An Oral Complement Factor D Inhibitor for Paroxysmal Nocturnal Hemoglobinuria by Antonio M

Home , Factor D

Danicopan: an oral complement factor D inhibitor for paroxysmal nocturnal hemoglobinuria by Antonio M. Risitano, Austin G. Kulasekararaj , Jong Wook Lee, Jaroslaw P. Maciejewski, Rosario Notaro, Robert Brodsky, Mingjun Huang, Michael Geffner, and Peter Browett

Haematologica 2020 [Epub ahead of print]

Citation: Antonio M. Risitano, Austin G. Kulasekararaj , Jong Wook Lee, Jaroslaw P. Maciejewski, Rosario Notaro, Robert Brodsky, Mingjun Huang, Michael Geffner, and Peter Browett. Danicopan: an oral complement factor D inhibitor for paroxysmal nocturnal hemoglobinuria. Haematologica. 2020; 105:xxx doi:10.3324/haematol.2020.261826

Publisher's Disclaimer. E-publishing ahead of print is increasingly important for the rapid dissemination of science. Haematologica is, therefore, E-publishing PDF files of an early version of manuscripts that have completed a regular peer review and have been accepted for publication. E-publishing of this PDF file has been approved by the authors. After having E-published Ahead of Print, manuscripts will then undergo technical and English editing, typesetting, proof correction and be presented for the authors' final approval; the final version of the manuscript will then appear in print on a regular issue of the journal. All legal disclaimers that apply to the journal also pertain to this production process. Title: Danicopan: an oral complement factor D inhibitor for paroxysmal nocturnal hemoglobinuria Short Title: Danicopan, oral factor D inhibitor for PNH

Antonio M. Risitano,1 Austin G. Kulasekararaj,2 Jong Wook Lee,3 Jaroslaw P. Maciejewski,4 Rosario Notaro,5,6 Robert Brodsky,7 Mingjun Huang,8 Michael Geffner,9 and Peter Browett10

1Federico II University of Naples, Naples, Italy and AORN Moscati, Avellino, Italy; 2King's College Hospital-NHS Foundation Trust, NIHR/Wellcome King’s Clinical Research Facility, London, UK and King’s College London, London, UK; 3Seoul St. Mary’s Hospital, The Catholic University of Korea, Seoul, Republic of Korea; 4Cleveland Clinic, Cleveland, OH, USA; 5Core Research Laboratory, Istituto per lo Studio, la Prevenzione e la Rete Oncologica (ISPRO), Firenze, Italy; 6Azienda Ospedaliera-Universitaria Careggi, Firenze, Italy; 7Johns Hopkins University School of Medicine, Baltimore, MD, USA; 8Achillion, Inc., A Subsidiary of Alexion Pharmaceuticals, Inc., New Haven, CT, USA; Alexion Pharmaceuticals, New Haven CT, USA; 9Achillion Inc., A Subsidiary of Alexion Pharmaceuticals, Inc., Blue Bell, PA, USA; 10University of Auckland, Auckland, New Zealand

Title character count: 88 Short title character count: 42 Abstract word count: 250 (250 maximum) Text word count: 3861 (4,000 maximum) Figures and tables: 2 tables and 4 figures Supplementary figures and tables: 3 supplementary figures (separate file) and 6 supplementary tables (separate file) References: 34

Presented as an oral presentation at the 24th European Hematology Association Congress, Amsterdam, NL, 15 June 2019. Presented as a poster presentation at the 61st annual meeting of the American Society of Hematology, Orlando, FL, 8 December 2019.

Correspondence to: Antonio M. Risitano, M.D., Ph.D. Hematology and Hematopoietic Stem Cell Transplant Unit AORN S. Giuseppe Moscati Contrada Amoretta 83100 Avellino, Italy Phone +39 347 5786759 [email protected]

KEY POINTS

• Danicopan, first in class, oral factor D inhibitor, showed efficacy notably LDH reduction

and rise in hemoglobin in untreated PNH patients

• C3-opsonized RBC clone size stayed low despite rise in GPI-deficient RBC clone size

supporting benefit of danicopan in control of extravascular hemolysis

ABSTRACT

Paroxysmal nocturnal hemoglobinuria (PNH) is characterised by complement-mediated intravascular hemolysis (IVH) due to absence of complement regulators CD55 and CD59 on affected erythrocytes. Danicopan is a first-in-class oral proximal, complement alternative pathway factor D (FD) inhibitor. Therapeutic FD inhibition was designed to control IVH and prevent C3-mediated extravascular hemolysis (EVH). In this open-label, phase 2, dose-finding trial, 10 untreated hemolytic PNH patients received danicopan monotherapy (100-200 mg thrice daily). Endpoints included change in lactate dehydrogenase (LDH) at day 28 (primary) and day

84 and hemoglobin. Safety, pharmacokinetics/pharmacodynamics, and patient-reported outcomes were measured. Ten patients reached the primary endpoint; two later discontinued: one for a serious adverse event (elevated aspartate aminotransferase/alanine aminotransferase coincident with breakthrough hemolysis, resolving without sequelae) and one for personal reasons unrelated to safety. Eight patients completed treatment. IVH was inhibited, demonstrated by mean decreased LDH (5.7 times upper limit of normal [ULN] at baseline vs 1.8 times ULN

[day 28] and 2.2 times ULN [day 84]; both p<0.001). Mean baseline hemoglobin, 9.8 g/dL, increased by 1.1 (day 28) and 1.7 (day 84) g/dL (both p<0.005). No significant C3 fragment deposition occurred on GPI-deficient erythrocytes. Mean baseline Functional Assessment of

Chronic Illness Therapy–Fatigue score, 34, increased by 9 (day 28) and 13 (day 84) points. Most common adverse events were headache and upper respiratory tract infection. This phase 2, monotherapy data shows proximal inhibition with danicopan provides clinically meaningful IVH inhibition and hemoglobin improvement in untreated PNH patients, without evidence of C3- mediated EVH. Registered at www.clinicaltrials.gov (#NCT03053102).

Introduction

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematologic disease characterised by chronic intravascular hemolysis (IVH), severe thrombophilia, and bone marrow failure.1 PNH is due to somatic mutations of the phosphatidylinositol N-acetylglucosaminyltransferase subunit A

(PIGA) gene in hematopoietic stem cells that impair biosynthesis of glycosylphosphatidylinositol

(GPI) anchors and surface expression of GPI-linked proteins.2-4 While bone marrow failure is secondary to T cell–mediated immune attack that spares PIGA-mutated hematopoietic stem cells,5,6 both hemolysis and thromboembolism are complement-mediated. GPI-deficient erythrocytes (and platelets) lack GPI-linked complement regulators CD557 and CD598 and are exquisitely vulnerable to complement activation, which occurs continuously and spontaneously due to “C3 tick-over”9 and acutely with specific triggers. PNH treatment was revolutionised by introduction of the terminal complement C5 inhibitor eculizumab, which has proven effective in addressing intravascular hemolysis10,11 and thromboembolism,12 with significant impact on long- term survival.13,14 Recently, a new long-acting C5 inhibitor dosed every eight weeks, ravulizumab, has demonstrated non-inferiority to eculizumab in controlling IVH.15,16 Although the significant benefits of C5 inhibition in the treatment of PNH patients has been clearly demonstrated, the hematologic benefit may be hampered by emergence of C3-mediated extravascular hemolysis (EVH) from early phases of complement activation,17,18 which C5 inhibition cannot address.19-21 Thus, alternative strategies of complement inhibition are required to improve PNH treatment, and agents are in development to address this and other unmet patient needs, including improved convenience.22

The complement cascade has three activating pathways (alternative, classical, and lectin- mannose) that merge at the key complement component C3; from this level (which is amplified

5 by alternative pathway [AP] proteins), the effector pathway starts, with generation of anaphylatoxins and the membrane attack complex (MAC).23 Novel strategies of therapeutic complement inhibition in development aim to intercept the complement cascade upstream of C5, some targeting upstream at pathway-specific initiating events.22 During AP initiation, the serine protease complement factor D (FD) cleaves factor B, leading to AP C3 convertase generation.

Danicopan (ACH-4471, ACH-044471, ALXN2040) is a first-in-class, oral, small-molecule FD inhibitor that prevents new AP C3 convertase formation.24 Consequently, proximal inhibition at the level of FD blocks AP-initiated upstream events and up to 80% of classical or lectin pathway initiated downstream events via amplification-loop inhibition.25 In vitro, danicopan inhibited both AP-mediated hemolysis and C3 fragment deposition on red blood cells (RBCs) from PNH patients.24 Phase 1 data from healthy human volunteers in single and multiple ascending dose trials showed that danicopan was generally well-tolerated and could achieve inhibition of AP complement activity.26 Further, this work identified thrice-daily danicopan 200 mg as a safe and effective dose/regimen.26 For PNH, targeting FD inhibition with a small molecule represents a potentially important treatment advancement because proximal AP inhibition may disable both terminal complement activation (inhibiting MAC–mediated IVH) and C3 fragment opsonization

(preventing EVH), with additional convenience of oral administration.

We investigated the FD inhibitor danicopan as single-agent treatment for untreated PNH, aiming to control IVH while preventing C3-mediated EVH.

METHODS

Study design

This international, open-label, single-arm, dose-finding, phase 2 study investigated danicopan in patients with hemolytic PNH not receiving complement inhibitor treatment. This trial was approved by regulatory agencies/local ethics committees and conducted according to

International Conference on Harmonisation and Good Clinical Practice Standards. Achillion,

Inc., a subsidiary of Alexion Pharmaceuticals, Inc., designed and sponsored the study, with advice from the investigators. All participants provided written informed consent. The trial is registered at www.clinicaltrials.gov as #NCT03053102.

