INTRODUCTION

Studies correlating hydroxychloroquine levels with control of lupus activity suggest that the association of higher levels with a favorable outcome may be both causal – resulting from benefit of achieving high levels – and that lower levels may indicate noncompliance. Conversely, studies of the association of levels with toxicity take into account levels achieved regardless of compliance. Articles from Petri et al. suggest there is no clear correlation between prescribed weight-based dosing of hydroxychloroquine and the resulting blood level in an individual (Fig. 1) emphasizing the potential utility of following blood levels of hydroxychloroquine (HCQ) rather than relying on administrating a calculated dose. This review summarizes new literature published since an earlier review published in this journal in 2018 [1]. The data available to date support the use of hydroxychloroquine levels in clinical practice both to help achieve therapeutic levels and reduce risk of toxicity. 

FIGURE 1: There is no strong correlation between a weight-based dose of hydroxychloroquine and the resultant hydroxychloroquine blood level. Reproduced with permission from Petri et al. [23▪].Box 1:

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MEASUREMENT OF HYDROXYCHLOROQUINE LEVELS

Hydroxychloroquine levels are measured by liquid chromatography or tandem mass spectrometry in isolation or in combination [2,3]. Levels can be measured in whole blood, serum or plasma, although whole blood is the preferred sample [4,5]. In a study of 26 lupus patients, whole blood had a concentration twice as high as serum or plasma (mean level 813 vs. 436 ng/ml for serum and 362 ng/ml for plasma) [4]. A strong correlation was noted between serum levels of hydroxychloroquine and whole blood levels (R2 = 0.82) but a poor correlation between plasma levels and whole blood levels (R2 = 0.46) suggesting that plasma levels should be avoided [4] as documented in the figure from Carlsson et al., which also corroborates the lack of a correlation between hydroxychloroquine dose and level, further strengthening the role of measuring hydroxychloroquine levels (Fig. 2). In this review, blood levels refer to whole blood levels as the default measure except when otherwise specified.

FIGURE 2: A strong correlation exists between hydroxychloroquine serum and whole blood levels while the correlation between plasma and whole blood is weaker and plasma measurements were less reproducible. (a) Graph of HCQ serum vs. HCQ whole blood levels. (b) Graph of HCQ plasma vs. HCQ whole blood levels. Various doses of HCQ are indicated as follows: circle = 1400 mg/week, triangle = 2000 mg/week, diamond 2800 mg/week. Adapted with permission from Carlsson et al. [4]. HCQ, hydroxychloroquine.APPROPRIATE TIMING OF HYDROXYCHLOROQUINE LEVELS

Because of the long half-life of hydroxychloroquine, it was initially thought that measurement of the level at any point after several months of use was valid. Al-Rawi et al. (2018), however, demonstrated that even after 6 months, whole blood levels of hydroxychloroquine measured at seven timepoints over a 12-h period varied for the same patient by 27%. Maximum levels occurred 2–6 h after administration. Knowing the time since the preceding hydroxychloroquine dose is important to properly interpret hydroxychloroquine levels, particularly when values are at the extremes of expected values [6]. Alternating daily doses, for example, alternating 400 mg with 200 mg every other day to achieve a mean daily dose of 300 mg, reduces accuracy of assessment.

MEASURING HYDROXYCHLOROQUINE LEVELS HELPS TO IDENTIFY NONADHERENCE

Nonadherence with hydroxychloroquine is a common problem [5,7–10] of which most rheumatologists are unaware [11▪▪,12▪]. In the US Medicaid population followed for 1 year after initiation of hydroxychloroquine, 36% of patients were persistently and 47% of were partially nonadherent (hydroxychloroquine use <80% days/month). Adherence declined over the course of a year [7].

Although there is no established threshold level to define nonadherence, less than 200 ng/ml has been most frequently utilized in the literature [5,12▪]. This value is affected by the use of different assays and may not be the appropriate cutoff for all laboratories. Blanchet et al.[5] suggested a serum hydroxychloroquine level of 106 ng/ml. Because of individual differences in metabolism, extremely low levels are most reliable indicators of nonadherence. Several studies have found that use of hydroxychloroquine levels to identify nonadherence and resulting physician–patient discussions improved patient adherence [12▪,13–15]. Additionally, questionnaires to assess for noncompliance have poorly correlated with blood levels [9]. In one study, 43% of the patients who were nonadherent when detected by drug levels would have qualified as being adherent based on the questionnaire [11▪▪].

