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A.M. Risitano et al.
treatment with clinical benefit entered a long-term extension study (ClinicalTrials.gov; NCT03181633).
Endpoints
The primary efficacy endpoint was change in LDH concentra- tion from baseline at day 28. Secondary efficacy parameters were change of hemoglobin concentration from baseline at days 28 and 84 and LDH change from baseline at day 84. Safety, tolerability, pharmacokinetics, and pharmacodynamics were also investigated. Laboratory assessments comprised hematology, chemistry and urinalysis. Patient-reported outcomes were measured at baseline and during the study via the validated Functional Assessment of Chronic Illness Therapy (FACIT)–Fatigue scale. Haptoglobin, bilirubin, reticulocytes, GPI-deficient clone size of erythrocytes (type III) and granulocytes (type II and III unless type III values provided), and percentage of C3 fragment-coated erythrocytes were also monitored. Soluble C5b-9 was evaluated as an exploratory endpoint (non-GLP); C5a was not monitored. Transfusion history up to 3 years prior to and during the study was collected.
Assay methods
Plasma danicopan concentrations were determined by liquid chromatography. Pharmacodynamics were determined by meas- uring serum AP activity with an AP Wieslab assay. Plasma Bb con- centration, serum factor D, C3, and C4 concentrations were also monitored. All aforementioned complement tests were conducted in a central laboratory using commercial kits. C3 fragment deposi- tion on erythrocytes was measured using flow cytometry with FITC-conjugated anti-human C3d antibody (see the Online Supplement for details).
Statistical analysis
This was a proof-of-concept, first-in-patients, exploratory, phase II study. The sample size was determined based on the very limited number of untreated PNH patients and the exploratory nature of this study to evaluate effectiveness of danicopan. Given the small sample size, only descriptive and exploratory statistics were utilized to present results for continuous biochemical and quality-of-life measurements. Patients who discontinued treat- ment during the trial were not replaced. Missing values were not imputed. To summarize categorical data, frequency counts and percentages are presented. The Pearson correlation coefficient (Pearson r) was used to examine the relationship between two variables. The quantitative analysis between pharmacokinetics (plasma danicopan concentration) and pharmacodynamics (AP inhibition) was conducted with nonlinear regression using the simple Emax dose-response equation (Prism 5.02, GraphPad Software, La Jolla, CA, USA).
Results
Patients’ characteristics
Eleven patients were screened. Ten untreated patients with hemolytic PNH were enrolled and received danico- pan. These patients’ baseline characteristics are presented in Table 1 and Online Supplementary Table S1. Their medi- an age was 33 years (range, 17-63 years) and median dis- ease duration was 5.9 years (range, 0-14 years). The mean GPI-deficient clone size was 79% for granulocytes and 32% for erythrocytes. Overt hemolysis was demonstrated by elevated LDH levels (1416±540 U/L, corresponding to 5.7±2.17 times ULN), increased reticulocyte count (154±69×109/L), increased total bilirubin (1.3±0.74 mg/dL), and reduced haptoglobin (5.8±2.9 mg/dL). The
baseline hemoglobin concentration was heterogeneous among patients (9.8±1.8 g/dL); two patients received transfusions in the 12 weeks preceding study entry, with one of these patients also having a medical history of aplastic anemia.
Study disposition and safety
The disposition of patients in the study is shown in Online Supplementary Figure S1. Two patients started dan- icopan at 100 mg tid and increased to 150 mg tid, and eight started at 150 mg tid. Increases to 175 mg and 200 mg tid were performed in eight and four patients, respec- tively. All ten patients reached day 28 and are included in the primary endpoint evaluation. Two discontinued before day 84: one because of a serious adverse event, elevated aspartate aminotransferase/alanine aminotrans- ferase coincident with breakthrough hemolysis, which resolved without sequelae; the other withdrew for per- sonal reasons unrelated to safety. All patients were eval- uated until they left the study or reached day 84 (n=8). Nine patients (90%) developed at least one adverse event during treatment; only one was serious (described above). In total, 38 unique treatment-emergent adverse events were recorded, of which four were considered possibly related and two probably related to danicopan. The most frequent events were PNH-related (hemolysis and its signs or symptoms) and infections, usually of the upper respiratory tract (Table 2). With few exceptions, adverse events were mild and resolved during the study. There were no clinically significant changes in other key laboratory parameters during treatment (Online Supplementary Table S2).
Pharmacokinetics and pharmacodynamics
Danicopan was bioavailable with dose-proportional exposure (peak serum concentration [Cmax] and area under the curve [AUC]) at 100, 150, and 175 mg tid doses, demonstrated by intensive pharmacokinetic and pharma- codynamic profiling on days 6, 13, and 20 (Figure 1A; Online Supplementary Table S3), whereas trough concentra- tions assessed biweekly at single time points from days 28 to 84 were variable (Online Supplementary Table S4). Trough drug concentration was much more variable than AUC and Cmax. For example, at the 175 mg tid dose, the concentrations varied from 62.6 to 223.1 ng/mL. There was appreciable inter-patient variability, as anticipated for a study with a small number of patients. One of two patients who received 200 mg tid was not included in day 56 analyses because the sample was not available (missed study visit). Per protocol, no patients were receiving 200 mg tid by day 20 (pharmacokinetic sampling was per- formed on days 6, 13, and 20). Plasma factor D concentra- tion did not change during treatment (Online Supplementary Figure S2A). As anticipated from its mecha- nism of action, treatment resulted in selective AP inhibi- tion (Figure 1B, upper panel; Online Supplementary Table S4) with no effect on classical pathway activity (Online Supplementary Figure S2B). Notably, AP activity ≤10% was observed at all time points except at 0 and 8 h when dan- icopan concentration was lowest (Figure 1B) indicating that the AP activity in patients may not be fully inhibited at the end of the 8 h dosing period. Pharmacokinetic/phar- macodynamic analysis showed a dose-response relation- ship between danicopan and AP inhibition (Online Supplementary Figure S2D).
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