Patients

This study was conducted from March 2017 to November 2018. Adult, untreated PNH patients were enrolled with hemoglobin <12 g/dL (and adequate reticulocytosis per investigator),

GPI-deficient granulocyte or Type III erythrocyte clone size ≥10%, lactate dehydrogenase

(LDH) ≥1.5 times upper limit of normal (ULN), platelets ≥50,000 µL, and willingness to be vaccinated for N. meningitidis, H. influenzae, and S. pneumoniae. Investigators used their clinical judgement to assess whether patients had enough bone marrow capacity to derive potential benefit by comparing the level of pre-entry hemoglobin to the absolute reticulocyte count. None of the subjects were receiving eculizumab due to lack of availability and/or patient willingness.

Treatment

Patients received oral danicopan at a starting dose of 100 mg or 150 mg, thrice daily

(TID). The starting dose was based on Phase 1 data showing that danicopan doses of 200 to 600 mg reached peak plasma level within 1 to 2 hours and were well-tolerated, and that 200 mg TID was effective on PNH RBCs.22,26 Dose escalations were permitted based on hemolysis control,

7 assessed by LDH, per investigator assessment in stepwise increments up to 200 mg thrice daily.

Dose escalation criteria for the first 28 days were specified in the study protocol with potential dose escalation points occurring at days 7 and 14 (Supplementary Appendix). Dose escalation was permitted thereafter and was done in consultation between the investigator and sponsor based on the hemoglobin response; absolute reticulocyte count, LDH, and indirect bilirubin were reviewed to evaluate evidence for possible additional clinical benefit of dose escalation. Because this study was the first treatment experience with danicopan in PNH patients, dose escalations were approached cautiously, especially when moving to the higher doses in the study (from 150 to 175 mg and 175 to 200 mg) or if ALT had fluctuated relative to baseline. Patients were instructed to take their medication approximately every 8 hours. On study center days when their labs were drawn, the morning dose was to be administered in the study center by the site personnel following safety and pharmacokinetic (PK) assessments. Additionally, patients were instructed to take each dose with food, including prior to intensive PK sampling labs on Days 1,

13, and 28. Treatment duration was 84 days; patients completing treatment with clinical benefit entered a long-term extension (ClinicalTrials.gov; NCT03181633).

Endpoints

The primary efficacy endpoint was change in LDH from baseline at day 28. Secondary efficacy parameters were hemoglobin change 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 were collected through hematology, chemistry and urinalysis. Patient-reported outcomes were measured at baseline and during 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

8 granulocytes (Type II and III unless Type III values provided), and percentage of C3 fragment coated erythrocytes were also monitored. sC5b-9 was evaluated as an exploratory endpoint (non-

GLP); C5a was not monitored. Transfusion history up to three years prior and during the study were captured.

Assay methods

Plasma danicopan concentrations were determined by liquid chromatography.

Pharmacodynamics were determined by measuring serum AP activity by AP Wieslab assay. Plasma Bb concentration, serum FD, C3, and C4 concentrations were also monitored. Plasma Bb concentration, serum CP activity, FD concentration and C3 concentration were also monitored. All aforementioned complement tests were conducted by a central lab using commercial kits. C3 fragment deposition on erythrocytes was measured using flow cytometry with FITC conjugated anti-human C3d antibody (see Supplement for details).

Statistical analysis

This is a proof of concept, first-in-patients exploratory Phase 2 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. Due to the small sample size, only descriptive and exploratory statistics were utilized to present results for continuous biochemical and quality-of-life measurements. Discontinued patients were not replaced. Missing values were not imputed. To summarise categorical data, frequency counts and percentages are presented.

The Pearson correlation coefficient (Pearson’s r) is used to examine the relationship between two variables. The quantitative analysis between pharmacokinetics (plasma danicopan concentration)

9 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).

Data sharing

Alexion will consider requests for disclosure of clinical study participant-level data provided that participant privacy is assured through methods like data de-identification, pseudonymization, or anonymization (as required by applicable law), and if such disclosure was included in the relevant study informed consent form or similar documentation. Qualified academic investigators may request participant-level clinical data and supporting documents

(statistical analysis plan and protocol) pertaining to Alexion-sponsored studies. Further details regarding data availability and instructions for requesting information are available in the

Alexion Clinical Trials Disclosure and Transparency Policy at http://alexion.com/research- development.

RESULTS

Patient characteristics

Eleven patients were screened. Ten untreated patients with hemolytic PNH were enrolled and received danicopan; baseline characteristics are in Table 1 and Supplemental Appendix

Table 1. Median age was 33 years (range 17-63 years) and median disease duration was 5.9 years

(range 0-14 years). 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,

10 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). Mean baseline hemoglobin 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

Study disposition can be found in Supplemental Figure 1 in the Appendix. Two patients started danicopan at 100 mg thrice daily and increased to 150 mg thrice daily, and eight started

150 mg thrice daily. Increases to 175 mg and 200 mg thrice daily were performed in eight and four patients, respectively. All 10 patients reached day 28 and are included in the primary endpoint evaluation. Two discontinued before day 84: one for a serious adverse event of elevated aspartate aminotransferase/alanine aminotransferase coincident with breakthrough hemolysis, which resolved without sequelae; the other withdrew for personal reasons unrelated to safety. All patients were evaluated 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 (identified above).

In total, 38 unique treatment-emergent adverse events were recorded, of which four were considered possibly related and two probably related to danicopan. 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 to other key laboratory parameters during treatment (Supplementary Appendix Table 2).

Pharmacokinetics and pharmacodynamics

Danicopan was bioavailable with dose proportional exposure (Cmax and AUC) at 100,

150, and 175 mg thrice daily doses demonstrated by intensive pharmacokinetic and pharmacodynamic profiling on days 6, 13, and 20 (Figure 1A; Supplementary Appendix Table

3), whereas trough concentrations assessed biweekly at single time points from days 28 to 84 were variable (Supplementary Appendix Table 4). 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 the protocol, no patients were receiving 200 mg TID by day 20 (PK sampling was performed on days 6, 13, and 20). Plasma FD concentration did not change during treatment (Supplementary

Appendix Figure 2A). As anticipated by its mechanism of action, treatment resulted in selective

AP inhibition (Figure 1B, the upper panel; Supplementary Appendix Table 4) with no effect on classical pathway activity (Supplementary Appendix Figure 2B). Notably, AP activity ≤10% was observed at all time points except at hours 0 and 8 when danicopan concentration was lowest

(Figure 1B) indicating that the AP activity in patients may not be fully inhibited at the end of the eight-hour dosing period. Pharmacokinetic/pharmacodynamic analysis showed a dose-response between danicopan and AP inhibition (Supplementary Appendix Figure 2D).

Efficacy

Primary endpoint

Change in LDH from baseline to day 28 was the primary efficacy endpoint. A significant reduction was observed among all 10 patients from a mean value of 5.7±2.17 times ULN at

12 baseline to 1.8±1.03 times ULN at day 28 (p<0.001; Figure 2A, Table 1), demonstrating achievement of the primary endpoint. The percentages of patients showing LDH <3 times ULN,

<2 times ULN, and <1 time the ULN were 90%, 60%, and 40%, respectively.

Secondary endpoints

Significant LDH reduction from baseline was sustained throughout the study: day 56,

2.3±1.41 times ULN (p<0.005); day 84, 2.2±1.04 times ULN (p<0.001; Figure 2A). The percentages of patients showing LDH <3 times ULN, <2 times ULN, and <1 times ULN were

71%, 43%, and 43% at day 56, and 75%, 37.5%, and 25% at day 84, respectively. Danicopan is a highly permeable drug and even in patients with high BMI, plasma concentrations do not appear to cause a clinically significant change in effect. Fluctuations in LDH indicated that low-level residual IVH remained in most patients, with possible transient exacerbations; this was due to transient weaker AP inhibition around predose periods, as described above, in addition to increase of susceptible GPI-deficient erythrocytes during treatment (see below). Nevertheless, only two breakthrough hemolytic events were recorded by the investigator as adverse events

(Table 2); a third patient experienced recurrent subclinical breakthrough episodes as a consequence of inadequate control of complement activation.

Treatment with danicopan translated to improvement of anemia: mean hemoglobin increased from 9.8 g/dL at baseline (range, 6.9 g/dL to 12.0 g/dL) to 10.9 g/dL at day 28 (range,

8.4 to 14.1; p<0.005), 10.9 g/dL at day 56 (range, 8.5 to 13.1; p<0.005), and 11.5 g/dL at day 84

(range, 8.7 to 13.7, p<0.005; Figure 2B and Table 1). Mean increase versus baseline was 0.9 g/dL at day 28 and 1.7 g/dL at day 84. In the 12 weeks preceding study entry, two patients received transfusions (Figure 3A). One of these patients, who had aplastic anemia (not receiving

13 immunosuppressive therapy), had received five transfusions totalling ten units. During treatment, this patient received three transfusions totalling seven units. The second patient had one transfusion (two units) during breakthrough hemolysis in the setting of viral infection; in this case, danicopan treatment was not detrimental to the course of infection, but (irrespective of its possible effect on other complement pathways) did not effectively counteract the transient acute complement activation (possibly due to PK/PD reasons). The remaining patients in the trial were transfusion independent through the 84 days of treatment. Accompanying control of IVH and improvement of anemia, patient-reported outcomes were assessed. The mean FACIT–Fatigue score at baseline was 34 and increased by 9 and 13 points at days 28 and day 84, respectively

(p<0.05; Figure 3B and Table 1).

