ALBERT

Description: Background FVIIIa acts as a cofactor in the intrinsic pathway in which FIXa activates FX. ACE910 is a FIXa/FX-recognizing bispecific antibody that was designed to be a replacement for FVIIIa. Because of its nature, ACE910 is not affected by FVIII inhibitor. A clinical trial is now being conducted for the potential effect in the prophylactic treatment for bleeding hemophilia A patients. Here we present the perioperative care of a patient who had incidentally suffered from appendicitis and underwent an emergency surgery during the clinical trial. Methods Plasma ACE910 concentration and FXIa-triggered thrombin generation assay (TGA) was obtained in the central measurement of the trial. An activated partial thromboplastin time (APTT), and the tissue factor (TF)-triggered TGA were conducted at our laboratory. TF-triggered TGA was performed by means of calibrated automated thrombogram (Thrombinoscope BV), in accordance with the manufacturer's instructions. We used PPP-reagent LOWTM and FluCa-KitTM in Fluoroscan Ascent FLTM (Thermo Fisher Scientific Inc.) and monitored the thrombin generation for 2 hours, set at an excitation wavelength of 390 nm and an emission wavelength of 460 nm, and ThrombinoscopeTM software (Thrombinoscope BV). ROTEM® was performed as manufactured (Tem Innovations GmbH). Case The patient is a 60-year-old man suffering from hemophilia A without inhibitors and had severe hemophilic arthropathy in the number of target joints. Even after biweekly prophylaxis had been introduced by 2000 units of rFVIII concentrates, the annualized bleeding rate remained to be 10.1 In November 2013, ACE910 was introduced by way of subcutaneous administration and the initial dose was 3 mg/kg, followed by weekly administration of 1 mg/kg. After that, he had not had any of joint or soft tissue bleeding. In the 63rd week after the initial administration, he had severe abdominal pain and diagnosed as acute appendicitis that required emergency surgery. His APTT was consistently normal since ACE910 administration, we selected to undergo the surgery without any additional FVIII replacement, although his previous product was set up to be administrated any time on demand. ACE910 had been administered as scheduled earlier on the day of the diagnosis of acute appendicitis, followed by the emergency appendectomy. Results The appendectomy was performed by pararectal incision. Although the patient's appendix was necrosed and perforated, it was easy to stop bleeding during surgery and the total amount of bleeding was only 45 mL. On postoperative day 11, a small amount of bleeding was found after the removal of drainage catheter placed subfascially, however, the bleeding stopped immediately after the bleeding site was sutured. No other issues on bleeding were found. Trough levels of plasma ACE910 concentration were maintained at 27-41 µg/mL during the period between the 12th week after the initiation of ACE910 and the time of preoperative stage. In FXIa-triggered TGA, lag time was remarkably improved after the initiation of ACE910 and remained stable throughout the course of emergency surgery (Table 1). Although peak thrombin levels were slightly decreased a week after surgery, APTT and several In-TEM values by ROTEM® remained at almost normal levels (Table 2). Discussion and Conclusion We successfully conducted the hemostatic management for appendicitis in the perioperative period without any additional administration of FVIII concentrate. The patient showed less bleeding under ACE910 prophylaxis. To date there are little information on appropriate use of FVIII concentrate in patients with acute bleeding or major surgery who are under ACE910 prophylaxis. Generally in bleeding hemophilic patients with major surgery, the loss of clotting factors due to hemodilution by fluid replacement should also be carefully monitored. In such condition, the optimum ACE910 concentration could not be well interpreted, however, the careful monitoring might be required especially in highly invasive surgeries. In our experience, TF-triggered TGA demonstrated a marginal change only between postoperative days 7 and 13, although it is not totally known whether these changes were affected by ACE910 pharmacodynamics. Further researches are needed to explore the suitable biomarkers to indicate hemostasis of hemophilic patients under the administration of ACE910. Disclosures Suzuki: Baxalta: Honoraria; Bayer Healthcare: Honoraria; Novo Nordisk Pharma: Honoraria. Kiyoi:Novartis Pharma K.K.: Research Funding; MSD K.K.: Research Funding; Pfizer Inc.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Yakult Honsha Co.,Ltd.: Research Funding; Alexion Pharmaceuticals: Research Funding; Teijin Ltd.: Research Funding; Taisho Toyama Pharmaceutical Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Japan Blood Products Organization: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; FUJIFILM RI Pharma Co.,Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Mochida Pharmaceutical Co., Ltd.: Research Funding. Kasai:Chugai Pharmaceutical Co., Ltd.: Employment. Matsushita:Asahi Kasei Pharma: Honoraria, Research Funding, Speakers Bureau; Sysmex: Speakers Bureau; Octapharma AG: Honoraria; Kyowa-Kirin: Honoraria, Research Funding; CLS-Behling: Research Funding; Biogen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bayer Healthcare: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Seamens: Speakers Bureau; Nihon Pharmaceutical: Honoraria, Research Funding, Speakers Bureau; Kaketsuken: Honoraria, Research Funding, Speakers Bureau; Eisai: Research Funding; Baxalta: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Japan Blood Products Organization: Honoraria, Research Funding; Novartis Pharma: Honoraria, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novo Nordisk Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Chugai Pharmaceutical Co., Ltd.: Research Funding.

