.gov/) and the ADME database (https://life-science.kyushu.fujitsu.com/admedb/).

Results:

Chloroquine and hydroxychloroquine are metabolized by P450 1A2, 2C8, 2C19, 2D6, and 3A4/5 in vitro and by P450s 2C8 and 3A4/5 in vivo by N-deethylation. Chloroquine effectively inhibited P450 2D6 in vitro; however, in vivo inhibition was not apparent except in individuals with limited P450 2D6 activity. Chloroquine is both an inhibitor and inducer of the transporter MRP1 and is also a substrate of the Mate and MRP1 transport systems. Hydroxychloroquine also inhibited P450 2D6 and the transporter OATP1A2.

Conclusions:

Chloroquine caused a statistically significant decrease of P450 2D6 activity in vitro and in vivo, inhibiting also its own metabolism by the enzyme. The inhibition indicates a potential for clinical drug-drug interactions when taken with other drugs that are predominant substrates of the P450 2D6. When chloroquine and hydroxychloroquine are used clinically with other drugs, substrates of P450 2D6 enzyme, attention should be given to substrate specific metabolism by P450 2D6 alleles present in individuals taking the drugs.

1. METABOLIC PROPERTIES OF CHLOROQUINE AND HYDROXYCHLOROQUINE

Chloroquine and its derivative hydroxychloroquine have been used for many years in the treatment and prevention of malaria and chronic inflammatory diseases (rheumatoid arthritis and systemic lupus erythrematosus) [1–6]. These drugs are also in vitro inhibitors of various viruses, including human immunodeficiency virus (HIV-1) [7, 8], and have strong antiviral effects on SARS-CoV infection of cultured primate cells [9]. Chloroquine and hydroxychloroquine have recently been proposed to be used for prevention and treatment of COVID-19 alone or in combination with other drugs (e.g., azithromycin, lopinavir) [10–15]. While some clinical trials have suggested some beneficial effects of chloroquine and hydroxychloroquine in COVID-19 patients, most of the reported data are still preliminary and need further elaboration. Clinical experience has shown that chloroquine has a narrow safety margin, as three times the adult therapeutic dosage for malaria can be lethal when given as a single dose, particularly in children, who are particularly sensitive to chloroquine toxicity, with one to two tablets being potentially fatal [16].

When given orally to treat rheumatic diseases, chloroquine and hydroxychloroquine are well absorbed (70–80% bioavailability), and between 21 and 47% of the dose is excreted unchanged. Chloroquine is well absorbed and distributed extensively, resulting in a large volume of distribution with apparent and terminal half-lives of 1.6 days and 2 weeks, respectively. There is great variability of plasma concentrations, with an 11-fold range of drug concentrations found following similar doses in treated patients, and the variation may depend on the presence of food, antacids, and iron salts. Protein binding ranges between 30 and 40%, with binding to both albumin and P-glycoprotein. Chloroquine phosphate binds to human serum albumin (HSA), and the number of binding sites and affinity constant were estimated to be 33 and 7.7 × 10³ M⁻¹, respectively. [17–19]. Chloroquine binding to plasma favors the (S)-(+)-chloroquine enantiomer (67% vs 35% for the (R)-(−)-enantiomer). Hence, unbound plasma concentrations are higher for (R-)-(−)-chloroquine. Following separate administration of the individual enantiomers, (R)-(−)-chloroquine produced higher and more sustained blood concentrations. The shorter half-life of (S)-(+)-chloroquine is related to its faster clearance. Blood concentrations of the (S)-(+)-forms of desethylchloroquine always exceeded those of the (R-)-(−)-forms, pointing to a preferential metabolism of (S)-(+)-chloroquine. However, both chloroquine and hydroxychloroquine are clinically administered as racemates [20, 21].

Following administration of chloroquine to patients and volunteers, its N-dealkylated metabolites (monodesethylchloroquine, didesethylchloroquine, 7-chloro-4-aminoquinoline), and in addition the chloroquine side chain N-oxide, and chloroquine di-N-oxide were identified by gas chromatography, thin-layer chromatography, and gas chromatography/mass spectrometry [22, 23]. The secondary amine metabolites may be of particular interest as they could be substrates in flavin monooxygenase (FMO)-catalyzed reactions, with formation of hydroxylamine metabolites which may contribute to inhibition of the target P-450 enzymes. However, no FMO reactions have bene reported to date.