Control of IVH by danicopan was further confirmed by changes in other laboratory parameters. Danicopan significantly increased the percentage of GPI-deficient erythrocytes

(56±19.9% at day 84 vs 32±24.6% at baseline; p=0.001), whereas no change was observed for

GPI-deficient granulocytes (Figure 4A). Total bilirubin decreased after danicopan treatment

(0.6±0.23 mg/dL at day 84 vs 1.3±0·74 mg/dL at baseline, p<0.05; Figure 4B). A sustained but not statistically increase in haptoglobin was observed (15.3±16.08 mg/dL at day 84 vs 5.8±2.89 mg/dL at baseline, p=0.15; Supplementary Appendix Figure 3A), as was a transient decrease in free hemoglobin (83±51 mg/dL at day 28 vs 138±132 mg/dL at baseline, p=0.26; Supplementary

Appendix Figure 3B). Absolute reticulocyte count decreased quickly and was sustained with treatment (81±33.6×109/L at day 84 vs 154±69×109/L at baseline, p<0.05; Figure 4C). Additional laboratory results can be found in Supplementary Appendix Table 6.

Exploratory endpoints

To investigate the mechanistic effect of proximal complement inhibition by danicopan on

PNH, complement biomarkers were monitored. Bb fragment, an activation product of factor B, tracks complement AP activation in vivo. Plasma Bb level was significantly elevated at baseline

(2.24±0.77 µg/mL) compared with healthy volunteers (0.84±0.212 µg/mL; p<0.05). After danicopan, Bb level was significantly reduced: day 28, 0.84±0.84 µg/mL (p<0.005); day 56,

0.47±0.09 µg/mL (p<0.005); day 84, 0.47±0.06 µg/mL (p<0.005; Figure 1C). In contrast to residual AP activity, Bb level remained consistently low irrespective of subtherapeutic plasma danicopan levels around predose periods (Figure 1B, middle panel). A strong positive correlation was found between Bb and LDH (Pearson’s r=0.80, p<0.0001; Supplementary Appendix Figure

2E), supporting Bb as a reliable biomarker of in vivo AP activation in PNH and, therefore, its value for monitoring efficacy. Danicopan also showed strong linear correlations with Bb and

LDH (negative), as did AP with Bb and LDH (positive); there was no correlation of classical pathway activity with any of these parameters (Supplementary Appendix Table 5), validating the role of danicopan in AP inhibition and subsequent in vivo changes of Bb and LDH. Additional laboratory results can be found in Supplementary Appendix Table 6.

There was a slight increase in serum C3 (114.2±17.3 mg/dL at day 84 vs 102.2±20.2 mg/dL at baseline, p=0.08; Supplementary Appendix Figure 2C), likely from reduced C3 consumption because of upstream complement blockade. Importantly, C3 fragment deposition on erythrocytes was very low (<0.5% of erythrocytes) throughout treatment (Figure 4A).

sC5b-9 was normal at baseline and remained relatively constant over time (data not shown).

DISCUSSION

Current PNH treatments target C5 inhibition.10-14 Novel complement inhibitors in development aim to address unmet needs of PNH patients.22 Here, we describe a novel approach to PNH treatment, which aims to change the current paradigm of PNH therapy by improving hemoglobin levels in addition to the reduction of hemolysis, with the added advantage and convenience of oral administration. We investigated danicopan, a first-in-class oral FD inhibitor, which blocks the proximal complement cascade upstream of C5 at the level of AP initiation and amplification. In untreated PNH patients, danicopan monotherapy resulted in inhibition of IVH, with significant LDH reduction at day 28 (primary endpoint) and throughout treatment duration.

In contrast with standard C5 inhibitor therapies, the inhibition of IVH observed during danicopan treatment was not associated with persistently increased bilirubin and reticulocyte count, or with the emergence of C3 deposition on surviving GPI-deficient erythrocytes, in agreement with its anticipated effect on EVH. Concomitant inhibition of IVH and prevention of C3-mediated EVH resulted in improvement of anemia, with a mean hemoglobin gain of 1.7 g/dL after 12 weeks of treatment. Consistent with these findings, all patients exhibited significant increases in the percentage of GPI-deficient erythrocytes, confirming their extended half-life in vivo.

Furthermore, all patients had improvement in FACIT-Fatigue quality-of-life measurements;

FACIT-Fatigue scores were used in this proof-of-concept study due to the lack of validated instruments for patient reported outcomes in PNH. Additional patient reported outcome instruments will be utilized in a Phase 3 trial. With the caveat of limited sample size and exposure, no safety concerns emerged during the study other than those described; in particular, infectious events were rare, clinically mild, and self-limiting, irrespective of unchanged danicopan treatment. Although the short study duration through the primary endpoint limits the

16 ability to draw robust conclusions, the data do not appear to support the postulated increased risk of infectious complications (due to upstream inhibition of the complement cascade)27, in agreement with in vitro data showing that killing of encapsulated and unencapsulated meningococci was nearly unaffected relative to eculizumab.28,29

Danicopan is the first oral complement inhibitor treatment demonstrating efficacy and safety in PNH patients as monotherapy in a Phase 2 study. The clinical effects observed were achieved irrespective of a low-level residual IVH, which remained detectable in some patients.

This residual IVH is likely the consequence of the increase of GPI-deficient erythrocytes susceptible to complement-mediated hemolysis and the pharmacokinetic/pharmacodynamic characteristics of danicopan. Danicopan plasma level and AP activity also correlated with in vivo biomarkers (LDH and Bb), and Bb seems a reliable indicator of complement activation in vivo during danicopan treatment. Based upon the results of the Phase 1 single and multiple ascending dose trials in healthy human volunteers, 200 mg TID was shown to be both safe and efficacious.26 However, in this patient cohort, full blockade of AP activity was not consistently achieved in all patients, irrespective of individual dose adjustment and the broad dose ranges used during the study. These observations suggest that residual IVH is due to low residual AP activity observed in some patients, which may be better inhibited by a more potent second- generation FD inhibitor analogue that will be assessed for safety and efficacy in a Phase 2 trial

(Clinicaltrials.gov, NCT04170023). In a concurrent Phase 2 trial, patients (n=12) on a stable regimen of eculizumab with hemoglobin <10 g/dL and who were transfusion dependent (≥1

RBC transfusion within 12 weeks of screening) received oral danicopan 100–150 mg TID, with possible response-based dose escalation to 200 mg TID at predefined time points. The addition of danicopan led to clinically and statistically significant reductions in frequency of RBC

17 transfusions and in the number of transfusion units in patients compared to a history of eculizumab treatment alone.30

Clinical development of proximal complement inhibitors has been motivated by the description of C3-mediated EVH as a mechanism driving significant anemia and limiting hematologic benefit in some PNH patients on eculizumab and other C5 inhibitors.17,31 Indeed, it is conceivable that, in combination with C5 inhibitors, proximal inhibitors can address C3- mediated EVH by preventing generation of C3 convertase, eventually leading to better hematologic response in PNH patients.17,31 Moreover, it has been hypothesised that proximal inhibitors, by preventing generation of downstream C5 convertases, can be effective even in absence of terminal inhibitors (i.e. eculizumab or other C5 inhibitors). Danicopan is the first compound demonstrating that proximal complement inhibitors can be used safely and effectively as monotherapy in PNH. By disabling the initiating event of complement activation, danicopan prevents generation of C5 convertases, obviating the need for downstream C5 inhibition.

Additionally, specific targeting of AP can preserve classical and lectin pathway–mediated anti- microbial activity. The landscape of complement inhibition in the treatment of PNH will continue to evolve with the availability of proximal inhibitors. In addition to danicopan, other oral agents targeting the AP, such as FB inhibitors, are under investigation for the treatment of

PNH.22,31,32 Proximal complement inhibitors also include subcutaneously administered agents targeting C3; one of these agents was reported to be effective in a recent Phase 3 trial in patients with PNH.33 All of these approaches look promising for the treatment of PNH and clinical data should tell us very soon what are the viable treatment options are in terms of safety and efficacy, and how we can best utilize in the appropriate patients (i.e., monotherapy vs add-on treatment).

Preclinical data seem to suggest that AP inhibitors may be as effective as terminal complement

18 blockade with clinical differences mostly due to the specific PK/PD profile of the individual inhibitor, rather than to its target in the complement cascade.22,31 Indeed targeting the AP, as

PNH is a disease due to AP dysregulation (i.e. continuous, spontaneous C3 tick-over, eventually exacerbated at time of additional complement activation), possible applications in other diseases will require an understanding of their pathogenic mechanisms.34

In conclusion, this study demonstrates that danicopan appears to be well-tolerated and showed clinically meaningful IVH inhibition and hemoglobin improvement in untreated PNH patients. This is the first evidence that a proximal complement inhibitor, used as monotherapy, can clinically impact PNH by inhibiting IVH and preventing EVH. While second-generation compounds with improved pharmacokinetics and pharmacodynamics are in development, this study paves the way to improved hematologic response and novel standards of care for hemolytic

PNH patients with an easier mode of administration.