WHAT HYDROXYCHLOROQUINE LEVEL DO WE TARGET?

A target therapeutic hydroxychloroquine level is not established in part as there is also no current standardization of hydroxychloroquine levels across various labs. Numerous studies have suggested levels of 500–1000 ng/ml as a therapeutic target [12▪].

In particular, several studies have focused levels needed for the prevention of lupus flares. In the PLUS study, a randomized control trial to evaluate the effects of targeting a hydroxychloroquine level at least 1000 ng/ml on the incidence of systemic lupus erythematosus (SLE) flares, increasing the dose of hydroxychloroquine to achieve levels at least 1000 ng/ml did not decrease the number of SLE flares over 7 months of follow-up. However, adherence increased in the control group and only 39% of the treatment group stayed above the 1000 ng/ml goal threshold [16]. Conversely, in two retrospective observational studies, Cunha et al.[17] reported that a hydroxychloroquine level greater than 600 ng/ml was associated with decreased likelihood of renal flares in patients with lupus nephritis, and Pedrosa et al.[18] reported that persistently low hydroxychloroquine levels less than 613.5 ng/ml best predicted risk of flares in 82 patients with lupus nephritis.

HYDROXYCHLOROQUINE LEVELS AND DISEASE ACTIVITY

Multiple studies have correlated higher disease activity with lower hydroxychloroquine levels [10,13,14,16–19]. This data is well synthesized in the excellent meta-analysis by Garg et al. Hydroxychloroquine levels reported varied widely with no consensus on appropriate level adequate for treatment. Hydroxychloroquine levels of at least 750 ng/ml predicted a 58% lower risk of active lupus [12▪].

Blanchet et al.[5] in 2020 reported that whole blood levels were higher in patients with low disease activity (SLEDAI ≤4) vs. high disease activity (SLEDAI > 4) (940.8 ± 448 vs. 765.9 ± 426 ng/ml, P = 0.001). Geraldino-Paradilla et al. (2019) evaluated 108 patients with SLE and found that patients who were nonadherent by their definition (defined as a hydroxychloroquine level ≤500 ng/ml) had a higher SLEDAI-2K score compared with those who were adherent (5.7 vs. 3.2) [10]. Iudici et al. in 2018 reported that the 5 of 83 lupus patients who flared during a 6 month follow-up period had a lower median hydroxychloroquine level at baseline compared with those who did not experience a flare (284 vs. 435 ng/ml) [20].

Two recent studies of lupus nephritis reported that persistent values above a target hydroxychloroquine level were correlated with lower likelihood of lupus nephritis flare. In the first study of 171 patients with class III, IV, or V lupus nephritis not on renal replacement therapy, no correlation between hydroxychloroquine levels and lupus nephritis flares was identified [17]. In patients with active nephritis at baseline the hydroxychloroquine levels of patients who went into remission were similar to those who continued to have active disease (P = 0.23). In patients with partial or complete remission at inclusion, the hydroxychloroquine levels were lower in those who experienced a renal flare during the follow-up period (0.59 vs. 0.81 mg/l, P = 0.005). The data suggested that a target hydroxychloroquine level of greater than 600 ng/ml reduces the likelihood of lupus nephritis flares [17]. Pedrosa et al.[18] reported that persistently low hydroxychloroquine levels were associated with higher risk of lupus nephritis flares in a study of 82 patients. A hydroxychloroquine level less than 613.5 ng/ml best predicted risk of flare in a study of 82 patients with lupus nephritis [18]. Table 1 summarizes recent study conclusions.

