Atypical hemolytic uremic syndrome (aHUS) is a rare form of thrombotic microangiopathy (TMA) arising from deregulation of the complement alternative pathway at the level of the surface of endothelial membrane, thereby causing uncontrolled complement activation.1

aHUS is characterized by a triad of non-autoimmune hemolytic anemia, thrombocytopenia, and acute kidney failure (AKI). Other complications may arise, including damage to various organs, such as the liver, lungs, heart, intestine, and the central nervous system.2,3 In 50%–60% of patients with aHUS, deregulation results from mutations in the genes encoding regulatory proteins of the complement alternative pathway, including factor H, factor I, factor B, C3, thrombomodulin, and membrane cofactor protein (MCP). These mutations favor the persistent activation of the C5b-9 complex, also called membrane attack complex (MAC), thereby resulting in endothelial damage.1,4 Deregulation can also be induced by antibodies against complement proteins, such as factor H, a phenomenon that is most frequently described in pediatric patients (25%–50%), as compared to adults (5%–10%).3,5

C3 glomerulopathy, including dense deposit disease (DDE) and C3 glomerulonephritis (C3GN), is a kidney disease caused by hyperactivation of the complement alternative pathway that induces the formation of C3-dominant glomerular deposits. Deregulation can result either from mutations in regulatory proteins or from the presence of autoantibodies against these proteins. C3 nephritic factor (C3NFs) is the most frequent autoantibody in this disease. The clinical manifestations of the 2 subtypes of C3-glomerulopathy include the presence of micro- or macrohematuria, with different levels of proteinuria, associated with progressive deterioration of kidney function.6

Eculizumab (Soliris®) is a recombinant humanized monoclonal antibody specifically targeted against the complement protein C5 that inhibits the cleavage of C5 by the C5 convertase into C5a (a proinflammatory anaphylatoxin) and C5b. This activity results in impaired cell lysis due to inhibition of MAC generation.7 In aHUS, eculizumab confers a rapid response by which hematological parameters normalize and kidney function improves.8,9 Eculizumab has demonstrated to be also a treatment option for C3 glomerulopathy, although the evidence available is more limited. Thus, this agent has been reported to cause a reduction of proteinuria and the stabilization of kidney function.10–12

To date, the efficacy of eculizumab is primarily assessed using markers of hemolysis (haptoglobin and LDH), platelet count, markers of kidney function, complement hemolytic activity (CH50), and circulating levels of the C3 complement. Despite the scarcity of data on the pharmacokinetics (PK) and pharmacodynamics (PD) of eculizumab, some studies associate eculizumab concentrations with biomarkers of complement activity, such as CH50, which measures total functional status of the classical complement pathway.13,14

According to the EPAR for eculizumab, serum pre-treatment or trough concentrations of 50–100 μg/mL are sufficient for essentially complete inhibition of terminal complement activity and maintenance of sustained free C5 concentrations.7 However, these therapeutic margin is controversial, as some studies report higher concentrations.8,9,13,15–17 In addition, pharmacokinetic studies show the impact of some covariates on the pharmacokinetics of eculizumab, such as weight and C5b9 concentrations.13,15 In the light of the high inter-individual variability of the pharmacokinetic profile of eculizumab, we conducted a study to assess and describe the association between eculizumab concentrations and complement inhibition in patients with aHUS and C3 glomerulopathy. A secondary goal was to determine a therapeutic margin that ensures therapeutic efficacy.

This study was conducted to determine and describe eculizumab concentrations in patients with aHUS and C3 glomerulopathy and establish an optimal therapeutic margin to achieve therapeutic efficacy (i.e., complete blockade of the complement). Overexposure was observed in most patients with aHUS, considering the therapeutic margin of a target pre-treatment concentration of 50–100 μg/mL, established in the EPAR.7

Based on the results obtained, eculizumab concentrations exceeded those considered to be within therapeutic margin according to the EPAR.7 This finding has been previously reported by other authors. Thus, Gatault et al. reported that 8 in 9 patients exhibited mean pre-treatment concentrations of eculizumab >100 μg/mL and 5 patients showed concentrations >300 μg/mL.13 The pilot study carried out by Reiss et al. revealed pre-treatment eculizumab concentrations >100 μg/mL in all patients, with high intra-individual variability.16 Volokhina et al. observed that the 11 patients included had concentrations of 36–459 μg/mL (n = 27) and 40–772 μg/mL (n = 90) during the induction and maintenance phases, respectively.17 Greenbaum et al. documented pre-treatment eculizumab concentrations >50 μg/mL (both, in the induction and maintenance phase) in the 22 patients included in their study, with post-treatment values <700 μg/mL.9 Finally, Passot et al. reported that all the 40 patients included had eculizumab concentrations ≥100 μg/mL, with concentrations ranging from 124 ± 9 to 1065 ± 121 μg/mL.15

The results obtained in our study on the association of eculizumab concentrations and complement inhibition during the maintenance phase consistently show that these concentrations exceed the standard, with a cut-off of 149.0 μg/mL. Jodele et al. reported that when eculizumab concentrations were >30 μg/mL, CH50 activity in enriched serum showed a plateau of CH50 inhibition.20 Volokhina et al. observed that all samples with eculizumab concentrations ≥50 μg/mL showed adequate complement inhibition (CH50≤12%).17

The results of this study show an inverse relationship between eculizumab concentrations and the effect of body weight. This finding is consistent with the results of previous studies, such as that conducted by Gatault et al. where weight was found to be the determining factor of observed inter-individual variability.13 In addition, clearance was lower and half-life was higher in patients with a lower body weight and a higher half-life, an effect frequently associated with other antibodies.22

In relation to genetic studies, carriers of MCP and CFI mutations had an improved prognosis, as compared to patients with pathogenic CFH variants, being the latter the most frequently reported mutations.4 Haplotypic homozygosity of the CHH (CFH tgtgt) and MCP (MCPggaac) genes has been demonstrated to be associated with a higher risk for aHUS.23 However, to date, the impact of pharmacogenetics on exposure to and pharmacokinetics of eculizumab has not yet been evaluated. In our cohort, complement genetic testing confirmed the presence of pathogenic variants, more specifically in the CFH, MCP, and CFI genes, in 29.4% of patients. Additionally, 70.6% had one or more aHUS risk polymorphisms in the CFH and/or MCP genes, 7 in homozygosity.