Acknowledgments

The study was sponsored and entirely supported by Achillion Inc., a subsidiary of

Alexion Pharmaceuticals, Inc. We thank the patients and investigators, as well as their staff, who participated in this trial: Serena Marotta, Luana Marano and Fabiana Cacace (Naples), Petra

Muus and Shreyans Gandhi (London), Federica Barone (Florence), Sung-Soo Park (Seoul), Paul

Hamilton (Auckland). In addition, we thank Heather Robison (employee of Achillion Inc., a subsidiary of Alexion Pharmaceuticals, Inc.) and Steven Podos, Danny Shin and Julia Catini

(Alexion employees and former employees of Achillion Inc., a subsidiary of Alexion

Pharmaceuticals, Inc.) for their assistance in writing this manuscript and The Curry Rockefeller

Group (funded by Achillion Inc., a subsidiary of Alexion Pharmaceuticals, Inc.) for their editorial assistance.

Authorship Contributions

MH and MG developed the study protocol, with the contribution of AMR, AGK, RN, RB and PB. AMR, AGK, JWL, and PB recruited and treated patients, and collected the data. AMR,

MH and MG analysed and interpreted the data, and wrote the manuscript; AGK, JWL, JPM, RN,

RB and PB contributed to the manuscript and approved its final version.

Correspondence: Antonio M. Risitano, Hematology and Hematopoietic Stem Cell Transplant Unit, AORN S. Giuseppe Moscati, Contrada Amoretta 83100, Avellino, Italy. Phone +39 347 5786759. [email protected]

Conflict of Interest Disclosures

AMR has received research support from Alexion, Novartis, Alnylam and Ra Pharma, lecture fees from Alexion, Novartis, Pfizer and Apellis, served as member of advisory/investigator board for Achillion, Alexion, Apellis, Biocryst, Novartis, Roche, and Samsung, and served as consultant for Amyndas.

AGK has served as on advisory boards for Alexion, Celgene, Novartis, Ra Pharma, and

Regeneron and has received travel grants from Achillion, Celgene, and Ra Pharma.

JWL has received grants from Alexion and Achillion, has served as a member of an advisory board for Alexion and Apellis, has received honoraria from Alexion, and has served as consultant to AlloVir.

JPM has received lecture honorarium from Alexion, Novartis, and Celgene and served as a consultant for Novartis.

RN has received lecture fees from Alexion.

RB has served on scientific advisory boards for Achillion and Alexion.

PB has served as a member of an advisory board for Merck, Janssen, Roche and AbbVie, has served as an associate editor for the Internal Medicine Journal, and received research funding from Roche, Beigene, and Amgen.

MH is an Alexion employee and have equity ownership in the same company.

MG was an Achillion, Inc., a subsidiary of Alexion Pharmaceuticals, Inc. employee and had equity ownership in the same company.

REFERENCES

1. Luzzatto L, Notaro R. Paroxysmal nocturnal hemoglobinuria. In: Handin RI, Lux SE,

Stossel TP, eds. Blood, principles and practice of hematology 2ed. Philadelphia: Lippincott

Williams & Wilkins; 2003:319-334.

2. Takeda J, Miyata T, Kawagoe K, et al. Deficiency of the GPI anchor caused by a somatic mutation of the PIG-A gene in paroxysmal nocturnal hemoglobinuria. Cell. 1993;73(4):703-711.

3. Miyata T, Yamada N, Iida Y, et al. Abnormalities of PIG-A Transcripts in Granulocytes from Patients with Paroxysmal Nocturnal Hemoglobinuria. N Eng J Med. 1994;330(4):249-255.

4. Medof ME, Gottlieb A, Kinoshita T, et al. Relationship between decay accelerating factor deficiency, diminished acetylcholinesterase activity, and defective terminal complement pathway restriction in paroxysmal nocturnal hemoglobinuria erythrocytes. J Clin Invest. 1987;80(1):165-

174.

5. Rotoli B, Luzzatto L. Paroxysmal nocturnal haemoglobinuria. Baillieres Clin Haematol.

1989;2(1):113-138.

6. Luzzatto L, Risitano AM. Advances in understanding the pathogenesis of acquired aplastic anaemia. Br J Haematol. 2018;182(6):758-776.

7. Nicholson-Weller A, March JP, Rosenfeld SI, Austen KF. Affected erythrocytes of patients with paroxysmal nocturnal hemoglobinuria are deficient in the complement regulatory protein, decay accelerating factor. Proc Natl Acad Sci U S A. 1983;80(16):5066-5070.

8. Holguin MH, Fredrick LR, Bernshaw NJ, Wilcox LA, Parker CJ. Isolation and characterization of a membrane protein from normal human erythrocytes that inhibits reactive lysis of the erythrocytes of paroxysmal nocturnal hemoglobinuria. J Clin Invest. 1989;84(1):7-

17.

9. Lachmann PJ, Halbwachs L. The influence of C3b inactivator (KAF) concentration on the ability of serum to support complement activation. Clin Exp Immunol. 1975;21(1):109-114.

10. Hillmen P, Young NS, Schubert J, et al. The Complement Inhibitor Eculizumab in

Paroxysmal Nocturnal Hemoglobinuria. N Engl J Med. 2006;355(12):1233-1243.

11. Brodsky RA, Young NS, Antonioli E, et al. Multicenter phase 3 study of the complement inhibitor eculizumab for the treatment of patients with paroxysmal nocturnal hemoglobinuria.

Blood. 2008;111(4):1840-1847.

12. Hillmen P, Muus P, Dührsen U, et al. Effect of the complement inhibitor eculizumab on thromboembolism in patients with paroxysmal nocturnal hemoglobinuria. Blood.

2007;110(12):4123-4128.

13. Kelly RJ, Hill A, Arnold LM, et al. Long-term treatment with eculizumab in paroxysmal nocturnal hemoglobinuria: sustained efficacy and improved survival. Blood. 2011;117(25):6786-

6792.

14. Loschi M, Porcher R, Barraco F, et al. Impact of eculizumab treatment on paroxysmal nocturnal hemoglobinuria: a treatment versus no-treatment study. Am J Hematol.

2016;91(4):366-370.

15. Lee JW, Sicre de Fontbrune F, Wong Lee Lee L, et al. Ravulizumab (ALXN1210) vs eculizumab in adult patients with PNH naive to complement inhibitors: the 301 study. Blood.

2019;133(6):530-539.

16. Kulasekararaj AG, Hill A, Rottinghaus ST, et al. Ravulizumab (ALXN1210) vs eculizumab in C5-inhibitor-experienced adult patients with PNH: the 302 study. Blood.

2019;133(6):540-549.

17. Risitano AM, Notaro R, Marando L, et al. Complement fraction 3 binding on erythrocytes as additional mechanism of disease in paroxysmal nocturnal hemoglobinuria patients treated by eculizumab. Blood. 2009;113(17):4094-4100.

18. Hill A, Rother RP, Arnold L, et al. Eculizumab prevents intravascular hemolysis in patients with paroxysmal nocturnal hemoglobinuria and unmasks low-level extravascular hemolysis occurring through C3 opsonization. Haematologica. 2010;95(4):567-573.

19. Luzzatto L, Risitano AM, Notaro R. Paroxysmal nocturnal hemoglobinuria and eculizumab. Haematologica. 2010;95(4):523-526.

20. Risitano AM, Notaro R, Luzzatto L, Hill A, Kelly R, Hillmen P. Paroxysmal nocturnal hemoglobinuria--hemolysis before and after eculizumab. N Engl J Med. 2010;363(23):2270-

2272.

21. Notaro R, Sica M. C3-mediated extravascular hemolysis in PNH on eculizumab:

Mechanism and clinical implications. Semin Hematol. 2018;55(3):130-135.

22. Risitano AM, Marotta S. Toward complement inhibition 2.0: Next generation anticomplement agents for paroxysmal nocturnal hemoglobinuria. Am J Hematol.

2018;93(4):564-577.

23. Taylor RP, Lindorfer MA. Mechanisms of Complement-Mediated Damage in

Hematological Disorders. Semin Hematol. 2018;55(3):118-123.

24. Yuan X, Gavriilaki E, Thanassi JA, et al. Small-molecule factor D inhibitors selectively block the alternative pathway of complement in paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. Haematologica. 2017;102(3):466-475.

25. Harboe M, Ulvund G, Vien L, Fung M, Mollnes TE. The quantitative role of alternative pathway amplification in classical pathway induced terminal complement activation. Clin Exp

Immunol. 2004;138(3):439-446.

26. Wiles JA, Galvan MD, Podos SD, Geffner M, Huang M. Discovery and Development of the Oral Complement Factor D Inhibitor Danicopan (ACH-4471). Curr Med Chem.

2020;27(25):4165-4180.

27. Sprong T, Roos D, Weemaes C, et al. Deficient alternative complement pathway activation due to factor D deficiency by 2 novel mutations in the complement factor D gene in a family with meningococcal infections. Blood. 2006;107(12):4865-4870.

28. Granoff DM, Kim H, Topaz N, MacNeil J, Wang X, McNamara LA. Differential effects of therapeutic complement inhibitors on serum bactericidal activity against non-groupable meningococcal isolates recovered from patients treated with eculizumab. Haematologica.

2019;104(8):e340-e344.

29. Konar M, Granoff DM. Eculizumab treatment and impaired opsonophagocytic killing of meningococci by whole blood from immunized adults. Blood. 2017;130(7):891-899.

30. Kulasekararaj A, Risitano AM, Maciejewski JP, et al. A Phase 2 Open-Label Study of

Danicopan (ACH-0144471) in Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH) Who

Have an Inadequate Response to Eculizumab Monotherapy. Blood.

2019;134(Supplement_1):3514.