Table 1 - Summary and conclusions of recent studies evaluating hydroxychloroquine levels in SLE patients Author (year) Study population Comparator groups Conclusion Blanchet et al. (2020) SLE patients on hydroxychloroquine for ≥6 months without dose modification in ≥2 months Low disease activity: SLEDAI ≤4High disease activity: SLEDAI >4 Whole blood levels were higher in patients with low disease activity (940.8 ± 448 vs. 765.9 ± 426 ng/ml, P = 0.001) Geraldino-Paradilla et al. (2019) SLE patients on hydroxychloroquine for ≥6 months who reported medication adherence Hydroxychloroquine level ≤500 ng/ml vs. hydroxychloroquine level >500 ng/ml SLEDAI-2K score higher in those who had level ≤500 ng/ml compared with adherent to hydroxychloroquine (5.7 vs. 3.2) Iudici et al. (2018) SLE patients in remission for >1 year and taking stable dose of hydroxychloroquine Patients with flare vs. no flare over 6-month period based on SELENA-SLEDAI score Median baseline hydroxychloroquine level lower in patients who experienced a flare over a 6 month period (284 vs. 435 ng/ml, P = 0.225) Cunha et al. (2018) Biopsy-proven class III, IV or V lupus nephritis on hydroxychloroquine for ≥3 months Patients with flare vs. patients with no flare In patients with active nephritis at baseline the hydroxychloroquine levels of those who received remission was similar to those who continued to have active disease. In patients in complete or partial remission at baseline, patients who experienced a renal flare had lower average hydroxychloroquine levels Pedrosa et al. (2020) Lupus nephritis patients Patients with flare vs. patients with no flare Flares were found to be more frequent in patients with hydroxychloroquine level <613.5 ng/ml (28 vs. 5%, P = 0.023)

SELENA-SLEDAI, Safety of Estrogens in Lupus Erythematosus National Assessment-Systemic Lupus Erythematosus Disease Activity Index; SLE, systemic lupus erythematosus; SLEDAI, Systemic Lupus Erythematosus Disease Activity Index.

In assessments of dermatologic disease with use of the CLASI, arguably the most objective lupus disease activity measurement, Chasset et al.[21] reported that increased HCQ dose and HCQ levels were associated with a statistically significant decrease in both CLASI and RCLASI score (P values <0.001 for both). Frances et al.[22] did not report a CLASI score but concluded that hydroxychloroquine levels correlated with cutaneous disease remission in both univariate and multivariate analyses.

SHOULD HYDROXYCHLOROQUINE LEVELS BE UTILIZED TO GUIDE AN INCREASE IN THE DOSE OF MEDICATION?

Aside from identifying patients at risk for toxicities, hydroxychloroquine levels can also be utilized to guide the need for increasing the dose of the medication. A study published in 2016 in Journal of American Academy of Dermatology included 34 patients with active cutaneous lupus (defined by CLASI or Revised CLASI score) and hydroxychloroquine level 750 ng/ml or less. The dose of hydroxychloroquine was increased by 200 mg per day and hydroxychloroquine levels were reassessed at 3 months. For those with persistently low levels, the dose was again increased by 200 mg with repeat level in 3 months. It should be noted that doses utilized in the trial exceeded those recommended by recent AAO guidelines with maximum daily dose of 800 mg/day in some study patients. With the increased hydroxychloroquine level, however, the investigators found a statistically significant decrease in both CLASI and RCLASI score (P values <0.001 for both). The hydroxychloroquine levels increased from 638 ng/ml at baseline to 1187 ng/ml [21]. This study supports the notion that an increased hydroxychloroquine level improves disease activity. Although such high doses of hydroxychloroquine are not currently recommended in clinical practice.

RETINOPATHY

Retinopathy is a feared complication of hydroxychloroquine use and the ability to identify patients at high risk for retinopathy is highly sought after by rheumatologists.

In a sentinel article, Petri et al.[23▪] in 2020 reported that blood levels of hydroxychloroquine helped predict retinal toxicity and should be used in clinical practice to guide dosing of hydroxychloroquine going forward. The study included 537 patients with SLE, 23 of whom (4.3%) developed retinal toxicity. Retinopathy was more common with higher hydroxychloroquine blood levels and usually occurred in patients treated for a duration greater than 5 years. The authors concluded that hydroxychloroquine levels should guide decreasing the dose of hydroxychloroquine to minimize toxicities [23▪].

