Absorption
Hydroxychloroquine is currently only available as an orally administered tablet, available in 200 mg as the sulfate salt form (equivalent to 155 mg base) [19]. There are numerous PK studies on the bioavailability of the oral tablet. However, most are in healthy volunteers or in patients with disease states other than COVID-19 [9,10,11,12,13]. Most studies have concluded that HCQ peak concentrations are estimated to be observed within 3–5 h [11,19,20]. In healthy males who received a single HCQ 200 mg oral dose, a mean peak blood HCQ concentration of 0.1296 mcg/ml was achieved in 3.26 h, while a peak plasma HCQ concentration of 0.0503 mcg/ml was achieved in 3.74 h. Tett and colleagues performed a randomized, crossover study in which the HCQ 155 mg oral tablet was compared to an intravenous infusion of racemic HCQ 155 mg to evaluate the absolute bioavailability of the commercially available HCQ tablet. These authors concluded that the mean (± SD) fraction of the oral dose absorbed (estimated from urine and blood) was 0.74 (± 0.13), while a wide range of absorption was calculated from plasma data [11,19].
Despite the lack of data and recommendations, current centers around the world have described crushing the HCQ tablets into suspensions and administering them via feeding tubes in patients otherwise unable to take oral medications, despite the Institute for Safe Medication Practices (ISMP) listing HCQ film-coated tablets on the “Do Not Crush” medication list [[21], direct communications]. Although this approach is commonplace in some inpatient centers among COVID-19 patients, there are no data on the impact of crushing HCQ tablets, administration via feeding tubes, and overall bioavailability or the timing of absorption. Given the uncertainty in PK with this approach, this further emphasizes the importance of understanding and optimizing PK and PD of HCQ against SARS-CoV-2.
Protein Binding
Most studies have shown that the binding of HCQ to protein is moderate (~ 40%) [9,22]. Albumin and alpha1-acid glycoprotein have been the two proteins associated with the majority of HCQ binding. HCQ exists as (R)- and (S)-enantiomers, and stereoselective protein binding has been documented [22].
Tissue Distribution
It has been shown that HCQ exhibits linear PK [23]. Due to HCQ’s sequestration in deep tissues, the volume of distribution (Vd) that HCQ displays is extremely large. Tett and colleagues reported a blood and plasma Vd of 5522 l and 44,257 l, respectively, following intravenous HCQ infusion in healthy volunteers [23]. HCQ and chloroquine, which show similar patterns of tissue distribution, have been shown to concentrate quite highly in the lungs, kidney, liver, and spleen in animal models [24]. Maisonnasse and colleagues found that HCQ concentrations in the lung were higher than in plasma, with lung:plasma ratios ranging from 27 to 177 in macaques [25]. Notably, these data may very well be quite different in humans, and lung:plasma ratios could be lower because of differences in the metabolic composition and lower drug recovery rates.
Table 1 Summary of in vitro EC50 values reported for hydroxychloroquine/chloroquine from selected studies Metabolism and Transport Mechanisms
CYP enzymes catalyze the dealkylation of HCQ to pharmacologically active metabolites, and HCQ/chloroquine has been documented to be metabolized through CYP 3A, 2D6, and 2C8 systems. The metabolism of HCQ leads to the three active metabolites, desethylhydroxychloroquine, desethylchloroquine, and bisdesethylhydroxychloroquine, although they have been shown to increase more significantly following chronic administration. It is anticipated that there are lower levels of these active metabolites present in the initial days of therapy for COVID-19 patients, and it is unclear how the various concentrations of active compounds translate to overall activity against SARS-CoV-2. Limited research has been conducted on investigating the association between genetic polymorphisms in CYP 3A, 2D6, and 2C8 and HCQ drug concentration levels [26,27,28,29,30].
Little is known about the role of membrane transporters on HCQ PK/PD. There is literature that suggests that HCQ inhibits uptake activity of human organic anion transporting polypeptide 1A2 [31]. Also, HCQ/chloroquine has been shown to be an inhibitor of p-glycoprotein [32].