31. Risitano AM, Marotta S, Ricci P, et al. Anti-complement Treatment for Paroxysmal

Nocturnal Hemoglobinuria: Time for Proximal Complement Inhibition? A Position Paper From the SAAWP of the EBMT. Front Immunol. 2019;10:1157.

32. Risitano AM, Roth A, Soret J, et al. LNP023 - A New Oral Complement Factor-B

Inhibitor Normalizes Hemoglobin in Paroxysmal Nocturnal Hemoglobinuria Patients With Poor

Response to Eculizumab, Both as Add-On and Monotherapy. The 46th Annual Meeting of the

European Society for Blood and Marrow Transplantation; August 29 - September 1, 2020; O019.

33. Hillmen P, Szer J, Weitz I, et al. Results of the Pegasus Phase III Randomized Trial

Demonstrating Superiority of the C3 Inhibitor, Pegcetacoplan, Compared to Eculizumab in

Patients With Paroxysmal Nocturnal Hemoglobinuria. The 25th European Hematology

Association Annual Congress; June 12, 2020; 295012; S192.

34. Holers VM. The complement system as a therapeutic target in autoimmunity. Clin

Immunol. 2003;107(3):140-151.

MAIN MANUSCRIPT TABLES

Table 1. Baseline Characteristics and Clinical Results

Patient 1* Patient 2 Patient 3§ Patient 4 Patient 5 Patient 6 Patient 7# Patient 8 Patient 9 Patient 10*

BL D28 D84 BL D28 D84 BL D28 D84 BL D28 D84 BL D28 D84 BL D28 D84 BL D28 D84 BL D28 D84 BL D28 D84 BL D28 D84 Danicopan 100 150 175 100 150 175 150 175 200 150 175 - 150 175 175 150 150 150 150 175 - 150 175 175 150 175 200 150 150 150 (mg po TID) Hgb (g/dL) 7.5 9.3 8.7 11.7 14.1 12.3 12.0 11.8 13.7 11.7 12.6 - 10.4 10.2 10.9 9.2 11.2 11.4 8.7 10.2 - 9.5 10.2 11.6 10.0 11.4 11.9 6.9 8.4 11.1 LDH (xULN) 3.76 2.64 2.36 7.39 2.22 3.27 3.60 0.77 0.94 5.09 1.84 - 1.63 0.89 0.90 7.93 0.90 2.50 6.68 3.95 - 8.55 1.51 3.26 6.08 2.29 2.98 6.02 0.80 1.02 Reticulocytes 84 76 69 150 71 85 80 42 45 170 109 - 45 55 49 236 38 105 137 50 - 217 74 48 171 78 118 249 109 130 (10^9/μL) Total bilirubin 0.53 0.70 0.41 2.34 0.88 0.47 2.40 0.94 1.05 0.58 0.35 - 0.41 0.41 0.41 1.17 0.23 0.53 0.83 1.04 - 1.88 0.88 0.74 1.32 0.54 0.71 1.64 0.35 0.41 (mg/dL) GPI-deficient RBC clone 20 31 36 11 41 43 36 49 58 24 38 - 18 33 42 78 82 78 20 - - 21 47 59 13 35 42 75 37 92 size (%) + C3d RBCs 0.1 -† 0.1 0.4 -† 0.1 0.1 -† 0.1 0.3 -† - 0.1 0.1 0.1 0.1 -† 0.4 0.5 0.1 - 0.2 -† - 0.1 -$ - 0.1 0.3 0.1 (%) FACIT- 22 33 31 32 47 49 42 43 52 23 36 - 22 23 40 20 52 52 49 51 - 44 49 50 39 46 46 47 52 52 Fatigue†† BL- baseline; D- day; Hgb- hemoglobin; FACIT- functional assessment of chronic illness therapy; GPI- glycosylphosphatidylinositol; LDH-lactic acid dehydrogenase; po- orally; RBCs- red blood cells; TID- three times a day; ULN- upper limit of normal. *Patient reported history of aplastic anemia. †C3 fragment deposition was not tested for D28. No data entry for this visit. ††Scores based on the Functional Assessment of Chronic Illness Therapy Fatigue (FACIT)-Fatigue Scale V4. Score range 0-52. A score of less than 30 indicates severe fatigue. §Patient received a protocol waiver to enter the study despite <12 g/dL Hgb inclusion criteria. Per sponsor and site investigator, the Hgb level did not represent a clinically meaningful difference relative to the threshold in the protocol and the patient had significant hemolysis as evidenced by their LDH values. Patient withdrew due to personal reasons not related to safety (Day 40). #Patient withdrew due to serious adverse event (Day 51). C3 fragment deposition was not tested at Day 84. No data entry for this visit. $Incorrect container type.

Table 2: Adverse events

Standard severity grade

% of Total Primary system organ class preferred term* n Mild Moderate Severe Life- threatening (N=10)

Number of subjects reporting 9 90 8 1 Number of unique TEAEs† 38 NA Number of subjects with SAEs 1 10 .. .. 1 .. Blood and lymphatic system disorders 2 20 ...... Hemolysis 2 20 1 .. 1 .. Gastrointestinal disorders 3 30 ...... Abdominal pain 1 10 .. 1 .. .. Mouth ulceration 1 10 1 ...... Nausea 1 10 1 ...... Vomiting 1 10 1 ...... General disorders and administration site 4 40 ...... conditions Fatigue 1 10 1 ...... Non-cardiac chest pain 1 10 1 ...... Edema, peripheral 1 10 1 ...... Vaccination site erythema 1 10 1 ...... Infections and infestations 5 50 ...... Pharyngitis 1 10 .. 1 .. .. Upper respiratory tract infection 4 40 3 1 .. .. Viral upper respiratory tract infection 1 10 1 ...... Injury, poisoning, and procedural complications 1 10 ...... Contusion 1 10 1 ...... Investigations 1 10 ...... Alanine aminotransferase increased 1 10 .. .. 1 .. Aspartate aminotransferase increased 1 10 ...... 1 Metabolism and nutrition disorders 1 10 ...... Iron deficiency 1 10 1 ...... Musculoskeletal and connective tissue disorders 3 30 ...... Back pain 2 20 2 ...... Myalgia 1 10 1 ...... Nervous system disorders 4 40 ...... Headache 4 40 4 ...... Psychiatric disorders 1 10 ...... Irritability 1 10 1 ...... Renal and urinary disorders 3 10 ...... Hemoglobinuria 2 20 2 ...... Paroxysmal nocturnal hemoglobinuria 1 10 .. 1 .. .. Reproductive system and breast disorders 1 10 ...... Dysmenorrhea 1 10 1 ...... Skin and subcutaneous tissue disorders 1 10 ...... Rash, papular 1 10 1 ...... *MedDRA Version 18.1. †The row represents the number of events; all other rows represent the number of subjects. NA=not available. SAEs=serious adverse events. TEAEs=treatment-emergent adverse events.

MAIN MANUSCRIPT FIGURE LEGENDS

Figure 1: Pharmacokinetic-pharmacodynamic evaluation of danicopan

(A) The mean plasma danicopan concentration by dose at hours 0 (predosing), 1, 1.5, 2, 2.5, 3, 4, 6, 8 (predosing) and 12 of days 6, 13, and 20. The number of patients is, respectively, two (100 mg thrice daily) and eight (150 mg thrice daily) at day 6 and day 13, and five (150 mg thrice daily) and five (175 mg thrice daily) at day 20. (B) The mean ± SD of ex vivo serum AP activity, plasma Bb concentration, and plasma danicopan concentration, combining all dosing groups together. Serum AP activity and plasma Bb concentration were determined for a subset of the aforementioned time points by AP Wieslab assay (Euro Diagnostica) and Bb ELISA, respectively. (C) Plasma Bb concentration (mean ± SD) at baseline (day 1) through the end of the study (day 84) with descriptive statistics. The dashed lines represent ULN and LLN which are derived from phase 1 studies in healthy volunteers (see Assay Methods in Supplement). N values of <10 for plasma Bb on days 42, 56, and 84 reflect the two early discontinuations and additional samples not collected. **p<0.005; LLN=lower limit of normal; SD=standard deviation; TID=thrice daily; ULN=upper limit of normal.

Figure 2: Effect of danicopan on LDH and Hemoglobin

(A) Change in LDH from baseline (day 1) to day 28 was the primary efficacy endpoint. LDH reduction per patient is shown here including the reduction from a mean value of 5.7±2.17 times ULN at baseline to 1.8±1.03 times ULN at day 28 (p<0.001) demonstrating achievement of the primary endpoint. Significant mean LDH reduction from baseline was sustained throughout the study at day 84, 2.2±1.04 times ULN (p<0.001). (B) Per patient effects on hemoglobin with a mean group increase from 9.8 g/dL at baseline (day 1) (range, 6.9 g/dL to 12.0 g/dL) to 10.9 g/dL at day 28 (range, 8.4 to 14.1; p<0.005), and 11.5 g/dL at day 84 (range, 8.7 to 13.7, p<0.005). Note, Patient 3 received a protocol waiver to enter the study despite <12 g/dL hemoglobin inclusion criteria. Per sponsor and site investigator, the hemoglobin level did not represent a clinically meaningful difference relative to the threshold in the protocol and the patient had significant hemolysis as evidenced by their LDH values. **p<0.005; SD=standard deviation.

