Supplementary Appendix

Transcription

1 Supplementary Appendix This appendix has been provided by the authors to give readers additional information about their work. Supplement to: Pasi KJ, Rangarajan S, Georgiev P, et al. Targeting of antithrombin in hemophilia A or B with RNAi therapy. N Engl J Med 217;377: DOI: 1.156/NEJMoa

2 Fitusiran, an investigational RNAi Therapeutic Targeting Antithrombin, in Hemophilia A or B K John Pasi, Savita Rangarajan, Pencho Georgiev, Tim Mant, Michael Desmond Creagh, Toshko Lissitchkov, David Bevan, Steve Austin, Charles R Hay, Inga Hegemann, Rashid Kazmi, Pratima Chowdary, Liana Gercheva-Kyuchukova, Vasily Mamonov, Margarita Timofeeva, Chang-Heok Soh, Pushkal Garg, Akshay Vaishnaw, Akin Akinc, Benny Sørensen and Margaret V. Ragni Table of contents for supplementary materials: Table S1. Patients participating in both Part B and Part C 2 Table S2. Demographics and clinical characteristics of the study population by dose cohort 3 Table S3. Summary of mean fitusiran plasma pharmacokinetic parameters after single subcutaneous administration in healthy volunteers (Part A) and multiple subcutaneous administration in hemophilia patients (Parts B and C) Summary of exploratory bleed analysis 5 Table S4. Exploratory analysis of bleed events in Part C 7 Summary of management of bleed episodes 8 Table S5. Management of bleed events with replacement factor while on fitusiran with 75% antithrombin lowering Table S6. Summary of all treatment-emergent adverse events in healthy volunteers 1 Table S7. Summary of treatment-emergent adverse events occurring in at least 2 patients with hemophilia Table S8. Peak alanine aminotransferase levels in hemophilia patients 12 Summary of SAE of reactivation of hepatitis C 13 Figure S1. CONSORT diagram 14 Figure S2. Dose-dependent antithrombin lowering in Parts A, B, and C 15 Figure S3. Analysis of thrombin generation by antithrombin lowering quartiles 16 Figure S4. Effect of fitusiran on liver levels of alanine transaminase 17 Figure S5. Effect of fitusiran on levels of bilirubin 18 Figure S6. Effect of fitusiran on liver levels of D-dimer 19 Figure S7. Effect of fitusiran on levels of fibrinogen 2 Figure S8. Effect of fitusiran on levels of prothrombin time (PT) 21 Figure S9. Effect of fitusiran on platelet levels 22 Figure S1. Effect of fitusiran on levels of activated partial thromboplastin time (APTT)

3 Table S1. Patients participating in both Part B and Part C. Each row represents one patient who participated in both Parts of the study and the doses they received. Part B Part C Patient ID Cohort Patient ID Dose Cohort B C5-2 8 mg B C B C B C B C

4 Table S2. Demographics and clinical characteristics of the study population by dose cohort Placebo (N=1) Part A Part B Part C.3 (N=3).15 (N=3).45 (N=6).75 (N=3).225 (N=3).45 (N=3).9 (N=3) 1.8 (N=3) Age (years) Mean Range Weight (kg) Mean Range Hemophilia, N Hemophilia A Hemophilia B Severe (<1% factor activity) Moderate (1 5% factor activity) Pre-study therapy, N On-demand Prophylaxis Pre-study annualized bleeding rate (bleeds per year) Median Range Previous or concomitant disease HIV 1 1 HCV HCV, hepatitis C virus; HIV, human immunodeficiency virus mg (N=6)

5 Table S3. Summary of mean fitusiran plasma pharmacokinetic parameters after single subcutaneous administration in healthy volunteers (Part A) and multiple subcutaneous administration in hemophilia patients (Parts B and C) Cohort Dose () Day C max (ng/ml) T max (hour) t 1/2 (hour) AUC -last (h ng/ml) CL_F (ml/h/kg) Vz_F (ml/kg) A (±.71) 5.39 (±1.2) 4.4 (±1.6) 43.6 (±8.41) 659 (±16) 49 (±151) B (±1.12) 3.3 (±1.2) 4.5 (±2.5) 2 (±1.3) 758 (±425) 413 (±165) B (±13.1) 5.2 (±1.1) 5. (±1.1) 65.3 (±37.3) 561 (±142) 388 (±767) (±8.13) 4. (±2.8) 4.8 (±1.5) 111 (±4.5) 447 (±229) 3375 (±253) B3 C1 C2 C3 C (±2.59) 8 (±3.5) (±47.6) (±1.7) 5.7 (±4.) (±76.6) (±5.52) 6. (±5.3) (±125) (±34.5) 3.3 (±1.15) (±31) (±13.) 6.7(±5.) (±265) (±1.3) 5.3 (±1.15) 5.3 (±1.74) 666 (±113) 647 (±111) 489 (±176) 124 (±19.3) 4.7 (±1.2) 4.2 (±1.) 149 (±181) 594 (±67.9) 363 (±118) (±17.9) 6. (±2.) (±297) (±122) 4.7 (±1.2) 4.9 (±1.4) 314 (±646) 347 (±259) 239 (±195) (±192) 6. (±3.5) (±739) C5 8* 152 (±36.6) 4. (±3.5) 5.5 (±1.6) 176 (±272) 45 (±34) 3765 (±371) (±16.2) 4.4 (±.9) 5.6 (±1.2) 1768 (±311) 583 (±125) 462 (±897) Cmax, maximum concentration; Tmax, time at maximum concentration; t½, half-life; AUC-last area under the curve from day to last measureable concentration; CL_F; apparent clearance; Vz_F, apparent volume of distribution * Fixed dose 8 mg was converted to corresponding dose for pharmacokinetic parameter estimation ± indicates SD. SD not reported where values represent N <3 4