Besides a number of potential adverse effects that might be caused by the use of chloroquine and hydroxychloroquine, alone or in combinations with other drugs [24–26], there are also other factors that might contribute to the clinically significant drug-drug interactions. For instance, it has been reported that inflammatory diseases such as Crohn’s disease increase mRNA expression of P450s 3A4 and 3A5 and the drug transporter P-glycoprotein (P-gp). Conversely, acute inflammation (an illness treated with chloroquine and hydroxychloroquine) and morbid obesity can lead to a decrease in P450 3A4 activity. In addition, morbid obesity my lead also to increased P450 2E1 activity and risk of toxic reactions [27–29]. These examples illustrate that changes of activity and/or expression of drug metabolism enzymes and/or transporters might result in clinically significant pharmacokinetic drug-drug interactions and/or adverse effects of drugs.

The present paper summarizes data published on the metabolism of chloroquine and hydroxychloroquine by human cytochrome P450 enzymes, their interactions with drug transporters, and the potential to cause drug-drug interactions in clinical practice.

2. CHLOROQUINE AND HYDROXYCHLOROQUINE METABOLISM BY P450 ENZYMES

In addition to factors that might influence the fate of chloroquine and hydroxychloroquine in the body, of particular interest is the interactions of these drugs with cytochrome P450 enzymes. It has been reported that P450 2C19 is, in addition to P450 3A4, the major enzyme that participates in chloroquine metabolism in vivo. In vitro, however, P450 2D6 was reported to catalyze chloroquine metabolism with only a minor contribution in vivo (N-dealkylation reaction). In addition, both chloroquine and hydroxychloroquine are inhibitors of P450 2D6 both in vivo and in vitro. The potency of inhibition has ranged from weak to medium (Tables 1 and 2 and references therein). Inhibition of P450 2D6 by chloroquine might be associated with minor participation of P450 2D6 in metabolism of chloroquine in vivo.

Table 1.

Metabolism of Chloroquine and Hydroxychloroquine with Cytochrome P450 Enzymes.

Enzyme	Drug/metabolite	N-Deethylation			References
		In vivo		In vitro
Human liver microsomes	Chloroquine			In vitro: Km 444 ± 121 μM, Vmax 617 ± 128 pmol/min/mg protein, reaction diminished when coincubated with quercetin (20–40% inhibition), ketoconazole, or troleandomycin (20–30% inhibition) and strongly inhibited (80% inhibition) by a combination of ketoconazole and quercetin [32]; Km 0.21 mM for low and Km 3.43 mM for high Km components, Vmax1.02 nmol/min/mg protein and 10.5 nmol/min/mg protein for low and high Vmax components, respectively [36]	[32,35,36]
P450 1A1	Chloroquine			Recombinant enzymes: microsomes, formation of desethylchloroquine	[32]
P450 1A2	Chloroquine			Recombinant enzymes: medium Km, high activity	[36]
P450 2C19	Chloroquine			Recombinant enzymes	[36]
P450 2C8	Chloroquine			Human liver microsomes and recombinant enzymes: 2C8 major enzyme in vivo suggested in addition to P450 3A4	[32,35, 36]
P450 2C8	Hydroxychloroquine		Patients taking drug for >3 months, genetic polymorphism discussed		[34]
P450 2D6	Chloroquine			Human liver microsomes and recombinant enzymes: low Km, low Vmax; minor participation in vivo expected [32]	[32,35, 36]
P450 2D6	Hydroxychloroquine		patients taking drug for >3 months, [desethylhydrochloroquine]: [hydroxychloroquine] ratio was related to CYP2D6 polymorphisms in Korean lupus patients		[34]
P450 3A4	Chloroquine			Human liver microsomes and recombinant enzymes: high Km, high Vmax system, significant participation and as major enzyme in human liver microsomes in addition to P450 2C8 proposed [32,36]	[32,35, 36]
P450 3A4	Hydroxychloroquine		Patients taking drug for >3 months, effect of genetic polymorphism could not be determined		[34]
P450 3A5	Hydroxychloroquine		Patients taking drug for >3 months, effect of genetic polymorphism could not be determined		[34]

Table 2.

Inhibition of Cytochrome P450 Catalyzed Metabolic Reactions by Chloroquine and Hydroxychloroquine.