Figure 3: Effect of danicopan on blood transfusions and FACIT-Fatigue

(A) Transfusions occurred in two patients throughout the trial totalling 9 units and 4 instances over 84 days. Transfusion history (84 days prior to screening through end of study; sum of all patients) are provided. (B) Mean FACIT–Fatigue score values (± SD) at baseline (day 1) through end of the study (day 84) with descriptive statistics. The range of FACIT–Fatigue score is 0 to 52; score of <30 indicates severe fatigue. *p<0.05; **p<0.005. Note, data were not obtained for one patient at day 56. FACIT= Functional Assessment of Chronic Illness Therapy. SD= standard deviation.

Figure 4: Additional clinical efficacy evaluation of danicopan

(A) The mean (± SD) clone size percentages displayed for GPI-deficient erythrocytes and granulocytes at baseline (day 1) through end of study (day 84) and mean (± SD) percentage of erythrocytes with C3 fragment deposition with descriptive statistics for GPI-deficient erythrocytes. Descriptive statistics for GPI-deficient granulocytes and erythrocytes with C3 fragment deposition are listed in table S6 in the Supplementary Appendix. Erythrocytes with C3 fragment deposition are defined as erythrocytes stained positive for anti-C3d antibody. (B) Total bilirubin at baseline (day 1) through end of study (day 84) with descriptive statistics. Data for one patient were not obtained at day 56. (C) Absolute reticulocyte count at baseline (day 1) through end of study (day 84) with descriptive statistics. Data for one patient were not obtained at day 56.

*p<0.05; **p<0.005; SD=standard deviation.

Title: Danicopan: an oral complement factor D inhibitor for paroxysmal nocturnal hemoglobinuria Short Title: Danicopan, oral factor D inhibitor for PNH

Antonio M. Risitano,1 Austin G. Kulasekararaj,2 Jong Wook Lee,3 Jaroslaw P. Maciejewski,4 Rosario Notaro,5,6 Robert Brodsky,7 Mingjun Huang,8 Michael Geffner,9 and Peter Browett10

1Federico II University of Naples, Naples, Italy and AORN Moscati, Avellino, Italy; 2King's College Hospital-NHS Foundation Trust, NIHR/Wellcome King’s Clinical Research Facility, London, UK and King’s College London, London, UK; 3Seoul St. Mary’s Hospital, The Catholic University of Korea, Seoul, Republic of Korea; 4Cleveland Clinic, Cleveland, OH, USA; 5 Core Research Laboratory, Istituto per lo Studio, la Prevenzione e la Rete Oncologica (ISPRO), Firenze, Italy; 6Azienda Ospedaliera-Universitaria Careggi, Firenze, Italy; 7Johns Hopkins University School of Medicine, Baltimore, MD, USA; 8Achillion, Inc., A Subsidiary of Alexion Pharmaceuticals, Inc., New Haven, CT, USA; Alexion Pharmaceuticals, New Haven CT, USA. 9Achillion Inc., A Subsidiary of Alexion Pharmaceuticals, Inc., Blue Bell, PA, USA; 10 University of Auckland, Auckland, New Zealand

Title character count: 88 Short title character count: 42 Abstract word count: 250 Text word count: 3861 Figures and tables: 2 tables and 4 figures Supplementary figures and tables: 3 supplementary figures (separate file) and 6 supplementary tables (separate file) References: 34

Presented as an oral presentation at the 24th European Hematology Association Congress, Amsterdam, NL, 15 June 2019. 31 Presented as a poster presentation at the 61st annual meeting of the American Society of Hematology, Orlando, FL, 8 December 2019.32

Correspondence to: Antonio M. Risitano, M.D., Ph.D.

Hematology and Hematopoietic Stem Cell Transplant Unit AORN S. Giuseppe Moscati Contrada Amoretta 83100 Avellino, Italy Phone +39 347 5786759 [email protected]

Supplementary Appendix

This appendix has been provided by the authors to give readers additional information about their work.

Supplement to: Risitano AM, et al. Danicopan: an oral complement factor D inhibitor for paroxysmal nocturnal hemoglobinuria.

Table of Contents

1. Assay Methods 2. Dose Escalation Criteria 3. Supplementary Figures and Tables

Supplemental Figure 1. Patient disposition

Supplemental Figure 2. Additional complement biomarker evaluation

Supplemental Figure 3. Additional clinical endpoint evaluation

Supplemental Table 1. Baseline Characteristics and Patient Disposition

Supplemental Table 2. Additional laboratory safety data

Supplemental Table 3. Danicopan plasma pharmacokinetic parameters determined by intensive PK profiling

Supplemental Table 4. Pre-dose AP activity and danicopan concentration from days 1 to 84

Supplemental Table 5. Correlation between PK, PD, and LDH

Supplemental Table 6. Summary of laboratory tests

1. Assay Methods

The plasma concentrations of danicopan were determined by an LC/MS-MS method. The complement biomarkers were measured by the clinical laboratories. Serum complement C3 and C4 concentrations and serum classical pathway (CP) activity were measured with the pre-existing methods established in the clinical laboratories by using the Cobas (Roche) and Complement Activation EIA (Diasorin) kits. Serum alternative pathway (AP) activity was measured with AP Wieslab assay (Euro Diagnostica); Bb concentration was measured with MicroVue Complement Fragment Bb EIA (Quidel); serum FD concentration was measured with Quantikine® ELISA (R&D Systems, Inc.) by a single central laboratory after validating the assays according to the manufacturers’ instructions. In normal individuals, the mean ± SD value of plasma Bb is 0.84±0.84 µg/mL, with a range of 0.553–1.357 µg/mL based on the data collected from three phase 1 studies in healthy volunteers (N=100; trial ID: ACTRN12617001521314, ACTRN12618001989235, ACTRN12618000896279) by the same single centralized lab with the same commercial kit.

C3 fragment deposition on erythrocytes was measured by a clinical laboratory with flow cytometry after validating the protocol described hereafter. After centrifugation of whole blood collected from patients, the pellet containing erythrocytes was transferred, washed (three times with phosphate buffer saline [PBS]), and resuspended in GVB0 buffer (Complement Technology) at a density of erythrocytes about 1-2×109/mL. Prior to the test, an aliquot (5×106) of erythrocytes was transferred to a fresh tube, washed once with flow cytometry (FC) wash buffer (PBS + 15 mM EDA + 1% BSA), and incubated with 100 µl FC wash buffer containing FITC conjugated human C3d antibody (Assay Pro, Cat# 11294-05041), and PE conjugated human CD59 antibody (NOVUS, Cat # MEM-43) in the dark at room temperature for 1 hour. In the meantime, another aliquot of erythrocytes was processed in the same way but the antibodies in FC wash buffer were replaced with isotopic controls. At the end of incubation, erythrocytes were washed three times with FC wash buffer, resuspended in 200 µl FC buffer wash, and finally submitted to flow-cytometry analysis. Intact erythrocytes were gated based on physical parameters on forward and side scatter; gating for the fluorescence dye-conjugated antibodies was established with the isotypic controls. The following values were reported:

erythrocytes stained negative for anti-CD59 antibody & positive for anti-C3d antibody × 100% total erythrocytes

2. Dose Escalation Criteria

1st Dose Escalation Point (Day 7): On a patient-by-patient basis, if the starting dose of 150 mg TID is well tolerated and the available safety data are satisfactory, a patient may be escalated to 175 mg TID if his/her Day 6 LDH level, as measured locally, is still greater than 50% of his/her baseline value, unless the patient has achieved <1× upper limit of normal (ULN) for LDH. Depending on when these data are received and reviewed, if the patient is going to dose escalate, the site should contact the patient as soon as possible (Days 6-8) to provide new dosing instructions. If necessary, the patient may be asked to return to the clinic for new drug supplies.

2nd Dose Escalation Point (Day 14): On a patient-by-patient basis, if the patient was not dose escalated at Day 7, the 150 mg TID dose is well tolerated, and the available safety data are satisfactory, a patient may be escalated to 175 mg TID if his/her Day 13 LDH level, as measured locally, is still greater than 20% of his/her baseline value, unless the patient has achieved <1× ULN for LDH. Depending on when these data are received and reviewed, if the patient is going to dose escalate, the site should contact the patient as soon as possible (Days 13-15) to provide new dosing instructions. If necessary, the patient may be asked to return to the clinic for new drug supplies. In addition to the dose escalation evaluations defined above, the investigator, in consultation with the sponsor, may escalate dosing in increments of 25 mg to a maximum of 250 mg TID after evaluating the

3 clinical benefit and the available safety, PK, and PD data (including laboratory test results) in order to improve control of hemolysis.

3. Supplementary Figures and Tables

Supplemental Figure 1: Patient disposition. TID=three times a day.