6 Summary of exploratory bleed analysis A post hoc exploratory analysis was conducted to explore the effect of monthly fitusiran administration on bleeding rates. A bleeding episode was defined as a bleed event that required treatment with a clotting factor. The treatment period was assessed separately as on onset period, the period during which fitusiran-mediated antithrombin lowering occurs (defined as day to 28), and the observation period, the period during which antithrombin levels are expected to be in the target range (defined as day 29 to the earlier of 56 days after last dose or last study visit). The annualized bleeding rate in each period was calculated as the number of bleeds in the period divided by the number of days in that period, multiplied by For the purpose of calculation of median annualized bleeding rates, the discontinued patient C5-4 was assumed to have the highest bleed rate on study. These data are summarized in Table S4. Across all dose cohorts in Part C (N=18), the median annualized bleeding rate (ABR) during the observation period was, compared to a median ABR of 3 during the pre-study period (Table S4). Ten patients (56%) reported no bleeds during the observation period. Across all dose cohorts in Part C (N=18), the median annualized spontaneous bleeding rate during the observation period was, with 12 patients (67%) reporting no spontaneous bleeds. For the 15 patients in dose cohorts achieving mean antithrombin lowering >75% (.45 mg per kilogram and higher), the median annualized bleeding rate was. Two patients among this group (2/15) had moderate hemophilia; however, both patients entered the study utilizing prophylactic therapy (which may be suggestive of a more severe clinical phenotype) and had pre-study annualized bleeding rates of and 6 on factor prophylaxis. The median annualized bleed rate excluding these moderate patients remained at., with 7/13 (54%) patients with no reported bleeds. 5

7 Evidence of reduced bleeding was observed in patients with hemophilia A and patients with hemophilia B. Given the mechanism of action of fitusiran and the similar observed relationship between antithrombin level and thrombin generation (Fig. 2E), we do not expect a difference in efficacy of fitusiran in the setting of hemophilia A or B. However, given the small patient numbers, particularly of hemophilia B patients, no conclusions can be drawn on effect in hemophilia subtypes. There are limitations of this exploratory analysis due to the short duration of treatment from which the observation period annualized bleeding rates are derived. Also, there is subjectivity in the number of bleed events and treatment as they are patient reported. Further, there was no minimum required bleeding frequency at study entry, and pre-study bleeding frequency was not balanced across dose cohorts. 6

8 Table S4. Exploratory analysis of bleed events in Part C Dose Patient Hemophilia type; moderate or severe Prior treatment Pre-study ABR * Onset ABR Maximum AT lowering (%) All bleeds, N Observation period ABR Spontaneous Bleeds, N C1-1 HA; severe PPx C1-2 HB; moderate OD 51 C1-3 HA; severe PPx C2-1 HA; severe PPx C2-2 HA; severe OD C2-3 HB; severe OD C3-1 HA; moderate PPx 83 C3-2 HA; severe OD C3-3 HA; severe OD C4-1 HA; severe OD 25 9 C4-2 HA; severe OD C4-3 HA; severe OD AsBR C5-1 HB; severe PPX C5-2 HB; severe PPx mg C5-3 HA; severe OD C5-4 HA; severe PPx C5-5 HB; moderate PPx C5-6 HA; severe PPx HA, hemophilia A; HB, hemophilia B; PPx, prophylaxis; OD, on-demand; ABR, annualized bleeding rate; AT, antithrombin; AsBR, annualized spontaneous bleeding rate. * Pre-study ABR derived from medical records Spontaneous bleeds defined as bleeding for no apparent/known reason, according to the WFH Guidelines for the Management of Hemophilia Patient C5-4 withdrew from study on day 45 Post-dose period was divided into an onset period (day to 28) and the observation period (day 29 to last visit or 56 days after last dose, whichever was earlier) 7