Interestingly, an article by Lenfant et al.[24▪▪] did not find a similar significant relationship between hydroxychloroquine levels and risk for toxicity. The study included 23 patients on hydroxychloroquine with retinal toxicity and 547 healthy controls on hydroxychloroquine for more than 6 months. The study did, however, identify a relationship between cumulative dose of hydroxychloroquine and longer duration of medication use and risk for retinopathy [25]. The author hypothesized that differences in control populations may have contributed to discordant findings between studies.

OBESITY

In the past decade, guidelines for appropriate dosing of hydroxychloroquine in the setting of obesity have fluctuated. The 2011 American Academy of Ophthalmology (AAO) Guidelines recommended the maximum dosage of hydroxychloroquine as 6.5 mg/kg/day (maximum dose of 400 mg), but in the setting of obesity, ideal body weight rather that real body weight should be utilized [26]. In 2016, the AAO changed the dosing recommendations to a maximum of 5.0 mg/kg of real body (maximum dose 400 mg daily) without changes in the setting of obesity [27].

Melles et al. (2014) concluded that real body weight is a better predictor of retinal toxicity. Their data suggested that with utilization of either 5 mg/kg/day real body weight and 6.5 mg/kg/day ideal body weight, the predicted rate of hydroxychloroquine retinopathy decreased with an increase in BMI [28].

Conversely, in a 2019 study of 537 lupus patients on hydroxychloroquine, Petri et al.[23▪] found that a higher BMI was associated with a higher risk of medication toxicity (P = 0.0160). Two percentage of patients with BMI less than 20 km/m2 experienced toxicity from hydroxychloroquine compared with 9.4% of patients with BMI greater than 35 kg/m2[23▪]. In a 2021 study of 108 lupus nephritis patients, Pedrosa et al. determined that obese patients were prescribed a lower daily dose of hydroxychloroquine based upon real body weight (4.4 vs. 4.9 mg/kg/day) but interestingly, the median hydroxychloroquine blood level was higher in the obese patients (BMI ≥30 kg/m2) (P = 0.002). Although they did not collect data on toxicity, the authors hypothesize that obese patients are, therefore, at an increased risk and posit that dosing based on ideal body weight should be considered [29].

This issue was addressed in the 2016 American Academy of Ophthalmology Hydroxychloroquine Guidelines for Short, Obese patients [30]. The authors identified 64 cases of hydroxychloroquine retinopathy in obese patients, 27% of whom had been treated with hydroxychloroquine with well tolerated doses per the 2016 AAO guidelines but still developed toxicity. The authors noted overdosing in women particularly is not uncommon. They recommended use of hydroxychloroquine to maximum of 6.5 mg/kg of ideal body weight rather than the widely utilized 5 mg/kg of real body weight [30]. These conflicting results raise the question: perhaps toxicity could be avoided if the dose utilized satisfied both of the recommendations (i.e. the dose was lower than upper limit recommended in both situations)?

We do not have full understanding of why hydroxychloroquine levels may be higher in obesity. It has been hypothesized that hydroxychloroquine does not deposit in adipose tissue but rather other connective tissues [31,32]. More recent literature, however, has suggested this may not be true although an alternative feasible hypothesis has not been confirmed to our knowledge [26,33].

In conclusion, while consensus on dosing hydroxychloroquine in the setting of obesity does not exist, the presence of obesity should prompt a provider to exercise caution when choosing dose of hydroxychloroquine and consider obtaining a hydroxychloroquine level.

THROMBOSIS

Higher hydroxychloroquine blood levels may convey a protective effect against the risk of thrombosis–a feared complication of SLE. A 2021 study by Petri et al. reported a correlation between a lower mean hydroxychloroquine whole blood level and thrombotic events (720 vs. 935 ng/ml, P = 0.0247). The study included 739 patients enrolled in the Hopkins Lupus cohort, with an overall incident thrombosis rate of 5.1% (38 patients). Levels found to be protective against thrombosis in their cohort included: a mean whole blood level of at least 1068 ng/ml and a most recent whole blood level of at least 1192 ng/ml [34▪].

