FormalPara Key Summary Points

Why carry out this study?

The pharmacokinetics of hydroxychloroquine (HCQ) are complex, and its blood concentrations vary greatly between treated patients, leading to differences in efficacy.

A low blood concentration of HCQ is a marker and predictor of disease progression in patients with SLE.

What was learned from the study?

Gender, age, weight, duration of HCQ use, systemic lupus erythematosus disease activity index (SLEDAI), platelet count, and immunoglobulin G levels might explain the differences in concentration.

Combining blood HCQ or desethylhydroxychloroquine (DHCQ) concentrations with individual patient characteristics to optimize dosage might help improve treatment outcomes in Chinese patients with SLE.

Introduction

Hydroxychloroquine (HCQ), a 4-aminoquinoline antimalarial agent, is the cornerstone of systemic lupus erythematosus (SLE) medical therapy [1, 2]. Long-term use of HCQ in patients with SLE can reduce disease activity, lower the risk of organ damage and thrombosis, improve blood glucose and lipid profiles, and increase patient survival [3].

The pharmacokinetics of HCQ are complex, and its blood concentrations vary greatly between treated patients, including healthy volunteers and adherent patients [4], leading to differences in efficacy [5]. Studies have shown a significant correlation between whole blood HCQ levels and clinical outcomes in patients with SLE [1, 6,7,8]. According to the study by Fasano et al., higher HCQ blood levels have a protective effect against disease attacks [9]. Poor compliance, as indicated by HCQ concentrations below 100 ng/ml, was found in 29% of patients with SLE [10] and can predict adverse outcomes [11]. Additionally, a low blood concentration of HCQ is a marker and predictor of disease progression in patients with SLE, with a negative predictive value of 96% at a whole blood HCQ concentration of 1000 ng/ml [12]. Therefore, investigating the factors that influence HCQ blood concentration is crucial for assessing patient compliance, evaluating treatment efficacy, and adjusting treatment plans.

HCQ is metabolized by cytochrome P450 (CYP450) enzymes to desethylhydroxychloroquine (DHCQ) and desethylchloroquine (DCQ), with DHCQ being the main active metabolite [13, 14]. Studies have suggested a correlation between blood DHCQ concentrations and HCQ efficacy [15]. So far, there have been few studies on the relationship between HCQ concentrations and clinical factors in patients with SLE, and few studies involving metabolites [16]. In this study, we analyzed various clinical factors to identify factors contributing to the wide inter-individual variation in blood HCQ and DHCQ concentrations, with the aim of guiding individual HCQ dose adjustment.

Methods

Study Design and Population

This study protocol was approved by the Ethics Committee of Nanjing Drum Tower Hospital (Approval Number: 2021-643-02). The study population was patients hospitalized in the Department of Rheumatology and Immunology at Nanjing Drum Tower Hospital from January 2020 to December 2022. Inclusion criteria were patients who met the 1997 American College of Rheumatology classification criteria [17], had been treated with oral HCQ for at least 3 months at a daily dose of 400 mg, and consented to participate in the study. Exclusion criteria were pregnant and lactating women, patients with incomplete data, patients at risk and need to use high-dose glucocorticoids (≥ 1 mg·kg−1·d−1 prednisone or equivalent doses of other hormones), and patients with poor adherence.

Data Collection

The collected data included patient basic characteristics (gender, age, weight, etc.), information on combination medication, and laboratory test results such as white blood cell (WBC), platelet, red blood cell (RBC), neutrophil percent (NEUT%), lymphocyte percent (LY%), alanine transaminase (ALT), aspartate transaminase (AST), C-reactive protein (CRP), albumin, creatinine (CR), estimated glomerular filtration rate (eGFR), blood urea nitrogen (BUN), total cholesterol (TC), triglycerides (TG), complement C3, complement C4, immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and erythrocyte sedimentation rate (ESR).

Measurement of HCQ, DHCQ Concentrations

Fasting venous blood was collected from patients early in the morning. Whole blood HCQ and DHCQ concentrations were quantified by high-performance liquid chromatography. Patients with HCQ concentration < 100 ng/ml were excluded for non-compliant with medication [6, 18, 19]. All measurements were performed at the Precision Medicine Center of Nanjing Drum Tower Hospital (Nanjing Drum Tower Hospital, Nanjing, China), using a method adapted from a previously published method [20].

