Unbound Fraction of Clozapine Significantly Decreases with Elevated Plasma Concentrations of the Inflammatory Acute-Phase Protein Alpha-1-Acid Glycoprotein

The current study consisted of (1) a spiking experiment and (2) a prospective patient study. Inclusion of study samples took place at the University Medical Center Utrecht, an academic teaching hospital in the center of the Netherlands, with annually approximately 28,000 clinical visits, 15,000 day-care hospitalizations, and 334,000 outpatient visits, and at the Diakonessenhuis, a community hospital in Utrecht, Zeist, and Doorn with annually approximately 24,000 clinical visits, 23,000 day-care hospitalizations, and 380,000 outpatient visits. The Institutional Review Board of the University Medical Center Utrecht determined that the study was not subject to the Medical Research Involving Human Subjects Act, and the University Medical Center Utrecht Biobank approved the use of anonymized remnant material for this study.

In the spiking experiment, blood sample aliquots (0.65 mL) from three randomly selected patients using clozapine were spiked with 0.025 mL (AGP spike 1) and 0.060 mL (AGP spike 2) of 20 mg/mL AGP (Sigma-Aldrich, St Louis, MO, USA) stock solution, simulating a final AGP concentration nearly 1.5- to 2.3-fold higher than the upper limit of the normal AGP concentration range. In this regard, the amounts of AGP in the spiking experiments reflect the in-vivo amount because AGP plasma concentrations could be up to 2- to 5-fold higher in acute-phase reactions. [21]

The AGP stock solution of 20 mg/mL was prepared in phosphate-buffered saline buffer. Aliquots were then incubated for 1 hour at room temperature to establish an equilibrium. The total and unbound clozapine concentrations were determined in the pre- and post-spiked sample aliquots. All samples were prepared and determined in duplicate.

For the prospective patient study, remnant EDTA plasma samples from all patients using clozapine aged 18 years or older were collected following routine therapeutic drug monitoring of total clozapine plasma concentrations at the University Medical Center Utrecht and the Diakonessenhuis Utrecht from October 2017 to December 2017. Samples were excluded if the blood sample was drawn within 8 hours after the last clozapine intake or in the case of a suspicion of intentional clozapine intoxication, ensuring that an adequate trough concentration had established and that only samples with non-intentional elevated concentrations were included. As anonymized remnant samples were used, multiple samples originating from the same patient were allowed for inclusion.

Samples were aliquoted and stored as plasma EDTA at − 80 °C for the measurement of total clozapine concentrations and at − 20 °C for the AGP determination. For the measurement of an unbound plasma concentration, aliquot samples were stored as ultra-filtrate at − 20 °C after the protein filtering step, as described below.

Total and protein unbound clozapine plasma concentrations were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) at the laboratory of clinical pharmacy and pharmacology of the University Medical Center Utrecht. To determine unbound clozapine plasma concentrations, plasma samples were ultra-centrifuged with a filter as an additional sample preparation step before LC-MS/MS quantitation. The liquid chromatography mobile phases consisted of mass spectrometry grade water mixed with formic acid (0.1%) and acetonitrile mixed with formic acid 0.1%, both ordered at Sigma-Aldrich. The LC-MS/MS system included a Thermofisher Hypersil GOLD PFP 50 × 2.1 mm column (Waltham, MA, USA) pressurized by a Surveyor MS Plus Pump (Waltham). Signal was detected with a Surveyor Autosampler plus (Waltham) and a Thermofisher TSQ Quantum access mass spectrometer with a Heated Electrospray Ionization Probe (Waltham). Data gathering and system control were performed by Xcalibur software (high-performance liquid chromatography) and Thermo Scientific Trace Finder software (mass spectrometry). Using Merck Millipore centrifree YM-30 cat4104 Amicon filters (Darmstadt, Germany), ultra-centrifugation took place at 2500 rpm (559×g) at 25 °C for 30 minutes using a Hettich Rotina 380R bucket swing centrifuge (Geldermalsen, the Netherlands). The ultra-filtrate and samples were stored in Eppendorf (Nijmegen, the Netherlands) micro-seed containers of 1.5 mL. The linearity of the assay ranged from 2.5 ng/mL to 570 ng/mL with R² values exceeding 0.99. Spiked quality-control samples at low, medium, and high concentrations were within 15% of the nominal values. The LC-MS/MS method was validated in accordance with the European Medicines Agency guideline on bioanalytical method validation [22]. Clozapine unbound fraction was calculated by dividing unbound plasma concentration by the total plasma concentration.

Alpha-1-acid glycoprotein plasma concentrations were determined using an immunoturbidimetric assay according to the manufacturer’s instruction (Roche Diagnostics GmbH, Mannheim, Germany) at the Clinical Laboratory of Chemistry and Hematology at Leiden University Medical Center. Spiked quality-control samples at low, medium, and high concentrations were within 15% of the nominal values. Samples with an AGP concentration ≥1.2 g/L and thus exceeding the upper limit of the reference concentration of normal physiologic AGP were assumed to be elevated.

Differences in mean unbound fraction between the pre- and spiked samples calculated from the spiking experiment were analyzed using a paired t test [Graphpad Prism 7.02 (La Jolla, CA, USA)]. Differences in mean unbound fraction and in total plasma concentrations between the normal and elevated AGP groups in the patient study were analyzed using an unpaired t test (Graphpad Prism 7.02). These results were plotted in scatter graphs and box-and-whisker plots using Graphpad Prism 7.02. An alpha level of 0.05 was considered statistically significant for all performed statistical tests.

In all three patients, the unbound fraction gradually decreased after each AGP addition (Fig. 1). After adding 0.025 mL of AGP solution (AGP spike 1), the unbound fraction relatively decreased by 30.7% in patient A (from 1.50% to 1.04%), by 30.2% in patient B (from 1.92% to 1.34%) and by 22.2% in patient C (from 1.35% to 1.05%) in comparison to the aliquots not spiked with AGP. The mean relative decrease in unbound fraction (28.3%) was statistically significant (p = 0.032). After adding 0.060 mL of AGP solution (AGP spike 2), the unbound fraction relatively decreased by 41.3% in patient A (from 1.50% to 0.88%), relatively decreased by 51.0% in patient B (from 1.92% to 0.94%), and relatively decreased by 34.1% in patient C (from 1.35% to 0.89%), in comparison to aliquots not spiked with AGP (Fig. 1). This mean relative decrease in unbound fraction (43.4%) was also significant (p = 0.048).

A total of 26 remnant samples from patients using clozapine were collected and included in the study (Table 1). Study samples with elevated AGP concentrations (n = 6) showed a 25% significantly lower mean unbound fraction [1.22% vs. 1.65%, mean difference = − 0.43% (95% confidence interval − 0.816 to − 0.0443), p = 0.03] in comparison to samples with normal AGP concentrations (n = 20). Both clozapine unbound concentrations and total clozapine plasma concentrations did not significantly differ between study samples with elevated AGP and those with normal AGP concentrations [7.2 ng/mL vs. 6.3 ng/mL (mean difference = 0.9 ng/mL, 95% confidence interval − 4.45 to 2.54, p = 0.590) and 525 ng/mL vs. 479 ng/mL (mean difference = 47 ng/mL, 95% confidence interval − 217 to 310, p = 0.72), respectively] (Fig. 2).