An inverse relationship was also observed between serum creatinine and pre- and post-treatment eculizumab concentrations, which reflects a potential improvement of kidney function. The hematocrit decreased as eculizumab concentrations increased, with optimal concentrations for the normalization of the hematocrit ranging from 100 to 200 μg/mL. The same occurred with hemoglobin and platelet count, although differences were not statistically significant. However, these findings might be considered an adverse effect of high eculizumab concentrations (with anemia and thrombocytopenia having been reported as adverse drug reactions in clinical trials).

Considering post-treatment concentrations, a direct relationship was observed between platelet count and post-treatment concentrations. Thus, high eculizumab concentrations are associated with an improvement in the hematological manifestations of the disease (in this case, post-treatment). Based on pre-treatment concentrations, an improvement was not observed in hematological parameters when concentrations were in the upper limit (>800 μg/mL). This finding led us to establish the optimal concentration range below that range (400–600 μg/mL for hemoglobin and the hematocrit and 600–800 μg/mL for platelet count). No statistically significant differences were observed either in pre- or post-treatment sC5b-9 concentrations, assumingly due to the low number of patients who underwent sC5b-9 testing. Volokhina et al. reported that eculizumab concentrations decreased below the target therapeutic margin 1 week after the first infusion in 2 patients with high baseline sC5b-9 concentrations.17 Jodele et al. documented that initial clearance of eculizumab was faster in patients with higher baseline sC5b-9 concentrations.20 Of note, a recent study revealed that eculizumab can bind to sC5b-9 in-vitro, which suggest that partial clearance of this agent may occur in a sC5b-9 complex in the first infusion.24 Fakhouri et al. reported that higher sC5b-9 concentrations at discontinuance of eculizumab were independently associated with aHUS relapse risk.25 For unclear reasons, SC5b-9 remained detectable in a significant proportion of patients despite optimal C5 blockade. This finding suggests C5 cleavage via other pathways different from the C5 convertase.24,26–29

Finally, we observed a high inter-individual variability in eculizumab clearance (48.9% CV), whereas intra-individual variability was lower (11.9% CV). Ter Avest et al. observed a higher intra-individual variability of 34.4%, which they explained by variations in the volume of C5 available, for example due to infections.30 In relation to pharmacokinetics, the median clearance in the study patients was 8.7 (6.4-11.2) mL/h; median volume of distribution was 3.6 (2.2–5.3) l; and median half-life was 12.3 (10.8–13.0), which are consistent with the values reported in the EPAR for eculizumab.7

This study has some limitations. Firstly, the number of patients with C3 glomerulopathy was very limited; therefore, conclusive results cannot be drawn on this population of patients. Anyway, it was assumed that the behavior of this agent could resemble that observed in patients with aHUS. Secondly, the lower limit of detection of the technique used for monitoring complement blockade was 13.49%, as compared to 10% reported in previous studies. However, the 2 limits make it possible to differentiate optimal from suboptimal values of complement blockade induced by eculizumab. Finally, more samples could not be obtained at time points other than pre- and post-treatment. This would have facilitated the characterization of eculizumab pharmacokinetics and determination of inter- and intra-individual variability. However, this enabled the inclusion of a higher number of patients, since participation in the study did not involve undergoing further procedures other than those included in standard care.

In conclusion, this study describes eculizumab pharmacokinetics in patients with aHUS and C3 glomerulopathy and establishes higher optimal concentrations to achieve complement blockade than those reported on the EPAR. To definitely determine the efficacy of eculizumab and personalize treatments, it is necessary that population pharmacokinetic models or algorithms are designed including eculizumab concentrations, CH50 blockade, body weight, and sC5b9 concentrations. Optimization of eculizumab dosing based on the monitoring of pharmacokinetics can only be achieved through close follow-up, which will ensure treatment efficacy.

The study protocol was approved by the Ethics Committee of Hospital Universitari Vall d'Hebron (protocol code EPA(AG)01/2020(5579).

This study was carried out with funds granted by the Spanish Foundation of Hospital Pharmacy (FEFH) and the Spanish Society of Hospital Pharmacy (SEFH) via the 2020–2021 call for grants for working groups.

Marta Miarons, Alba Pau Parra and Natalia Ramos contributed to study design and conception; Alba Pau Parra, Natalia Ramos, Silvia Manrique-Rodríguez, Monica Climente, Laura García Quintanilla, Ángel Escolano and Marta Miarons contributed to patient selection and data collection; data analysis and interpretation were carried out by Alba Pau Parra, Janire Perurena-Prieto and Marta Miarons; Alba Pau Parra and Marta Miarons drafted the manuscript. All authors reviewed critically and contributed to improve the manuscript and approved the final version for publication.

This study describes eculizumab pharmacokinetics in patients with aHUS and C3 glomerulopathy and reveals higher concentrations than those documented, and establishes pre-treatment concentrations >149.0 μg/mL as optimal to achieve complement blockade.

The results obtained demonstrate that complement blockade increases at higher pre-treatment concentrations, and serum creatinine decreases at higher pre- and post-treatment concentrations. Finally, we observed a high inter-individual variability in eculizumab clearance, whereas intra-individual variability was lower.