Enzyme	Drug/metabolite	Method		References
		In vivo	In vitro
P450 1A2	Chloroquine	No effect on caffeine metabolism [33]	Fluorescence-based high throughput screening assays with recombinant P450, no inhibition	[33,42]
P450 1A2	Chloroquine (+)-(S)-		Fluorescence-based high throughput screening assays with recombinant P450: no inhibition	[42]
P450 1A2	Chloroquine (−)-(R)-		Fluorescence-based high throughput screening assays with recombinant P450: no inhibition	[42]
P450 2C19	Chloroquine	No effect on mephenytoin metabolism		[33,45]
P450 2C19	Chloroquine (+)-(S)-		Fluorescence-based high throughput screening assays with recombinant P450: no inhibition	[46]
P450 2C19	Chloroquine (−)-(R)-		Fluorescence-based high throughput screening assays with recombinant P450: no inhibition	[42]
P450 2C8	Chloroquine		Recombinant enzymes: no inhibition or very weak inhibition	[35,36,42]
P450 2C9	Chloroquine		Human liver microsomes and recombinant enzymes: no inhibition or very weak inhibition	[38]
P450 2C9	Chloroquine (−)-(R)-		Fluorescence-based high throughput screening assays with recombinant P450: no inhibition	[42]
P450 2C9	Chloroquine (+)-(S)-		Fluorescence-based high throughput screening assays with recombinant P450: no inhibition	[42]
P450 2D6	Chloroquine	In black Zimbabweans and 12 white Swedish healthy volunteers no P450 2D6 inhibition was observed no statistically significant difference on the urinary debrisoquine to 4-hydroxydebrisoquine metabolic ratio [45]; chloroquine inhibited P450 2D6 debrisoquine metabolism in healthy Zambians, medium and concentration dependent inhibition observed [47]; chloroquine inhibited metabolism of debrisoquine by 2D6 in humans, the effect was modest and progressive with repeated doses, clinically significant pharmacokinetic drug-drug interaction proposed [33]	Fluorescence-based high throughput screening assays with recombinant P450 Ki 12 μM [42] inhibition of bufuralol hydroxylation Ki 13 μM, human liver microsomes competitive inhibition [42]; moderate inhibition of primaquine metabolism [31]; weak inhibition IC50 127 μM for bufuralol 1´-hydroxylation in extensive metabolizer liver [44]; Ki 0.18 μM for inhibition of metoprolol hydroxylation in rat liver microsomes [40]; Ki 15 μM in human liver microsomes [43]; IC50 17 μM with Echerichia coli recombinant enzyme, IC50 39 μM with β-lymphoblastoid microsomes, IC50 21 μM with human liver microsomes [30]	[30,31,33,40,42,43,44,45,47,48]
P450 2D6	Chloroquine (−)-R-		Fluorescence-based high throughput screening assays with recombinant P450s, Ki 20 μM	[45]
P450 2D6	Chloroquine (+)-S-		Fluorescence-based high throughput screening assays with recombinant P450s, Ki 6.2 μM	[45]
P450 2D6	Monodesethylchloroquine		Human liver microsomes and recombinant enzymes, medium inhibition	[30,42]
P450 2D6	Hydroxychloroquine	Increased bioavailability of metoprolol observed, one individual, who was heterozygous for a mutant CYP2D6 allele, was converted to a poor metabolizer by hydroxychloroquine administration		[46]
P450 2E1	Chloroquine	No effect on chlorzoxazone metabolism		[33]
P450 3A4	Chloroquine	No effect on dapsone metabolism [33]	Human liver microsomes, inhibition of quinine 3-hydroxylation IC50 >200 μM [48], inhibition of midazolam hydroxylations [49]	[33,48,49]

Major in vitro approaches to predicting roles of individual P450s in the metabolism of chloroquine and hydroxychloroquine include (i) comparison of rates measured with recombinant P450s (normalized to P450 content in liver), (ii) correlation of the catalytic activity of interest with marker activities of individual P450s (or immunochemically determined levels) in multiple human tissue samples, and (iii) analysis of the extent of inhibition of the catalytic activity in liver (or other tissue) microsomes with or without selective chemical inhibitors and/or P450-specific antibodies. In vivo methods include non-invasive pharmacokinetic measurements and their relationship with (i) genetics, (ii) treatment with an established P450 inducer, or (iii) treatment with a selective P450 inducer or inhibitor [30, 31].