9 Summary of management of bleed episodes There was a total of 172 bleeding episodes in Parts B and C requiring treatment with clotting factor (factors VIII or IX). All bleeding episodes were successfully treated, with 164 of the episodes (95%) treated with 1 or 2 doses. The range of dose of factor concentrates used was IU/kg for factor VIII and IU/kg for factor IX. Of particular interest is the management of breakthrough bleeding events while antithrombin levels are pharmacologically lowered. There was a total of 9 bleeding episodes requiring treatment with replacement factors VIII or IX during which antithrombin lowering was 75%. All bleeding episodes were successfully treated, with 8 episodes (89%) treated with 1 or 2 doses of factor. The dose range of factor concentrates used per administration was IU/kg for factor VIII and 6.7 IU/kg for all administrations of factor IX. 8

10 Table S5. Management of bleed events with replacement factor while on fitusiran with 75% antithrombin lowering Treatment of bleeds Factor VIII Factor IX Recommended management per protocol No more than 3 IU/kg; re-dose after 24 hours if symptoms not relieved No more than 5 IU/kg; re-dose after 24 hours if symptoms not relieved Total patients with bleeds, N 4 1 Total bleeds, N 6 3 Total administrations, N 9 3 Mean administrations per bleed, N Mean dose per injection, IU/kg Median dose per injection, IU/kg Dose range per injection, IU/kg Mean total amount of factor per bleed, IU/kg

11 Table S6. Summary of all treatment-emergent adverse events (AEs) in healthy volunteers AE Event Severity Event Serious Outcome Headache Mild No Non-cardiac chest pain Limited symptom panic attack Mild Mild No No Rhinitis Mild No Upper respiratory tract infection Mild No Recovered/ Resolved Recovered/ Resolved Recovered/ Resolved Recovered/ Resolved Recovered/ Resolved Relationship to Study Drug Possibly related Unlikely related Unlikely related Not related Not related Discontinue Study Drug No No No No No A total of 5 mild AEs were reported. Four of these events were considered unlikely/not related and 1 was possibly related (headache). All AEs resolved spontaneously. 1

12 Table S7. Summary of treatment-emergent adverse events (AEs) occurring in at least 2 patients with hemophilia AE, number of patients (%).15 N=3 Part B: Weekly dosing.45 N=6.75 N=3.225 N=3.45 N=3 Part C: Monthly dosing.9 N=3 1.8 N=3 8 mg fixed N=6 All treated patients N=25* At least 1 AE 2 (66.7) 4 (66.7) 2 (66.7) 1 (33.3) 3 (1) 1 (33.3) 3 (1) 6 (1) 19 (76.) Injection site pain 1 (16.7) 5 (83.3) 6 (24.) Injection site erythema 1 (33.3) 3 (5.) 4 (16.) Arthralgia 1 (33.3) 2 (33.3) 1 (33.3) 1 (16.7) 4 (16.) Upper respiratory tract infection Alanine aminotransferase increased Joint range of motion decreased 1 (33.3) 2 (33.3) 3 (12.) 1 (33.3) 1 (16.7) 2 (8.) 1 (33.3) 1 (33.3) 1 (16.7) 2 (8.) Nasopharyngitis 1 (16.7) 1 (33.3) 2 (8.) Headache 1 (33.3) 1 (16.7) 2 (8.) Osteoarthritis 1 (16.7) 1 (33.3) 2 (8.) Pain in extremity 1 (33.3) 1 (33.3) 2 (8.) Viral upper respiratory tract infection 2 (33.3) 2 (8.) *Five patients participated in both Parts B and C 11

13 Table S8. Peak alanine aminotransferase levels in hemophilia patients Part B: Weekly dosing Part B: Monthly dosing Dose N Peak ALT <1.5x ULN* Peak ALT 1.5-3x ULN* Peak ALT >3x ULN* (67%) 1 (33%) (83%) 1 (17%) (1%) (67%) 1 (33%) (67%) 1 (33%) (67%) 1 (33%) (33%) 2 (67%) 8 mg 6 4 (67%) 1 (17%) 1 (17%) *ULN = upper limit of normal; levels relative to ULN (or baseline for patients with baseline values >ULN) 12

14 Summary of SAE of reactivation of hepatitis C The SAE of reactivation of hepatitis C occurred in a patient with prior history of hepatitis C virus infection receiving.45 fitusiran monthly. This SAE was deemed unrelated to study drug by the investigator and did not lead to discontinuation of study drug. The patient was asymptomatic, but had an elevated alanine aminotransferase of 14 U/L on day 7 (compared to 29 U/L at baseline). Quantitative PCR revealed a hepatitis C virus RNA level of 55, IU/mL. Although a baseline viral RNA value was not available, the event was categorized as a reactivation of hepatitis C by the investigator. The patient was later admitted to hospital (7 days after last dose of fitusiran) for further evaluation, resulting in the event being considered an SAE. On admission, the patient remained asymptomatic, with ALT 49 U/L. Abdominal ultrasound was performed and was unremarkable. 13