Statistical Analysis

Categorical variables were expressed as frequencies and percentages, and continuous variables were expressed as means and standard deviations. Chi-square and Fisher’s exact tests were used to analyze qualitative data. Independent samples t test was used to compare the means of two independent groups. The association of influencing factors and low HCQ and DHCQ blood concentrations was assessed with binary logistic regression analysis. Statistical analysis was performed using IBM SPSS Statistics (version 26, IBM Corporation, Armonk, NY, USA). P < 0.05 was considered statistically significant.

Results

Patient Characteristics

This study analyzed 272 patients who received a daily dose of 400 mg HCQ, and their characteristics were presented in Table 1. The mean patient age was 40.63 ± 14.60 years, with 88.24% being female, and the mean body mass index (BMI) was 22.67 ± 4.26 kg/m2. 86.40% of patients were taking glucocorticoids and 49.63% patients were taking immunosuppressants. The mean HCQ concentration was 690.90 ng/ml and the mean DHCQ concentration was 431.84 ng/ml.

Table 1 Characteristics of 272 patients with systemic lupus erythematosus
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Univariate Analysis

Patients were divided into two groups based on the mean blood concentrations of HCQ and DHCQ. Patients with HCQ blood concentrations ≤ the mean (690.90 ng/ml) were classified as the low concentration group (n = 158), and patients with HCQ blood concentrations > the mean (690.90 ng/ml) were classified as the high concentration group (n = 114). For HCQ concentrations, statistically significant differences were found between the two groups in gender (P = 0.039), age (P = 0.041), weight (P = 0.029), duration of HCQ use (P < 0.001), SLEDAI (P < 0.001), platelet count (P < 0.001), RBC count (P = 0.046), complement C3 levels (P < 0.001), complement C4 levels (P < 0.001), and IgG levels (P = 0.021) (Table 2). Patients with DHCQ blood concentrations ≤ the mean (431.84 ng/ml) were categorized as the low concentration group (n = 176) and patients with DHCQ blood concentrations > the mean (431.84 ng/ml) were categorized as the high concentration group (n = 96). Gender (P = 0.013), duration of HCQ use (P < 0.001), SLEDAI (P = 0.003), platelet count (P < 0.001), ALT (P = 0.008), albumin (P = 0.034), BUN (P = 0.010), CR (P = 0.011), eGFR (P = 0.014), CRP (P = 0.026), complement C4 levels (P < 0.001), and IgG levels (P = 0.004) were related to DHCQ concentrations (Table 2).

Table 2 Results of univariate analysis of HCQ and DHCQ concentrations
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Multivariate Analysis

To determine factors independently associated with low blood HCQ and DHCQ concentrations, binary logistic regression was used to examine factors that showed significant differences (P < 0.05) in univariate analysis. Table 3 and Fig. 1 shows the data analysis results. Gender (OR = 0.23; P = 0.015; 95% CI = 0.07–0.75), age (year) (OR = 0.96; P < 0.001; 95% CI = 0.94–0.98), weight (kg) (OR = 1.04; P = 0.013; 95% CI = 1.01–1.06), duration of HCQ use (month) (OR = 0.92; P < 0.001; 95% CI = 0.89–0.95), SLEDAI (OR = 1.41; P < 0.001; 95% CI = 1.19–1.67), platelet count (× 109/l) (OR = 0.99; P < 0.001; 95% CI = 0.99–1.00), and IgG levels (g/l) (OR = 1.07; P = 0.014; 95% CI = 1.01–1.12) were associated with low HCQ concentrations. Gender (OR = 0.20; P = 0.006; 95% CI = 0.06–0.64), duration of HCQ use (month) (OR = 0.95; P < 0.001; 95% CI = 0.92–0.97), SLEDAI (OR = 1.27, P = 0.007, 95% CI = 1.07–1.51), and platelet count (× 109/l) (OR = 0.99; P < 0.001; 95% CI = 0.99–1.00) were associated with low DHCQ concentrations. In addition, we performed linear regression (Supplementary Table 1), the results also confirmed the influence of the above factors.

Table 3 Factors associated with low HCQ and DHCQ concentrations
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Fig. 1
figure 1

Binary logistic regression model results. HCQ hydroxychloroquine, DHCQ desethylhydroxychloroquine, Duration duration of HCQ use, SLEDAI systemic lupus erythematosus disease activity index, IgG immunoglobulin G

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Discussion

HCQ is commonly used as a first-line treatment for SLE and other rheumatic diseases. Its pharmacokinetic profile is complex, and blood levels vary widely between individuals. Blood concentrations of HCQ have been shown to correlate with clinical efficacy and can indicate patient compliance [6,7,8, 10]. In this study, we investigated potential factors that may affect HCQ and DHCQ concentrations by comparing patient characteristics between high and low concentration groups. Binary logistic regression analysis revealed that gender, age, weight, duration of HCQ use, SLEDAI, platelet count, and IgG levels were influencing factors for low blood HCQ concentrations. Duration of HCQ use, platelet count, SLEDAI and gender were influencing factors for low blood DHCQ concentrations.