The major metabolites of chloroquine and hydroxychloroquine in humans are formed by the same metabolic reactions both in vitro and in vivo, i.e. N-delakylation(s) (Fig. 1, Table 1). Following administration chloroquine is rapidly N-dealkylated to the pharmacologically active desethylchloroquine and bis-desethylchloroquine by P450 enzymes. Desethylchloroquine and bis-desethylchloroquine concentrations reach 40% and 10% of chloroquine concentrations, respectively, and both chloroquine and desethylchloroquine concentrations decline slowly, with elimination half-lives of 20 to 60 days. Parent drug and metabolite can be detected in urine months after a single dose. It was also reported that ratio of desethylhydroxychloroquine:hydroxychloroquine is in patents with systemic lupus erythematosus related to P450 2D6 polymorphism [21,32,33,34,35]. In vitro metabolism of chloroquine and hydroxychloroquine was investigated by using both human liver microsomes and recombinant human enzymes (Table 1). Using microsomes from a panel of 16 human livers phenotyped for 10 different P450 enzymes, desethylchloroquine formation from chloroquine was highly correlated with testosterone 6β-hydroxylation (r = 0.80; p < 0.001), a P450 3A4-mediated reaction, and P450 2C8-mediated paclitaxel 6β-hydroxylation (r = 0.82; p < 0.001). In human liver microsomes the deethylation pathway was inhibited by quercetin (20–40% inhibition), ketoconazole, and troleandomycin (20–30% inhibition); additional inhibition was seen by using a combination of ketoconazole and quercetin, which strongly inhibited (80%) the reaction [32,35]. It was also shown that desethylchloroquine formation was significantly correlated with P450 3A4/5-catalyzed midazolam 1´-hydroxylation (r²= 0.76) and P450 2C8-catalyzed paclitaxel 6β-hydroxylation (r²= 0.81). Experiments using human liver microsomes confirmed that the major metabolite of chloroquine is desethylchloroquine. Eadie-Hofstee plots were biphasic and suggested the potential involvement of multiple enzymes, with apparent K_m and V_max values of 0.21 mM and 1.02 nmol/min/mg protein, and 3.4 mM and 10.5 nmol/min/mg for low and high K_m components, respectively. Of the cDNA-expressed P450s examined, P450s 1A2, 2C8, 2C19, 2D6, and 3A4/5 had significant activity for desethylchloroquine formation [32,36]. The major contribution to metabolism of chloroquine in vivo is attributed to P450s 2C8 and 3A4, which have both been characterized as high V_max/high K_m systems. P450 2D6, which also catalyzes the deethylation of chloroquine, had a lower K_m but also a significantly lower V_max. Therefore, P450 2C8, P450 3A4, and, to a much lesser extent also P450 2D6, are expected to account for most of the in vivo chloroquine N-deethylation [32] and in human liver microsomes P450s 2C8 and 3A4/5 are the major enzymes responsible for the chloroquine N-deethylation (Table 1 and references therein).

Figure (1).

Structures of Chloroquine, Hydroxychloroquine, and Metabolites Formed by Cytochrome P450 Enzymes.

Hydroxychloroquine is a substrate of P450s 2C8, 2D6, and 3A enzymes in vivo (Table 1 and references therein). The metabolites of hydroxychloroquine reported are desethylhydroxychloroquine, desethylchloroquine, and bis-desethylchloroquine [34, 37]. As already mentioned, it was shown that formation of desethylhydroxychloroquine a major metabolite and pharmacologically active form of hydroxychloroquine was, in Korean lupus patients taking drug orally, related to CYP2D6 polymorphisms. It was suggested, therefore, that the role of an individual’s CYP polymorphisms should be considered when prescribing oral hydroxychloroquine. However, the polymorphisms related to CYP3A5*3 and CYP3A4*18B did not show any significant association with the ratios of blood concentrations of hydroxychloroquine to desethylhydroxychloroquine, or desethylhydroxychloroquine to hydroxychloroquine [34]. For example, the mean maximal blood concentrations of hydroxychloquine in rheumatoid arhrtritis patients after 6 weeks of aplication was ~1400 ng/ml, ~2600 ng/ml, ~5400 ng/ml, following application of 400 mg, 800 mg, or 1200 mg of the drug, respectively. For the same time period maximal blood concentrations of the metabolite desethylhydroxychloroquine were ~750 ng/ml, ~1200 ng/ml and ~1450ng/ml with large interindividual variations [5].

3. CHLOROQUINE AND HYDROXYCHLOROQUINE AS INHIBITORS OF P450 ENZYMES

Chloroquine has been tested for its inhibitory potency in P450-catalyzed reactions (Table 2). An in vitro study with the substrates 17α-ethinylestradiol and tolbutamide (test/marker substrates for P450 3A4 and 2C9 enzymes, respectively), revealed that chloroquine has essentially no effect on metabolism of these drugs [38,39]. However, in vivo studies that included metoprolol [40,41,42], bufuralol [43], and debrisoquine [33] (as test/marker compounds for P450 2D6) indicated that chloroquine and hydroxychloroquine inhibit P450 2D6-mediated reactions both in vitro and in vivo. In one study using human liver microsomes, chloroquine only weakly inhibited 1´-hydroxylation of bufuralol (IC₅₀ 127 ± 12 μM) [44].