Description: Introduction: Emicizumab (Emi) is an anti-factor (F)IXa/FX bispecific antibody that mimics FVIIIa cofactor function. The clinical trials on Emi prophylaxis for hemophilia A patients with inhibitor (HA-Inh) demonstrated marked reduction in bleeding rates. However, Emi at clinically relevant plasma concentrations (~50 mg/ml) corresponding to ~15% of FVIII equivalent activity may not provide enough hemostasis at severe bleeding or major surgical intervention. In these circumstances, additional infusions of bypassing agents (BPAs) may be needed for hemostatic management. In the HAVEN 1 study, thrombotic events/ thrombotic microangiopathy occurred in Emi-treated HA-Inh receiving consecutive infusions of aPCC in higher doses. After these reports, guidance was provided to avoid the use of aPCC as first choice during Emi prophylaxis. If aPCC is the only available BPA, use of the lowest dose expected to achieve hemostasis with the initial dose ≦50 U/kg was recommended. To further support these recommendations, more investigation is required for the use of BPAs under Emi prophylaxis. Objective: We examined the coagulation effects by spiking BPAs to whole blood or plasma samples from Emi-treated HA. Methods: Samples from eight Emi-treated HA-Inh and two hemophilia A patients without inhibitors (HA-Inh(-)) in phase 1/2 and HAVEN 1 were tested. The Emi doses were 0.3-3.0 mg/kg/w or 1.5 mg/kg/w, respectively. Either aPCC or rFVIIa was spiked to patients' whole bloods or plasmas, and assessed by rotational thromboelastometry (ROTEM; Ca2+ trigger) and clot waveform analysis (CWA; ellagic acid/tissue factor-mixed trigger; Nogami, JTH2018). Furthermore, samples in Emi-treated HA-Inh of phase 1/2 (n=1) or HAVEN 2 (n=2) receiving BPAs infusion for hemostatic management were assessed by both assays. Results and Discussion: The baseline levels, before spiking BPAs, in ROTEM parameters, such as clot time (CT) and clot formation time (CFT), of Emi-treated HA were 1,380/521sec(s) (reference range 762-1,127/207-511s), respectively. Spiking aPCC markedly shortened CT/CFT to 167/81.5s and 148/78.0s at 0.65 and 1.3 U/mL corresponding to 50 and 100 U/kg infusion, respectively. Even at 0.13 U/mL (10 U/kg), CT/CFT shortened to 290/92.6s, which was shorter than that of reference range. Its effect was dose-dependent. Spiking rFVIIa also improved CT/CFT to 726/179s, 551/141s at 32.3 and 112 μg/mL (90 and 270 μg/kg). In CWA, the baseline levels of adjusted-|min1| (ad|min1|), indicative of maximum coagulation velocity, in Emi-treated HA were 5.53 (normal control 7.98±0.24). Ad|min1| was improved to 6.48 and 8.04 by spiking aPCC (10 and 100 U/kg), or 7.11 and 7.12 by spiking rFVIIa (90 and 270 μg/kg), respectively. For rFVIIa its effect was not dose-dependent. According to the investigation for in vivo effect by BPAs infusion in a total of 9 treatment events of Emi-treated HA-Inh, CT/CFT (1,812/553s) was improved to 1,151/346s, of which was within normal range. Ad|min1| (5.7) was improved to 6.6 30 min(m) after aPCC infusion (44-74 U/kg). By rFVIIa infusion (90-119 μg/kg), CT/CFT (1,962/758s) improved to 912/207s, and ad|min1| (4.9) also improved to 6.5 30m after infusion, of which improvement remained within normal range. The hemostasis effects were clinically satisfying and no thrombosis occurred in all cases. The ROTEM parameters by spiking aPCC were apparently hypercoagulant relative to those by infusion at same dose, supporting our previous report (Furukawa JTH2015). The shortening effect on the ROTEM parameters by spiking aPCC 100 U/kg in Emi-untreated HA seemed to be similar to that by spiking aPCC 10 U/kg in Emi-treated HA. In addition, ad|min1| by spiking aPCC 10 U/kg with Emi also improved as well as ~100 U/kg without Emi in our previous report (Nogami JTH2018). Taken together, it is suggested that the hemostatic effect of aPCC infusion (10 U/kg) to Emi-treated HA could be comparable to that of 100 U/kg to Emi-untreated HA-Inh, though there is a limitation to predicting the hemostatic effect of aPCC in a clinical setting from these