N=11

Patients screened

N=10 1 screen failure due to elevated liver function Patients received study medication tests and creatinine

N=2 N=8

100 mg TID 150 mg TID

N=1 N=1 N=1 N=1 N=1 N=1 N=1 N=1 N=1 N=1 ↑150 mg ↑175 mg ↑150 mg ↑175 mg ↑175 mg Continue ↑175 mg ↑175 mg ↑175 mg Continue TID day 13 TID day 13 TID day 13 TID day 13 TID day 13 150 mg TID TID day 13 TID day 20 TID day 13 150 TID ↑175 TID ↑200 mg ↑175 mg ↑200 mg ↑200 mg ↑200 mg day 74 TID day 76 TID day 70 TID day 28 TID day 28 TID day 36

Extension Extension Extension Early Extension Extension Withdraw Extension Extension Extension treatment treatment treatment withdraw treatment treatment day 50 for treatment treatment treatment day 40 for break- personal through reasons hemolysis

Supplemental Figure 2: Additional complement biomarker evaluation. The additional complement biomarkers as indicated in panels A, B, and C were monitored from baseline (day 1, predose) through the end of the study (day 84). The mean ± SD value at each time point is plotted over the study duration (baseline to day 84) along with more detailed statistical analyses at baseline and a few critical time points shown at the bottom of the chart. Panel D presents the results of quantitative relationship analysis between pharmacokinetic (PK; danicopan plasma concentration) and pharmacodynamic (PD; percent inhibition of AP activity relative to baseline in corresponding serum samples). In total, there are 211 paired data points with both the plasma danicopan concentration and the corresponding serum AP activity available from all patients enrolled in the study. Best fit values and 95% confidence intervals (CIs) are in ng/mL. Panel E displays the correlation between Bb concentration and LDH concentration. AP=alternative pathway. CAE=complement activity enzyme. CP=classical pathway. EC50=half maximal effective concentration. LDH=lactate dehydrogenase. PD=pharmacodynamics. PK=pharmacokinetics. SD=standard deviation. *p<0.005.

A. Mean (± SD) of complement factor D

Baseline (Day 1) Day 28 Day 56 Day 84 N 10 10 5 8 Mean (SD) 3088 (883.5) 3122 (818.2) 2986 (668.3) 2816 (785.3) Range 2019, 4976 1907, 4665 2125, 3620 1686, 3808

B. Mean (± SD) of CP activity

Baseline (Day 1) Day 28 Day 56 Day 84 N 10 8 5 6 Mean (SD) 126 (35.4) 129 (28.3) 110 (32.9) 119 (47.3) Range 87, 195 89, 180 68, 155 66, 204

C. Mean ± SD of serum C3 concentration from days 1 to 84

Baseline (Day 1) Day 28 Day 56 Day 84 N 10 10 5 6 Mean (SD) 102.2 (20.21) 115.5 (24.00) * 106.8 (9.15) * 114.2 (17.31) Range 76.0, 144.0 83.0, 173.0 97.0, 120.0 99.0, 148.0

D. PK (danicopan plasma concentrations) and PD (AP activity inhibition) analysis

E. Correlation between plasma Bb concentration and LDH concentration

Supplemental Figure 3: Additional clinical endpoint evaluation. Additional clinical endpoints as indicated were monitored from baseline (day 1, predose) through the end of the study (day 84). The mean ± SD value at each time point is plotted over the study duration (baseline to day 84) along with more detailed statistical analyses at baseline and a few critical time points shown at the bottom of the chart. SD=standard deviation.

A. Mean (± SD) haptoglobin

Baseline (Day 1) Day 28 Day 84 N 10 10 8 Mean (SD) 5.8 (2.90) 11.8 (14.81) 15.3 (16.08) Range 4.0, 10.0 4.0, 53.0 4.0, 42.0

B. Mean (± SD) free hemoglobin

Baseline (Day 1) Day 28 Day 42 Day 56 Day 84 N 10 10 9 7 8 Mean (SD) 138 (132.8) 83 (51.1) 109 (77.7) 173 (166.4) 250 (212.2) Range 50, 500 40, 195 40, 270 30, 515 48, 670

Supplemental Table 1: Baseline Characteristics and Patient Disposition

Parameters (N=10) Value Sex, n (%) Female 5 (50) Male 5 (50) Ethnicity, n (%) White 7 (70) Asian 1 (10) Indian 1 (10) Native Hawaiian or other 1 (10) Age, years Mean (SD) 35.9 (13.57) Median 32.5 Range 16.9*–62.5 Body mass index (kg/m2) Mean (SD) 25.1 (4.40) Median 25.3 Range 18–34 Disease history Aplastic anemia and PNH 2 (20) PNH 8 (80) PNH duration, years Mean (SD) 5.7 (4.41) Median 5.9 Range 0–14 Transfusion history in the 3 years prior to the first dose Number of patients 4 Unit of RBC transfusions .. Mean (SD) 14.8 (13.65) Median 14.5 Range 2–28 Subject disposition, n (%) Completed 28-day treatment 10 (100) Completed 84-day treatment 8 (80) Reason for discontinuation .. Adverse event 1 (10) Withdrew consent 1 (10) *Patient considered adult in country of enrollment.

Supplemental Table 2: Additional laboratory safety data

Test Statistic Baseline, Day 1 Day 28 Day 42 Day 56 Day 84

AST, U/L N 10 10 9 7 8

Mean (SD) 77 (31.1) 36 (24.6) 28 (13.3) 29 (13.0) 27 (11.5)

Median 82 30 23 27 25

Range 25–111 13–93 15–56 14–53 14–45

ALT, U/L N 10 10 9 7 8

Mean (SD) 27 (11.3) 26 (18.0) 27 (27.9) 20 (8.3) 15 (3.5)

Median 27 20 19 21 14

Range 12–43 10–58 9–98 9–32 11–22

Alk. phos., U/L N 10 10 9 7 8

Mean (SD) 68 (20.9) 72 (22.2) 76 (23.1) 74 (16.9) 84 (25.9)

Median 64 70 66 74 82

Range 42–113 42–119 45–125 53–102 45–112

GGT, U/L N 10 10 9 7 8

Mean (SD) 21 (22.8) 21 (22.4) 18 (14.1) 17 (10.4) 17 (10.2)

Median 12 14 13 13 14

Range 5–79 7–82 11–55 10–40 6–37

Creatinine, mg/dL N 10 10 9 7 8

Mean (SD) 0.76 (0.182) 0.77 (0.212) 0.75 (0.180) 0.84 (0.242) 0.78 (0.194)

Median 0.69 0.71 0.73 0.79 0.68

Range 0.50–1.02 0.47–1.07 0.49–0.99 0.50–1.18 0.61–1.14 eGFR, N 9 9 8 6 7 mL/min/1·73m2

Mean (SD) 102.0 (19.09) 100.2 (23.17) 100.8 (20.97) 89.0 (23.28) 100.2 (24.48)

Median 101.0 97.0 94.9 80.6 97.0

Range 76.0–131.9 72.0–135.0 78.0–129.9 69.0–130.0 67.0–146.0

INR, ratio N 10 10 ·· ·· ··

Mean (SD) 1.34 (0.428) 1.45 (0.574) ·· ·· ··

Median 1.14 1.12 ·· ·· ··

Range 1.00–2.19 1.00–2.40 ·· ·· ··

Test Statistic Baseline, Day 1 Day 28 Day 42 Day 56 Day 84

APTT, seconds N 10 10 ·· ·· ··

Mean (SD) 31.8 (6.71) 34.0 (8.68) ·· ·· ··

Median 30.5 34.3 ·· ·· ··

Range 24.0–44.4 23.0–47.9 ·· ·· ··

D-Dimer, ng/mL N 10 10 ·· ·· 6

Mean (SD) 50.9 (41.05) 48.1 (47.68) ·· ·· 103.3 (194.49)

Median 35.0 28.0 ·· ·· 20.0

Range 20.0–153.0 20.0–170.0 ·· ·· 20.0–500.0

C-reactive protein, N 10 10 9 7 8 mg/dL

Mean (SD) 2 (1.3) 2 (1.9) 1 (1.8) 1 (1.3) 3 (2.9)

Median 1 1 1 1 1

Range 0–4 0–6 0–6 0–4 0–9 Alk. phos. =alkaline phosphatase. ALT=alanine aminotransferase. APTT=activated partial thromboplastin time. AST=aspartate aminotransferase. eGFR=estimated glomerular filtration rate. GGT=gamma glutamyl transferase. INR=prothrombin international normalized ratio. SD=standard deviation.

Supplemental Table 3: Danicopan plasma pharmacokinetic parameters determined by intensive PK profiling

C (ng/mL) T (hr) AUC (hr•ng/mL) Dose* Parameters max max last C8 (ng/mL) 100 mg TID Mean 349 4.17 1476 47.7 (N=2, day 6) SD 33.2 0.707 291 23.3 CV% 9.5 17.0 19.7 48.9 Median 349 4.17 1476 47.7

150 mg TID Mean 465 3.30 2019 108.9 (N=8, day 6†; SD 189 1.20 780 45.2 N=5, day 13; CV% 40.6 36.7 38.6 41.6 N=4, day 20) Median 438 3.67 1940 100.5

175 mg TID Mean 543 3.60 2231 96.2 (N=5, day 13; SD 200 1.30 861 31.3 N=6, day 20) CV% 36.7 34.5 38.6 32.5 Median 498 4.48 1894 86.9 *One of two patients who received 200 mg TID was not included on Day 56 because the sample was not available (missed study visit). PK sampling was performed as specified in the protocol on Day 6, 13, and 20; no patients were on 200 mg TID danicopan by day 20. † One patient on day 7; N for C8 is 7 as a patient left the site prior to hour 8.

AUClast, area under the curve time 0 to last measurable time; C8, trough concentration at hour 8; Cmax, peak concentrations; CV%=percent coefficient of variation. PK=pharmacokinetics. SD=standard deviation. Tmax=peak time. TID=three times a day.