The inhibitory effects of chloroquine on P450 2D6 and 2C19 activity have been difficult to demonstrate in vivo, and contradictory results have been obtained. An experiment using a cocktail of drugs consisting of caffeine (substrate of P450 1A2), mephenytoin (substrate of P450 2C19), debrisoquine (substrate of P450 2D6), chlorzoxazone (substrate of P450 2E1), and dapsone (substrate of P450 3A4) showed that chloroquine caused a progressive and statistically significant decrease in P450 2D6 activity over 14 days following chloroquine administration. These results showed that chloroquine, being a substrate of P450 2D6, is also an inhibitor of the enzyme. The effect was modest but indicates the potential for drug-drug interactions when taken with other drugs that are predominant substrates of 2D6 enzyme, inhibiting also its own metabolism [33]. An in vivo study in healthy black Zambians showed that an inhibitory effect was sustained for one week after therapeutic doses of the drugs and the authors suggested that the drug should not be used in combination with other drugs that prolong the QT time interval, particularly those that are metabolized by P450 2D6. In addition, the median debrisoquine/4-hydroxydebrisoquine (0–8 h urinary) ratio in healthy Zambians was increased (from 1.96 to 3.91; P <0.01; 95% confidence intervals 1.34–2.66) when debrisoquine was given 2 h after treatment with chloroquine, causing significant inhibition of P450 2D6 activity [47]. However, the effects of chloroquine on debrisoquine hydroxylation (catalyzed by P450 2D6) and S-mephenytoin hydroxylation (catalyzed by P450 2C19), as assessed in 11 black Zimbabwean and 12 white Swedish healthy volunteers, indicated that chloroquine does not inhibit either P450 2D6 or P450 2C19 in vivo and that it is unlikely to compromise the metabolism of substrates for these two enzymes [45]. It was also reported that chloroquine inhibited recombinant P450 2D6-catalyzed metabolism of primaquine in cryopreserved human hepatocytes [31], but not quinine 3-hydroxylation catalyzed by P450 3A4 [48]. However, chloroquine inhibited quinine 3-hydroxylation in rat and dog liver microsomes but not in human liver microsomes [49].

Hydroxychloroquine has been shown to increase the bioavailability of metoprolol in homozygous P450 2D6 extensive metabolizers (EMs) in that significant increases in the area under the plasma concentration-time curve (AUC 65 ± 5%) and maximal plasma concentration (72 ± 7%) of metoprolol were observed. It was concluded that hydroxychloroquine inhibits metoprolol metabolism, most probably by inhibiting P450 2D6. However, the inhibitory effect of hydroxychloroquine on dextromethorphan metabolism was not apparent except in an individual with low P450 2D6 activity [46].

These studies indicate that chloroquine is low to medium potency inhibitor of P450 2D6 in vitro (K_i ~12 μM for recombinant enzyme), but the IC₅₀ ranged from 39 μM (for β-lympoblastoid-generated P450 2D6) to 127 μM (for liver microsomes) showing that the data are at least somewhat dependent on the experimental model used. Inhibition of P450 2D6 activity in vitro by chloroquine enantiomers was stereoselective (K_i 6.21 μM for the (S)-(+)-enantiomer vs. K_i 20 μM) for the R-(−)-enantiomer [42]. No other P450 enzymes tested were significantly affected (Table 2). In vivo P450 2D6 inhibitory activity has been reported, ranging from medium, concentration-dependent inhibition to no inhibition. In individuals showing progressive inhibition with repeated doses, clinically significant pharmacokinetic drug-drug interaction potential was reported and it was suggested that inhibition was not apparent except in individuals with limited P450 2D6 activity [39]. These results may be connected to substrate-specific metabolism by polymorphic P450 2D6 alleles present in individuals participating in the experiments, in that the activity profile of the P450 2D6 alleles can be substrate specific and manifest in pharmacokinetics consequences for individuals [50]. This information should be considered in the context of the participation of P450 enzymes, and in partcular P450 2D6 in the metabolism of marketed drugs [51,52].

4. INTERACTION OF CHLOROQUINE AND HYDROXYCHLOROQUINE WITH DRUG TRANSPORTERS

The multiple drug resistance (MDR) transporter was first discovered for its role in imparting resistance to drugs in cancer cells by pumping them out [53]. As knowledge about this system, together with P450 3A4, developed in the late 1980s, the overlap in substrate specificity was recognized and the role of the MDR1 protein (also called P-170, P-glycoprotein, P-gp, or ABCB1) as a drug export protein was realized in the late 1990s. With time the number of proteins involved in these processes has grown. The overall view today is that passive diffusion of drugs in and out of cells does occur in many cases but that transporters can have important roles in many cases and can have major effects on tissue selectivity of drugs. The transporters can be considered in the context of their own activities and also their interactions with P450s in that the regulation of many transporters involves the same systems as P450s, e.g. P-gp and some of the P450 3A and 2C subfamily enzymes are regulated by the pregnane X receptor (PXR). Another issue is that P-gp export transporter can also lower the level of a drug in a cell and accordingly slow the rate of its metabolism [27,54].