ex vivo results. Conclusion: Spiking tests in Emi-treated HA-Inh by ROTEM and CWA are useful to evaluate clinical coagulation potentials and dose finding of BPAs in Emi-treated HA receiving BPAs. Additionally, under Emi prophylaxis, a lower dose of aPCC infusion might be a possible option to treat breakthrough bleed in Emi-treated HA-Inh. Disclosures Furukawa: CSL Behring: Research Funding. Nogami:Bayer: Consultancy, Honoraria, Research Funding; Shire: Consultancy, Honoraria, Research Funding; Novo Nordisk: Consultancy, Honoraria, Research Funding; Sysmex: Consultancy, Honoraria, Research Funding; Chugai Pharmaceutical Co., Ltd: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Anti-FIXa/X bispecific antibodies, Research Funding, Speakers Bureau. Matsumoto:Shire Japan Co. Ltd: Research Funding. Kasai:Chugai Pharmaceutical Co., Ltd: Employment. Shima:F. Hoffmann-La Roche Ltd: Membership on an entity's Board of Directors or advisory committees; Chugai Pharmaceutical Co., Ltd: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Anti-FIXa/X bispecific antibodies , Research Funding, Speakers Bureau.

Description: Background: ACE910, a humanized bispecific antibody to factor (F) IXa and FX mimicking the functions of FVIII, exerts tenase activities without FVIII (a) (Kitazawa et al. Nature Medicine. 2012;18, 1570). In primate hemophilia A (HA) models, the hemostatic enhancing effect of ACE910 has been reported and the clinical study investigating the effect and safety of ACE910 for human HA patients is on-going. However, the hemostatic effect of ACE910 remains unquantified and difficult to be evaluated. Objectives: In this study, we evaluated the viscoelastmetric parameters in the whole blood obtained from HA patients under long-term treatment of ACE910 in phase 1 and its extension studies, utilizing rotation thromboelastometry (ROTEM), in order to investigate global hemostatic function of HA patients under treatment with ACE910. Methods: Ca2+-triggered hemostatic functions were assessed by ROTEM in the citrated whole blood samples obtained and stabilized at the indicated points from five severe HA cases with inhibitors (Case 1 : 39 BU/ml, 2 : 41 BU/ml, 3 : 17 BU/ml, 4 : 11 BU/ml and 5 : 111 BU/ml ) and two severe cases without inhibitors (Case 6 and 7) under subcutaneous administration of ACE910, with the different types of dosing regimen (group A with 0.3 mg/kg/week subcutaneous injection (loading dose of 1 mg/kg) for Case 1, 2, group B with 1 mg/kg/week (loading dose of 3 mg/kg) for Case 3, 4, group C with 3 mg/kg/week for Case 6 and group D changing from group A to group C for Case 5, 7) in a course of clinical study. The samples from the subjects prior to administration of ACE910 were spiked ex vivo with ACE910 and the hemostatic functions of them were also evaluated by ROTEM. The parameters of clot time (CT), clot formation time (CFT), maximum clot formation (MCF) and alpha angle were evaluated. The hemostatic potentials in the samples from twenty healthy volunteers and other ten HA patients with the various FVIII:C were evaluated by ROTEM as controls and the correlation between FVIII:C and CT was obtained. Annualized bleeding rates (ABR) of each case were calculated. The studies were approved by local ethics committee and the informed consent was obtained from each patient. Results: Addition of ACE910 (f.c. 10 or 30 μg/ml) into the sample from each case prior to administration of ACE910 shortened CT from 5,562 ± 374 sec (median 5,924 sec) to 1,475 ± 138 or 1,131 ± 82 sec, equivalent to FVIII:C 3.3 or 12.2 IU/dL, respectively, in an ACE910 concentration dependent manner. In group A, the plasma concentration of ACE910 got to 12 ± 5 μg/ml at 12 week. CT was shortened to 1,443 ± 24 sec, equivalent to FVIII:C 3.7 IU/dL maximally at 47 week consistent with the result of the spiked result. ABR decreased from 38.6 ± 25.8 to 1.1 ± 0.8, showing 97% decrease. As for Case 3 in group B (ACE910: 31 μg/ml at 12 week), CT was shortened to 1,164 sec, equivalent to FVIII:C 11.9 IU/dL maximally at 47 week consistent with the spiked