A letter to the editor in Arthritis and Rheumatology by Kao et al. pointed out that although high whole blood levels of hydroxychloroquine may be protective against thrombosis, this increases the risk of retinopathy [35]. Petri et al.[23▪] in 2020 reported that by dividing hydroxychloroquine levels into tertiles, most toxicity occurred with blood levels of 1177–3513 ng/ml. If the level required to mitigate risk of thrombosis is 1068 ng/ml, this leaves only a very narrow therapeutic window between treatment and toxicity.

HYDROXYCHLOROQUINE IN PREGNANCY

The guidelines for the treatment of SLE in pregnancy recommend hydroxychloroquine as first-line treatment. It is highly likely that the use of hydroxychloroquine in lupus pregnancies has contributed to lower rates of preterm delivery [36], intra-uterine grown restriction [36,37], preeclampsia [37], and lupus flares [36,38]. Additionally, lupus flares are predicted by hydroxychloroquine discontinuation during pregnancy [39].

In addition to controlling disease activity and decreasing flares, hydroxychloroquine has the potential benefit of decreasing the risk of congenital heart block based on observational data from the PATCH study in which +Ro/SSA mothers with a prior pregnancy complicated by complete heart block were treated with hydroxychloroquine 400 mg daily [40]. Hydroxychloroquine is also recommended for use with any history of obstetric or thrombotic APS to decrease the risk of thrombosis [41].

A recent observational analysis of 50 patients with rheumatic diseases enrolled in the Duke Autoimmunity in Pregnancy registry evaluated the association between hydroxychloroquine levels and premature delivery. Fifty-six percent of the patients included had underlying lupus. Of the patients with SLE, premature deliveries occurred with both hydroxychloroquine levels less than 100 ng/ml and greater than 500 ng/ml although the frequency of premature birth was much greater in the group with the lower hydroxychloroquine level (83 vs. 21%) [42]. In an ACR abstract published around this time by the same research group, the authors hypothesize that hydroxychloroquine levels of 101–500 ng/ml by their assay is ideal. They did also note that hydroxychloroquine levels decline as pregnancy progresses, with a nadir in the third trimester [43]. Given the wide variety of levels reported in the literature, further work is needed to investigate this issue.

Another article from the Duke Autoimmunity in Pregnancy registry data sought to understand the complex physiology of hydroxychloroquine levels during pregnancy including 50% increase in blood volume, increased adipose tissue, and changes in glomerular filtration, which can have marked effects on drug metabolism. The authors concluded that although the volume of distribution of hydroxychloroquine increased with the changes of progression through pregnancy, the total drug exposure did not change when compared with the same group postpartum [44].

CONCLUSION

The use of hydroxychloroquine levels can help to advance our clinical use of a medication that has been the cornerstone of lupus treatment for decades. Although the ultimate therapeutic target for hydroxychloroquine levels remains unclear, we believe that there is utility in routinely obtaining levels on at least one occasion in lupus patients. The results must be interpreted in the context of the local clinical laboratory and therapy adjusted according to the risk for undertreatment or toxicity. Additionally, the identification of nonadherent patients and attempting to improve adherence with counseling and subsequent monitoring of hydroxychloroquine levels can lead to better care of lupus patients.

On the basis of the literature available, we believe that prospectively following hydroxychloroquine levels might help avoid medication toxicity. Toxicity of hydroxychloroquine increases over time and following levels may enable appropriate surveillance and decreasing of medication dose in an effort to avoid long-term toxicity particularly after 5–10 years of exposure.

Acknowledgements

The authors wish to thank Emily Lewis for assistance in manuscript preparation.

Financial support and sponsorship

W.J.M. was supported by the Michael and Marcia Klein Professorship and Mary Piazza lupus research fund.

Conflicts of interest

There are no conflicts of interest.

REFERENCES AND RECOMMENDED READING

Papers of particular interest, published within the annual period of review, have been highlighted as:

▪ of special interest

▪▪ of outstanding interest

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