Transporters discussed in the present paper belong to the ATP binding cassette (ABC) transporter family and solute carrier family (SLC) transporters (Table 3). ABC transporters are membrane-bound proteins and are present in all prokaryotes, as well as plants, fungi, yeast, and animals. In humans the ABC transporters participate in transport of a wide variety of compounds (drugs, environmental, and physiological compounds) with the use of the energy of ATP. The most important drug transporters of the ABC protein family are P-glycoprotein (P-gp/ABCB1), bile salt export pump (BSEP/ABCB11), multidrug resistance-associated proteins (MRP1–4/ABCC1–4), breast cancer resistance protein (BCRP/ABCG2), and the organic anion transporter polypeptide family (OATPs). OATP1B1, OATP2B1, and OATP1B3 are influx transporters expressed by hepatoytes, facilitating drug access to the liver, while P-glycoprotein (P-gp) and BCRP are efflux transporters in intestinal epithelial cells, liver, kidney, and brain. ABCC1 and multidrug resistance-associated proteins 1 and 2 (MRP1 and MRP2) are efflux transporters. MRP1 has wide tissue distribution, and MRP2 is an efflux transporter in hepatocytes. MRP1 has wide substrate transport specificity, including important drugs. The main roles of the MRP1 transporter are the efflux of xenobiotics and endogenous metabolites, transport of inflammatory mediators (e.g., LTC4), and defense against oxidative stress. In addition, MRP1 plays a role in the development of drug resistance of various types of cancer and contributes to inflammatory responses [55,56].

Table 3.

Interaction of Chloroquine and Hydroxychloroquine with human transporters.