Excretion
Reports have described a median of ~ 20% of HCQ being excreted renally as unchanged drug in humans [33,34]. Urinary elimination of HCQ as metabolites and unchanged drug has been reported to be between 6% and 60% [9,29,34]. Lim and colleagues reported HCQ clearance to be 15.5 l/h (two-compartment model best described these data). Most reports have described a terminal elimination half-life of 30–60 days (in contrast to a terminal blood half-life reported by Carmichael and colleagues of 43.3 h) [10,13]. Using plasma data following administration of the oral HCQ tablet, Tett and colleagues calculated a mean (± SD) terminal elimination half-life of 32 (± 9) days [11].
Hydroxychloroquine/Chloroquine Mechanism, Concentration, and In Vitro Inhibitory Activity AGAINST SARS-CoV-2
Although the mechanism of action of hydroxychloroquine and chloroquine against SARS-CoV-2 has not been fully elucidated, it is thought that these agents may prevent terminal glycosylation of its functional entry receptor (angiotensin-converting enzyme 2 receptor), thus inhibiting viral entry [6,7]. Furthermore, it has been shown that these agents can alkalinize intracellular compartments through incorporation of lysosomes and endosomes, leading to inhibition of viral replication and infection [8].
The majority of in vitro analyses of HCQ and chloroquine have utilized Vero cell lines (Table 1). Liu and colleagues evaluated the antiviral effects of HCQ against SARS-CoV-2 compared to chloroquine at four different multiplicities of infection (MOI) 48 h post-infection. At MOIs (0.01, 0.02, 0.2, and 0.8), the half maximal effective concentration (EC50) values for chloroquine (2.71, 3.81, 7.14, and 7.36 µM) and HCQ (4.51, 4.06, 17.31, and 12.96 µM), respectively, were determined. Furthermore, the half cytotoxic concentration (CC50) values were not found to be statistically significant from each other (chloroquine: 273.20 µM vs. HCQ: 249.50 µM) [35].
Wang and colleagues evaluated chloroquine against SARS-CoV-2 (nCOV-2019BetaCoV/Wuhan/WIV04/2019) and demonstrated potent in vitro activity in Vero E6 cells, which were infected at a MOI of 0.05. At 48 h, chloroquine was shown to potently inhibit SARS-CoV-2 with a EC50 of 1.13 µM and CC50 > 100 µM. This evaluation also showed that chloroquine worked at the “entry” and “post-entry” stages of SARS-CoV-2 infection [36].
Yao and colleagues performed an in vitro evaluation of HCQ and chloroquine against SARS-CoV-2-infected Vero cells at a MOI of 0.01 for 2 h, followed by treatment concentrations for 24 or 48 h. The authors concluded that HCQ was more potent than chloroquine, with EC50 values of 6.14 µM and 23.90 µM, respectively, at 24 h and EC50 values of 0.72 µM and 5.47 µM, respectively, at 48 h. Furthermore, HCQ was shown to exhibit a superior antiviral effect compared to chloroquine when cells were pre-treated prior to viral challenge [37].
An evaluation from Maisonnasse and colleagues analyzed the in vitro activity of HCQ against SARS-CoV-2 in VeroE6 cells (MOI: 0.01) and a model of reconstituted human airway epithelium (MOI: 0.1). In VeroE6 cells at 48 and 72 h, the half maximal inhibitory concentration (IC50) values were 2.19 µM and 4.39 µM, respectively. However, HCQ at 1 µM or 10 µM was not shown to significantly reduce viral titers in the reconstituted human airway epithelium at 48 h compared to untreated control [25].
Despite these in vitro evaluations, it is important to note that very little is known regarding the relevance of in vitro EC50 values in optimizing the PK/PD of HCQ in humans. Furthermore, depending on testing conditions, the reported studies have reported a ~ 24-fold range in EC50 values. Finally, it is currently unknown which cell line is optimal for showing activity, and the discordant results between different cell lines introduce additional uncertainty in the relevance of these values. These limitations further emphasize the need to define the “optimal target” and how to correlate this target to efficacious HCQ exposures.