A PK/PD relation for HCQ has been shown in both serum and whole blood from SLE patients [5,6,7, 10,11,12, 17, 18, 20] but the interest of each approach has never been assessed. This study shows, as detailed below, that monitoring whole-blood levels appears more suitable than using serum levels for assessing the PK/PD relation in daily clinical practice. It also shows that serum and whole-blood HCQ levels correlate strongly (and better than for DCQ) and that the mean ratio of serum/whole-blood levels for HCQ were 0.53 ± 0.15. Additionally, it proposes for the first time serum HCQ cut-off levels to assess severe non-adherence, based on data from a large cohort of patients.
Studies addressing the PK/PD relation found that higher whole-blood HCQ levels were associated with less SLE activity and fewer flares [5,6,7,8,9,10,11,12, 29]. Using serum levels, Mok et al. also reported that SLE patients with serum HCQ levels > 500 ng/mL tend to have lower mean disease activity scores and a lower incidence of disease flares . In agreement with this result, our study shows that patients with SLEDAI scores < 4 had higher HCQ serum levels than other patients (P = 0.008). However, in the multivariate analysis, only whole-blood HCQ levels were independently associated with active SLE (P = 0.023), an indication that whole-blood HCQ levels are more informative than the serum level about the PK/PD relation. It has been suggested that whole-blood measurements might be more reproducible and stable than serum measurements [8, 29]. In general, serum levels are valuable when the drug is not sequestered in red blood cells. Given that HCQ diffuses into these cells , the handling of samples, for example centrifugation, could influence HCQ partitioning between red blood cells and serum and produce misleading serum HCQ levels and thus a false pharmacological interpretation. The duration and force of centrifugation are known to significantly influence the levels of HCQ and DCQ in serum . Red blood cell partitioning is also sensitive to temperature, pH, and blood collection procedures . In addition, autoimmune haemolytic anaemia, which can occur in SLE, would probably modify serum HCQ levels considerably. All of these elements point out the need to minimise analytical variation by rigorous standardisation of centrifugation when serum is used for drug monitoring. Here, we observed substantial interindividual variability in HCQ and DCQ levels in both serum and whole blood. The magnitude of this variability was quite similar between the two biological matrices, probably because of the rigorous standardisation of centrifugation requested for the clinical trial. In this context, our study might have underestimated the interindividual variability in serum HCQ levels in daily clinical practice.
Since PLUS study failed to demonstrate the benefit of adapting daily HCQ dose to its whole-blood levels , drug monitoring is mainly recommended today to assess non-adherence to HCQ treatment in SLE patients . We previously reported that patients with very low whole-blood HCQ levels admitted severe non-adherence to the treatment, and we proposed a cut-off of 200 ng/mL that has proved to be effective in our daily practice since then. Others have chosen different cut-offs (500, 100, < 15 ng/mL, or undetectable levels) or have used serum levels [5, 11, 12, 14, 16,17,18,19]. As far as we know, the present study is the first to propose serum HCQ cut-off points corresponding to our cut-off of 200 ng/mL (or 100 ng/mL as an alternative definition) to identify non-adherent patients. The strength of our study is that the ratio of serum/whole-blood HCQ could be determined from the data of 573 patients. At a serum HCQ cut-off of 106 ng/mL, the sensitivity was 0.87 (95% CI 0.76–0.94) and the specificity 0.89 (95% CI 0.72–0.98). HCQ levels undetectable by one method were also undetectable by the other. Further research to validate the best cut-off point for clinical practice requires confirmation in a larger cohort of SLE patients.
In this study, the best correlation of HCQ levels with dose per kg was observed with weight measured as TBW and LBM. Among patients weighing more than 90 kg, neither the dose per kg of LBM (P = 0.60) nor that of TBW (P = 0.18) was statistically associated with whole-blood HCQ. Nonetheless, this result should be interpreted with caution given the low number of patients (n = 33). LBM is known to correlate better with the pharmacokinetics of hydrophilic drugs than TBW does, especially with their volume of distribution, while TBW is a better parameter for lipophilic drugs . The lipophilicity of HCQ may explain in part why the relation between whole-blood HCQ level and dose per kg of LBM is no better than that of dose per kg of TBW. Finally, the relation between whole-blood HCQ and dose per kg of IBW was worse than that with dose per kg of TBW. A French multicentre prospective study in patients with cutaneous lupus erythematosus similarly did not observe a relation between whole-blood HCQ and dose per kg of IBW. Interestingly, it has been recently shown that TBW also correlates better with retinal toxicity than IBW, which suggests that TBW should be used to prevent the onset of this ocular toxicity . Taken together, these results confirm that TBW is more appropriate than IBW for determining the HCQ dose to be prescribed in SLE patients.
Our study has some limitations. First, it was necessary to enrich our PLUS cohort to obtain patients with different levels of SLE activity (since patients with severe SLE were not included in the PLUS study) and to have patients with severe non-adherence. Second, whole-blood HCQ levels were measured in 2 different laboratories but we have previously reported that the methods used by both laboratories are comparable . Third, the evaluation of sensitivity and specificity of HCQ cut-offs in serum was based on data from a small cohort of non-adherent patients (n = 68). We note that our estimates are conservative: we used only patients with whole-blood HCQ levels between 200 and 300 ng/mL to calculate the specificity; it would have been much higher had we used patients with higher levels, since none of them had serum levels lower than 106 ng/mL.
In conclusion, our data support the use of whole blood rather than serum as the matrix for drug monitoring of HCQ levels in SLE patients to assess the PK/PD relation. However, when whole blood is not available, our results support the use of serum HCQ to assess non-adherence with a cut-off of 106 ng/mL, corresponding to 200 ng/mL and undetectable levels by one method also undetectable by the other.