Introduction: Accumulation of the photoreactive heme precursor protoporphyrin IX (PPIX) is a signature of the disease pathophysiology for both erythropoietic protoporphyria (EPP) and X-linked protoporphyria (XLP), and PPIX levels correlate with disease severity and light tolerance. Additionally, an elevated ratio of circulating metal-free PPIX vs. metal-bound PPIX is observed in patients with protoporphyrias, and the extent of metal-free elevation is distinguishable between EPP (85-100%) and XLP (50-85%). Therefore, the accurate determination of metal-free PPIX and metal-bound PPIX is of particular interest in patients with EPP or XLP.
Methods: Novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays were developed and validated to quantify circulating metal-free and zinc (Zn)-complexed PPIX in whole blood and plasma. Each of the four assays were developed and validated according to applicable regulatory guidance for bioanalytical assay validation with regard to accuracy, precision, sensitivity, and selectivity. The minimum required sample volume for each assay was 200 µL, compared to >1 mL for most currently available protoporphyria-related assays.
During bioanalytical method development, rapid degradation of metal-free PPIX (≥90%) and Zn-PPIX (up to 96%) was observed in plasma samples within 24 hours under ambient light conditions. In contrast, whole blood was less susceptible to photodegradation (i.e., ≤20% for both metal-free PPIX and Zn-PPIX) under the same conditions. The most effective method for maintaining sample integrity was reduction of light exposure during sample collection, handling, and processing. Implementation of light-reduction controls was used during method validation and facilitated improved stability of metal-free PPIX and Zn-PPIX in plasma and whole-blood samples. For the metal-free PPIX plasma assay validation, the effect of hemolysis was evaluated due to the apparent sequestration of metal-free PPIX in red blood cells. The bioanalytical work informed specific sample-collection procedures to mitigate instability and hemolysis, such as the use of opaque consumables for sample collection, controls for plasma aliquoting, and operating under low-light conditions for all sample handling and processing.
BEACON is a Phase 2, randomized, open‐label, parallel‐arm trial (ACTRN12622000799752) of approximately 22 participants who will receive oral, once‐daily administration of 20 or 60 mg of bitopertin for 6 months. Baseline light tolerance was assessed using 2 patient-reported outcomes measures during a 2- to 4-week screening period. Participants were asked to conduct weekly sun exposure challenges and time to first prodromal symptom (itching, burning, tingling, etc.) was averaged for the screening period.
Results: More than 600 clinical samples from 17 participants in the BEACON study have been collected as of 06 June 2023 and analyzed using the validated porphyrin assays. Measured metal-free PPIX baseline values ranged from 1440 - 34,100 ng/mL (2.6 - 61 µM) in whole blood and 177 - 2560 ng/mL (0.31 - 4.5 µM) in plasma ( n=16). Baseline percentage of intra-patient plasma/whole-blood concentrations ranged from 2.5 - 16%. Each participant's baseline values were converted to erythrocyte metal-free PPIX concentrations using measured hematocrit levels and ranged from 3590 - 88400 ng/mL (6.4 - 157 µM). These values were comparable to data previously reported in the literature (6-139 µM 1). Additionally, higher baseline whole-blood metal-free PPIX concentrations were associated with decreased averages for time to prodrome (Table 1).
Conclusion: Fully-validated LC-MS/MS assays to quantify metal-free PPIX and Zn-PPIX in plasma and whole blood were used to analyze clinical samples from the BEACON study evaluating bitopertin in EPP. PPIX values were consistent with previously reported values for patients with EPP. The methods also provided improved accuracy and specificity for metal-free vs. metal-complexed PPIX and reduced sample volume requirements. In addition, higher baseline whole-blood PPIX values were associated with decreased light tolerance within the trial population, supporting its use as a measure of disease severity in EPP.
Disclosures
Mangus:Bristol-Myers Squibb: Current equity holder in publicly-traded company; Disc Medicine: Current Employment, Current equity holder in publicly-traded company; Agios: Current equity holder in publicly-traded company. Mensing:Disc Medicine: Current Employment, Current equity holder in publicly-traded company. Savage:Disc Medicine: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months. Chin:Disc Medicine: Current Employment, Current equity holder in publicly-traded company. Chen:Disc Medicine: Current Employment, Current equity holder in publicly-traded company. Liu:Disc Medicine: Current Employment, Current equity holder in publicly-traded company. Yang:Disc Medicine: Current Employment, Current equity holder in publicly-traded company.