Supplemental Table 4: Pre-dose AP activity and danicopan concentration from days 1 to 84

PD: AP activity (%) PK: danicopan concentration (ng/mL) Time 100 mg TID 150 mg TID 175 mg TID 200 mg TID 100 mg TID 150 mg TID 175 mg TID 200 mg TID (Day) Mean SD N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD N

Day 1 75.7 7.8 2 62.6 13.9 8 ·· ·· ·· ·· ·· ·· 0.0 0.0 2 0.0 0.0 8 ·· ·· ·· ·· ·· ··

Day 6 14.3 18.2 2 11.6 9.3 8 ·· ·· ·· ·· ·· ·· 91.6 79.8 2 169.3 92.0 8 ·· ·· ·· ·· ·· ··

Day 13 11.4 15.2 2 12.6 12.2 8 ·· ·· ·· ·· ·· ·· 149.6 120.8 2 155.5 80.4 8 ·· ·· ·· ·· ·· ··

Day 20 ·· ·· ·· 16.9 18.1 5 9.4 10.6 5 ·· ·· ·· ·· ·· ·· 159.7 79.6 5 193.8 71.9 5 ·· ·· ··

Day 28 ·· ·· ·· 11.6 13.2 4 13.5 16.3 6 ·· ·· ·· ·· ·· ·· 153.8 56.4 4 155.1 68.5 6 ·· ·· ··

Day 42 ·· ·· ·· 27.7 32.1 2 4.8 1.50 2 59.0 30.6 2 ·· ·· ·· 194.5 77.1 2 279.7 157.2 3 82.9 8.3 2

Day 56 ·· ·· ·· 5.6 2.7 2 2.6 3.3 2 3.8 -- 1 ·· ·· ·· 149.5 31.8 2 202.0 63.6 2 297.0 -- 1

Day 70 ·· ·· ·· 11.6 9.50 3 8.3 11.3 3 ·· ·· ·· ·· ·· ·· 203.3 65.2 3 299.0 223.1 3 ·· ·· ··

Day 84 ·· ·· ·· 16.9 20.8 2 10.5 14.9 4 19.9 22.9 2 ·· ·· ·· 152.0 50.9 2 263.3 112.4 4 132.5 44.5 2 AP=alternative pathway. PD=pharmacodynamics. PK=pharmacokinetics. SD=standard deviation. TID=three times a day.

Supplemental Table 5: Correlation between PK, PD, and LDH

Pearson Correlation Coefficients, N=40 Prob > |r| under H0: Rho=0

Danicopan CP AP Bb LDH

Danicopan, ng/mL 1.00000 -0.11470 -0.83666 -0.62271 -0.65284 0.4810 <0.0001 <0.0001 <0.0001

CP activity, CAE -0.11470 1.00000 0.09553 0.15734 0.29287 0.4810 0.5576 0.3322 0.0667

AP activity, % -0.83666 0.09553 1.00000 0.59026 0.67100 <0.0001 0.5576 <0.0001 <0.0001

Bb, µg/mL -0.62271 0.15734 0.59026 1.00000 0.82860 <0.0001 0.3322 <0.0001 <0.0001

LDH, IU/L -0.65284 0.29287 0.67100 0.82860 1.00000 <0.0001 0.0667 <0.0001 <0.0001 AP=alternative pathway. CP=classical pathway. LDH=lactate dehydrogenase. PD=pharmacodynamics. PK=pharmacokinetics.

Supplemental Table 6: Summary of laboratory tests

Laboratory Tests Statistics Baseline (Day 1) Day 28 Day 42 Day 56 Day 84 LDH, U/L N 10 10 9 7 8 Mean (SD) 1416 (540.3) 444 (255.8) ** 435 (222.9) ** 574 (351.9) ** 537 (260.4) ** Median 1509 417 389 610 607 Range 407–2130 193–983 198–815 228–1131 226–818 LDH, x ULN N 10 10 9 7 8 Mean (SD) 5.7 (2.17) 1.8 (1.03) ** 1.7 (0.89) ** 2.3 (1.41) ** 2.2 (1.04) ** Median 6 1.7 1.6 2.4 2.4 Range 1.6–8.6 0.8–3.9 0.8–3.3 0.9–4.5 0.9–3.3 AP functional N 10 10 6 5 8 activity, % Mean (SD) 65.2 (13.71) 12.7 (14.37) ** 30.5 (31.39) 4.1 (2.59) ** 14.4 (15.82) ** Median 71.4 6.0 21.6 3.9 4.0 Range 44.0–81.2 1.4–36.6 3.7–80.6 0.3–7.5 2.2–36.1 CP functional N 10 8 ·· 5 6 activity, CAE Mean (SD) 126 (35.4) 129 (28.3) ·· 110 (32.9) 119 (47.3) Median 111 123 ·· 101 111 Range 87–195 89–180 ·· 68–155 66–204 C3, mg/dL N 10 10 ·· 5 6 Mean 102.2 (20.21) 115.5 (24.00) ** ·· 106.8 (9.15) ** 114.2 (17.31) (SD) Median 99.5 108.0 ·· 103.0 109.5 Range 76.0–144.0 83.0–173.0 ·· 97.0–120.0 99.0–148.0 Bb, µg /mL N 10 10 7 5 6 Mean (SD) 2.24 (0.774) 0.84 (0.838) ** 0.49 (0.205) ** 0.47 (0.086) ** 0.47 (0.059) ** Median 2.29 0.58 0.45 0.49 0.47 Range 1.15–3.24 0.41–3.18 0.33–0.94 0.37–0.56 0.38–0.54 Factor D, ng/mL N 10 10 ·· 5 8 Mean (SD) 3088 (883.5) 3122 (818.2) ·· 2986 (668.3) 2816 (785.3) Median 2953 3144 ·· 3243 2887 Range 2019–4976 1907–4665 ·· 2125–3620 1686–3808 Hemoglobin, g/dL N 10 10 9 7 8 Mean 9.8 (1.76) 10.9 (1.65) ** 11.0 (1.72) ** 10.9 (1.71) ** 11.5 (1.41) ** (SD) Median 9.8 10.7 11.0 11.1 11.5 Range 6.9–12.0 8.4–14.1 8.2–13.3 8.5–13.1 8.7–13.7 Reticulocytes, N 10 10 9 7 8 9 10 /L Mean (SD) 154 (69.0) 70 (24.9) ** 80 (32.5) * 101 (61.7) 81 (33.6) * Median 160 73 73 85 77 Range 45–249 38–109 47–141 47–225 45–130 Haptoglobin, N 10 10 ·· ·· 8 mg/dL Mean (SD) 5.8 (2.90) 11.8 (14.81) ·· ·· 15.3 (16.08) Median 4.0 8.5 ·· ·· 8.5 Range 4.0–10.0 4.0–53.0 ·· ·· 4.0–42.0 Free hemoglobin, N 10 10 9 7 8 mg/dL Mean (SD) 138 (132.8) 83 (51.1) 109 (77.7) 173 (166.4) 250 (212.2) Median 105 67 90 160 180 Range 50–500 40–195 40–270 30–515 48–670 Total bilirubin, N 10 10 9 7 8 mg/dL Mean (SD) 1.3 (0.74) 0.6 (0.29) * 0.6 (0.24) * 0.7 (0.32) * 0.6 (0.23) * Median 1.2 0.6 0.6 0.7 0.5 Range 0.4–2.4 0.2–1.0 0.3–1.0 0.2–1.1 0.4–1.1

Laboratory Tests Statistics Baseline (Day 1) Day 28 Day 42 Day 56 Day 84 Direct bilirubin, N 10 10 9 7 8 mg/dL Mean (SD) 0.3 (0.15) 0.2 (0.10) 0.2 (0.05) 0.2 (0.09) 0.2 (0.07) * Median 0.2 0.2 0.2 0.3 0.2 Range 0.2–0.6 0.1–0.4 0.2–0.3 0.1–0.4 0.1–0.3 C3d+ of total N 10 3 9 ·· 6 erythrocytes, % Mean (SD) 0.2 (0.15) 0.2 (0.12) 0.1 (0.03) ·· 0.2 (0.12) Median 0.1 0.1 0.1 ·· 0.1 Range 0.1–0.5 0.1 –0.3 0.1–0.2 ·· 0.1–0.4 ANC, 109/L N 10 10 9 7 7 Mean (SD) 2.1 (0.83) 2.0 (0.62) 1.9 (0.50) 1.9 (0.58) 2.4 (1.24) Median 2 2 1.8 2 2.2 Range 0.8–3.8 1.1–3.0 1.3–2.7 1.1–2.5 1.2–4.4 Platelets, 109/L N 10 10 9 7 8 Mean (SD) 162 (61.7) 175 (70.9) 168 (78.6) 163 (78.4) 165 (83.8) Median 174 166 172 171 161 Range 37–245 47–290 43–322 35–270 35–320 FACIT-FATIGUE N 10 10 ·· 7 8 score Mean (SD) 34 (11.5) 43 (9.6) * ·· 39 (13.2) * 47 (7.5) ** Median 36 47 ·· 46 50 Range 20–49 23–52 ·· 18–51 31–52 GPI-deficient N 10 9 7 5 8 clone, erythrocytes Mean (SD) 32 (24.6) 44 (15.6) 60 (20.1) ** 59 (21.0) 56 (19.9) ** % Median 21 38 55 59 51 Range 11–78 31–82 39–89 39–91 36–92 GPI-deficient N 10 9 7 5 8 clone, Mean (SD) 79 (16.9) 71 (23.5) 77 (21.9) 74 (20.3) 79 (13.3) granulocytes % Median 82 88 86 85 82 Range 46–99 38–96 42–97 49–91 57–96 *p<0.05. **p<0.005. ANC=absolute neutrophil count; AP=alternative pathway; CAE=complement activity enzyme; CP=classical pathway; FACIT=Functional Assessment of Chronic Illness Therapy; GPI- glycosylphosphatidylinositol; LDH=lactate dehydrogenase; ULN=upper limit of normal.