15 Figure S1. CONSORT diagram *54 volunteers not eligible based on inclusion and exclusion criteria; 4 volunteers screened, but not included as Part A was subsequently closed and study moved on to Part B; 4 volunteers withdrew consent between screening and enrollment. Five patients were assessed twice for eligibility, for Parts B and C. Five of the 25 hemophilia patients participated in Parts B and C. Only patients treated with fitusiran were included in the safety analysis for Part A (N=3). # Patient discontinued study on day 45 due to non-cardiac chest pain. 14

16 Figure S2. Dose-dependent antithrombin lowering in Parts A, B, and C. Bars represent mean (± standard error of the mean) nadir levels of antithrombin achieved in each dose cohort. 15

17 Figure S3. Analysis of thrombin generation by antithrombin lowering quartiles. For all patients, at each time point an antithrombin level and a corresponding thrombin generation measurement were recorded. All available thrombin generation values were associated with an antithrombin activity level relative to baseline and were binned into antithrombin lowering quartiles. Peak thrombin levels measured in healthy volunteers (Part A) are shown for comparison. Boxes denote median and interquartile ranges. An antithrombin lowering dependent increase in thrombin generation is shown, with median peak thrombin increasing from 17 nm (interquartile range = 12 22) in the lowest quartile (<25% antithrombin lowering) to 67 nm (interquartile range = 5 87) in the highest quartile ( 75% antithrombin lowering) Peak Thrombin Generation (nm) N=4 Healthy Volunteers AT Lowering < 25% (N=3) AT Lowering 25 <5% (N=25) AT Lowering 5 <75% (N=25) Patients with Hemophilia AT Lowering 75% (N=16) 16

18 Figure S4. Effect of fitusiran on liver levels of alanine aminotransferase. The time courses of alanine aminotransferase (ALT) shown for patients in Part B (A) and Part C (B). A) 5 Part B ALT (U/L) weekly: red.45 weekly: blue.75 weekly: green B) ALT (U/L) * Part C.225 monthly: red.45 monthly: blue.9 monthly: green 1.8 monthly: yellow 8 mg monthly: purple *Patient discontinued on day

19 Figure S5. Effect of fitusiran on levels of bilirubin. The time courses of bilirubin shown for patients in Part B (A) and Part C (B). A) 5 Part B Bilirubin ( mol/l) weekly: red.45 weekly: blue.75 weekly: green B) 5 Part C Bilirubin ( mol/l) monthly: red.45 monthly: blue.9 monthly: green 1.8 monthly: yellow 8 mg monthly: purple 18

20 Figure S6. Effect of fitusiran on levels of D-dimer. The time courses of D-dimer shown for patients in Part B (A) and Part C (B). A) D-Dimer ( g/ml) Part B weekly: red.45 weekly: blue.75 weekly: green B) D-Dimer ( g/ml) * Part C *Patient discontinued on day monthly: red.45 monthly: blue.9 monthly: green 1.8 monthly: yellow 8 mg monthly: purple 19

21 Figure S7. Effect of fitusiran on levels of fibrinogen. The time courses of fibrinogen shown for patients in Part B (A) and Part C (B). A) 2 Part B Fibrinogen ( mol/l) weekly: red.45 weekly: blue.75 weekly: green B) 2 Part C Fibrinogen ( mol/l) monthly: red.45 monthly: blue.9 monthly: green 1.8 monthly: yellow 8 mg monthly: purple 2

22 Figure S8. Effect of fitusiran on levels of prothrombin time (PT). The time courses of PT shown for patients in Part B (A) and Part C (B). A) 25 Part B PT (s) weekly: red.45 weekly: blue.75 weekly: green B) 25 Part C PT (s) monthly: red.45 monthly: blue.9 monthly: green 1.8 monthly: yellow 8 mg monthly: purple 21

23 Figure S9. Effect of fitusiran on platelet levels. The time courses of platelets shown for patients in Part B (A) and Part C (B). A) 5 Part B Platelets (1^9/L) weekly: red.45 weekly: blue.75 weekly: green B) 5 Part C Platelets (1^9/L) monthly: red.45 monthly: blue.9 monthly: green 1.8 monthly: yellow 8 mg monthly: purple

24 Figure S1. Effect of fitusiran on activated partial thromboplastin time (APTT). The time courses of APTT shown for patients in Part B (A) and Part C (B). A) 15 Part B APTT (s) weekly: red.45 weekly: blue.75 weekly: green B) 15 Part C APTT (s) monthly: red.45 monthly: blue.9 monthly: green 1.8 monthly: yellow 8 mg monthly: purple