Transporter	Compound	Substrate	Inhibition	Induction	Remarks/Model	References
BCRP1; BCRP; ABCG2; MRX; MXR; ABCP; BMDP; MXR1; ABC15; CD338; CDw338; EST157481; MGC102821	Chloroquine		X		Chloroquine (100 μM) caused reduced transport of estrone-sulfate in membrane vesicles isolated from HEK293 cells over expressing transport protein, reduced to 69%	[60]
BSEP, ABCB11	Chloroquine			X	No significant inhibition; 100 μM induced transport of taurocholic acid in membrane vesicles isolated from HEK293 cells over expressing transport protein, increased to 117% [61]	[61,62,63]
hOCT2, SLC22A2	Chloroquine		X		Inhibition of [3H]MPP(+)-uptake in HEK293 cells stably expressing human OCT2	[64]
hSERT; SLC6A4; HTT; 5HTT; OCD1; 5HTT; 5HTTLPR	Chloroquine		X		Ki values for hSERT using HeLa cells to inhibit [3H]5-HT uptake was 122 μM [66]	[65,66]
MATE1, SLC47A1	Chloroquine	X	X		Substrate and competitive inhibitor of MATE1-mediated metformin uptake (Ki 2.8 μM) in HEK293-MATE1 cells, suggest drug-drug interaction potential	[67]
MRP1, MRP,	Chloroquine	X	X	X	Chloroquine/protease inhibitor combinations exerted combined inhibitory effects on P-gp and MRP1 function [72]; inhibited the photoaffinity labeling of MRP by IAAQ, cell growth assays showed MRP-expressing multidrug-resistant cells (H69/AR and HL60/AR) to be more resistant to chloroquine than their parental cells (i.e., IC50 of 121 versus 28 μM chloroquine for H69/AR and H69MK 571, an LTD4 receptor antagonist reversed the resistance of H69/AR cells to chloroquine, direct binding to MRP suggested [68]; induction of expression after long-term exposure of CEM T cells, resulting in resistance to chloroquine and co-administered drugs [70]; molar excesses of chloroquine inhibited the photoaffinity labeling of MRP1 by IAAQ [69]; chloroquine resistance was associated with the specific over expression of MRP1 [68,69,70]; rate constant for MRP1-mediated calcein efflux from L23/R cells 12 x 10−4 s−1 at 50 μM chloroquine, control value 11 x 10−4 s−1; little effect on MRP1-associated ATPase activity, inhibition of uptake of 3.3 μM [3H]cGMP into inside-out human erythrocyte membrane vesicles IC50 86 μM [71]	[68,69,70, 71,72]
MRP4, MOATB, MOATB, ABCC4, EST170205	Chloroquine		X		Little or no effect on MRP4-associated ATPase activity	[71]
OATP1A2, OATP-A, OATP1, OATP, SLC21A3, SLCO1A2	Chloroquine		X		IC50 1.0 μM for estrone-3-sulfate using transporter-over expressing Madin-Darby canine kidney (MDCKII) cells [73]; potently inhibited the uptake activity of all-trans-retinol in overexpressing human embryonic kidney (HEK293) cells and human retinal pigment epithelium cells [60]	[60.73]
OATP1A2, OATP-A, OATP1, OATP, SLC21A3, SLCO1A2	Hydroxychloroquine		X		Potent inhibitor of OATP1A2 function, 1.0 μM using MDCKII cells and the model substrate estrone-3-sulfate [72]; potently inhibited the uptake activity of all-trans-retinol in over expressing HEK293 cells and in primary human retinal pigment epithelium cells [60]	[60,72]
OATP1B1, OATP-C, OATP2, LST-1, SLC21A6, SLCO1B1	Chloroquine			X	Pre-incubation with chloroquine at clinically relevant concentration(s) significantly decreased [3H]E217G and [3H]pitavastatin accumulation in HEK293-OATP1B1 cells and [3H]pitavastatin accumulation in human SCH cells; chloroquine pretreatment (25 μM, 2 h) resulted in increased OATP1B1 total protein levels and ∼1.9-fold decrease in Vmax without affecting the Km for OATP1B1-mediated [3H]E217G transport in HEK293-OATP1B1 cells; potential to cause OATP-mediated drug-drug interactions downregulating transport activity	[74]
OATP1B3; SLCO1B3; LST3; OATP8; SLC21A8; LST-3TM1	Chloroquine			X	Treatment with chloroquine (25 μM, 5 h) increased OATP1B3 protein levels to 1.85-fold vehicle in HEK293-OATP1B3 cells and human SCH cells	[75]
OATP2B1, OATP-B, SLC21A9, SLCO2B1	Chloroquine		X		No effect or only moderate inhibitor effects observed using MDCKII cells and the model substrate estrone-3-sulfate	[73]
P-glycoprotein (P-gp), ABCB1	Chloroquine		X		Chloroquine (100 μM) reduced methyl quinidine transport to 50% in membrane vesicles isolated from HEK293 cells overexpressing ABC transport proteins [61]; chloroquine competed poorly for the binding site of photoreactive analogs of verapamil on P-glycoprotein in membranes of human tumor cells of the CEM/VBL100 line [62]; Ki,app 54 μM for taxol transport inhibition in Caco-2 cells [63]; weak inhibitor of Calcein-AM in MDR1-MDCKII cells [76]; poor inhibitor of photolabeling with vinblastine analog, partial reversal activity on drug resistance [79]; enhanced vinblastine cytotoxicity in CEM/VLB1K cells by 10- to 15-fold, inhibited [125I]vinblastine analog labeling of P-gp [80]; chloroquine/protease inhibitors combination exerts combined inhibitory effects on P-gp function [72]	[61,62,63,72,75,76,77,78,79,80]

A compound (drug) may interact with a transport protein as a substrate, inducer (by induction of the transporter expression), and/or inhibition of protein function (Table 3). In the clinic, as a consequence, the pharmacokinetics of co-administered drugs can be critically altered to result in drug-drug interactions. The result of drug-drug interactions on the level of drug transport may lead to either an unexpected high plasma concentrations and potential toxic effects or, alternativley, subtherapeutic concentrations at the site of action lowering therapeutic effects. Drug-drug interactions at the level of transporters may reduce systemic exposure to drug by blocking influx transporters in the intestine but increase it by modulating influx and efflux transporters in the liver and efflux transporters in the small intestine [56].

In clinical practice chloroquine is often co-administerd with other drugs in the treatment of malaria [57,58], as well as for treatment of COVID-19 [59]. Therefore, the effect of chloroquine and hydroxychloroquine and co-administered drugs on ABC-mediated transport should be considered in order to provide the most effective treatments for patients.

Reported data on effects of chloroquine and hydroxychloroquine on the activities and properties of drug transporters are presented in Table 3 and references therein. The results presented were obtained either by using human cells or recombinant transporters in different in vitro assays. The drugs were tested for their activitiess as substates, inhibitors, or inducers of a particular transporter activity. The in vitro assays used have involved various types of cells overexpressing transporters, e.g. HEK293 (embryonic human kidney cell line), MDCKII (epithelial Madin-Darby canine kidney cell line), human tumor cells, Caco-2 cells (human epithelial colorectal adenocarcinoma cells), CEM/VLB1K (human leukemic cell line9), CEM T cells (human T lymphoblastic leukemia cell line), HeLa (immortal cell line derived from cervical cancer cells), and human SCH cells (human gastric choriocarcinoma cell line) (Table 3).