Although several studies have investigated the relationship between HCQ concentrations and clinical factors in patients with rheumatic diseases, most have not examined DHCQ concentrations [5, 16, 21]. Our study was one of the few in which both HCQ and DHCQ concentrations were measured in the Chinese population with SLE. In addition, to avoid the confounding effects of dose, we only included patients taking a daily dose of 400 mg HCQ.

We found higher concentrations of HCQ and DHCQ in female patients. This gender difference in drug concentration may be attributed to differences in CYP-mediated metabolism, the influence of sex hormones on absorption, and the difference of fat percentage in body composition [22]. Estrogen has been shown to downregulate CYP3A4 expression [23].

HCQ has a large apparent volume of distribution (over 2000 l) due to poor plasma protein binding (about 50%) and high tissue binding (including blood cells) [7, 24]. Our study results were consistent with this finding. We observed higher HCQ blood levels in patients with low body weight, and the expected delay in reaching steady-state concentrations (3–4 months) due to the accumulation of HCQ in tissues may explain the positive correlation between HCQ and DHCQ concentrations and duration of HCQ use [25, 26].

HCQ is primarily metabolized by CYP450 enzymes in the liver to a variety of active metabolites, with approximately one-quarter of the prototype being cleared through the kidneys [27,28,29]. A previous study that included 111 patients with SLE receiving long-term HCQ therapy observed higher HCQ concentrations in patients with renal insufficiency [5]. In our study, we did not find an association between renal function and HCQ or DHCQ concentrations, as it was challenging to recruit patients with SLE on long-term HCQ due to their clinical severity and use of higher doses of immunosuppressive drugs and glucocorticoids. However, we did observe higher HCQ blood levels in older patients, which may be due to decreased renal function.

The SLEDAI score, which measures lupus disease activity, is strongly correlated with HCQ concentration [21]. The effective concentration threshold of HCQ in clinical practice is still controversial [30]. Costedoat-Chalumeau et al. [12] recommended a whole-blood HCQ target concentration of 1000 ng/ml for patients with SLE. In our study, only 19.49% of patients (n = 53) achieved this target. For the goal of 750 ng/ml in a meta-analysis by Garg et al. [30], 37.5% (n = 102) of patients achieved it. Our study showed that higher HCQ concentrations were associated with lower SLEDAI in the population with a mean HCQ concentration of 690.90 ng/ml. Thus, we found that higher HCQ concentrations were beneficial in reducing disease activity even in the range below previously recommended concentration. In addition, higher DHCQ concentrations were also found to be beneficial in reducing disease activity.

Our study also found a significant association between low platelet counts and low HCQ and DHCQ blood concentrations. We believe this finding may be related to the fact that we measured the whole blood rather than serum concentration. HCQ has been shown to bind to platelets and other blood cells, which can result in its retention in the blood and limit its distribution to eliminated organs [31]. This may explain why low platelet count was associated with lower HCQ and DHCQ blood concentrations in our study.

This study has some limitations. First, this study was limited by patient compliance, which may lead to bias. In our study, the mean HCQ concentration was significantly lower than that in other populations. Although we excluded patients with blood levels below 100 ng/ml, some patients may still be noncompliant with medication. One explanation for this could be that the study population were hospitalized patients, who tended to have more active SLE and were more likely to be nonadherent. Second, the sample size of this study was relatively small, and only hospital attenders were included, which may limit the generalizability of the findings to the broader population. Third, Lee et al. showed that HCQ and DHCQ blood concentrations may be influenced by genetic polymorphisms in CYP450 enzymes [32], but our study did not consider genetic factors. Future studies with larger sample sizes and more diverse populations, as well as consideration of genetic factors, may provide further insights into the factors that influence HCQ and DHCQ blood concentrations in patients with SLE.

Conclusions

In summary, this study found that several factors, including gender, age, weight, duration of HCQ use, SLEDAI, platelet count, and IgG levels, influenced the blood concentrations of HCQ. The duration of HCQ use, platelet count, SLEDAI, and gender were found to be influencing factors for low blood DHCQ concentrations. Notably, higher HCQ and DHCQ blood concentrations were beneficial in reducing disease activity, even if the concentrations were maintained below the recommended concentration. These findings suggested that examining the related factors and optimizing the dose according to individual characteristics might help to improve the efficacy of HCQ in Chinese patients with SLE.