The investigations cover effects of chloroquine on P-gp-mediated transport of drugs that are substrates of the transporter and/or on expression of the transporter. Depending on the model and competing drug used, chloroquine was found to be a weak or poor inibitor of P-gp-mediated transport (quinidine, taxol) or of the binding of photoreactive analogs of verapamil and vinblastine (methyl) to the transporter. However, enhanced vinblastine toxicity was observed in CEM/VLB1K cells. In addition, using peripheral blood mononuclear cells, combinatory inhibition effects of chloroquine/protease inhibitors (Table 3) was observed. Chloroquine increased the blockage of the dye Rh123 and also carboxyfluorescein efflux activity exerted by protease inhibitors (Table 3). These results show that chloroquine itself is a weak inhibitor of P-gp transport; however, in combination with other drugs (i.e., inhibitors of the transport system) inhibitory effects might be significantly enhanced.

Studies with MRP-type transporters [68–71] showed that chloroquine is substrate of the MRP1 transporter because MRP1-expressing multidrug-resistant cells accumulate less drug than the parental drug-sensitive cells, and direct binding to the transporter was suggested [68]. It was also shown that long term exposure of CEM T cells to chloroquine induced expression of the transporter and resulted in resistence to chloroquine and coadministered drugs [70]. However, a molar excess of chloroquine inhibited photolabeling of MRP1 by the reagent IAAQ and chloroquine resistance was associated with specific overexpression of MRP-1 [68–70]. As with P-gp, a combination of chloroquine and protease inibitor drugs exerted combined inhibitory effects on MRP1. These data show that chloroquine can acts as a substrate, inducer, and inhibitor of MRP1. However, it was reported that chloroquine excerts little or no effect on MRP4-associated ATPase activity [71].

Potent inhibition of OATP1A2 by chloroquine and hydroxychloroquine [60,73] but only moderate inhibition (BCRP1, hSERT, OATP2B1) or no inhibition (BSEP) of other transporters has been reported [61–63]. Increased protein levels of OATP1B1 and OATP1B3 were reported after treatment of the cells with chloroquine, indicating induction of expression [74,75]. For details see Table 3.

As already discussed, in addition to induction and/or inhibition of activity of P450 enzymes and drug transporters, plymorphism might have additional effect on pharmacokinetic and consequently therapeutic properties of chloroquine and hydroxychloroquine [81,82].

The present data show the complexity of interactions of chloroquine with drug transporting systems, in that it can exert both inhibitory and inducing activity, but in some cases chloroquine is also substrate of the transporters (Mate and MRP1) (Table 3). Co-administration of chloroquine with other drug types is highly anticipated when treating patients for human immunodeficiency virus (HIV) and other viral infections (including COVID-19), and malaria co-infections, and the effect of chloroquine on ABC-mediated transport systems should be of particular attention in order to develop the most effective treatment strategies for patients receiving multiple drug regimens.

CONCLUSION

Major and pharmacologicaly active metabolites of chloroquine and hydroxychloroquine are formed by N-dealkylation reactions catalyzed by P450 enzymes both in vivo and in vitro. P450s 1A2, 2C8, 2C19, 2D6, and 3A4/5 exhibited significant formation of desethyl metabolites with chloroquine as substrate in vitro. The major contributions to in vivo metabolism of chloroquine is attributed to P450s 2C8 and 3A4, and that of hydroxychloroquine to P450 2C8, 2D6, and 3A4/5. Chloroquine and its desethyl metabolite inhibited P450 2D6 both in vivo and in vitro although not strongly. Chloroquine, in interaction with drug transporting systems, can exert both inhibitory and inducing activity (BSEP, MRP1), but in some cases chloroquine is also substrate of the transporters (Mate, MRP1).

Formation of N-desethylhydroxychloroquine from hydroxychloroquine in vivo has been related to CYP2D6 polymorphisms, but hydroxychloroquine also inhibited P450 2D6 in vivo as shown by inhibiting metoprolol bioavailability. Hydroxychloroquine was reported to inhibit also organic anion transporter OATP1A2, shown by inhibition of the uptake activity of all-trans-retinol.

The interaction of chloroquine and hydroxychloroquine with both, P450 enzymes (2D6) and drug transporter systems may by inhibiting their activity require particular attention when these drugs are clinically applied in order to develop the most effective treatment strategies for patients receiving multiple drug regimens.