data. ABR apparently decreased from 38.6 to 3.6. In Case 4 who dropped out the clinical study at 4 week, CT was shortened to 977 sec, equivalent to FVIII:C 22 IU/dL, maximally at 3 week, and continued to be shortened till 29 week (1,758 sec, equivalent to FVIII:C 1.1 IU/dL). In group C, the shortening of CT from 1,594 ± 103 sec, equivalent to FVIII:C 2.0 ± 1.0 (median 3.6) IU/dL (before administration of ACE910), to 1,226 ± 71 sec (8.5 ± 2.5 (median 10.4) IU/dL) (at steady state) was observed in consistent with the decrease of ABR from 8.1 to 1.1. In group D, CT was shortened from 3,405 ± 386 sec to 1,409 ± 85 sec, equivalent to FVIII:C 4.2 IU/dL after middle to high dose escalation in consistent with decrease of ABR from 22.3 ± 9.6 to 3.8 ± 2.6. Among all cases except Case 4, CT and ABR were of clinically relevant difference between before and after administration of ACE910. Conclusions: The comprehensive hemostatic function evaluated by ROTEM in hemophilia A patients irrespective of presence of inhibitors was improved after administration of ACE910 in a dose dependent fashion, resulting in the reduction of ABR, which suggested the hemostatic effectiveness of ACE910 for HA patients. Disclosures Yada: Chugai Pharmaceutical Co., Ltd: Research Funding. Nogami:Bayer, Novo Nordisk, Baxalta. Biogen: Research Funding; Chugai: Membership on an entity's Board of Directors or advisory committees; Bayer, NovoNordisk, Baxalta, Chugai, Kaketsuken, Pfizer, Biogen: Honoraria. Shida:Chugai Pharmacoceutical Co. Ltd.: Research Funding. Takeyama:Chugai Pharmaceutical Co., Ltd.: Research Funding. Kasai:Chugai Pharmaceutical Co., Ltd.: Employment. Shima:Chugai Pharmaceutical Co., Ltd: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biogen: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Baxalta: Honoraria, Research Funding; Novo Nordisk: Honoraria, Research Funding; Kaketsuken: Honoraria; Bayer: Honoraria, Research Funding.

Description: Emicizumab is a factor (F) VIII(a) mimicking bispecific antibody to FIX(a) and FX, which is a new therapeutic agent for hemophilia A patients (PWHA) with inhibitor. Although the clinical effect and safety were confirmed in the clinical studies (NEJM 2016, NEJM 2017, Blood Adv. 2017), there exists difficulty in monitoring of emicizumab by APTT-based assay. In the present study, we challenged to establish a hemostatic monitoring system convertible to FVIII activity (FVIII:C) for PWHA under the emicizumab prophylaxis by utilizing non-activated rotational thromboelastometry (ROTEM). The studies were approved by local ethics committee and the informed consent was obtained from each patient. We firstly prepared the FVIII-deficient whole blood samples treated with a neutralizing anti-FVIII polyclonal IgG (30BU/mL), and compared the in vitro hemostatic response in the presence of emicizumab (0, 5 and 50µg/mL) spiked into these samples among three modes of ROTEM such as NATEM, EXTEM and INTEM. According to CaCl2 triggered-NATEM without any other activator, the sum of clot time and clot formation time (CT+CFT) was 1269 ± 197 sec (mean ± SD) for normal control and 6713 ± 855 sec for the samples without emicizumab. The value of CT+CFT was significantly shortened in the presence of emicizumab at 5 µg/ml and 50 µg/ml to 2157 ± 192 sec and 1521 ± 313 sec, respectively (p = 0.025). By contrast, measured by EXTEM utilizing tissue factor (0.5 pM) and CaCl2 as triggers, the values of CT+CFT for control, 0, 5 and 50 µg/mL of emicizumab were 552 ± 97 sec, 771 ± 186 sec, 658 ± 135 sec and 778 ± 90 sec, respectively. There was no significant difference (p = 0.44) among them. By INTEM, triggered by ellagic acid with CaCl2, the values of CT+CFT for control, 0, 5, and 50 µg/mL of emicizumab were 662 ± 202 sec, 5854 ± 705 sec, 951 ± 170 sec and 814 ± 216 sec, respectively. The value of CT+CFT was shortened in the presence of emicizumab, however, little difference was observed between two doses. The other parameters, maximum clot firmness and alpha angle, were not suitable for quantitative evaluation irrespective of the mode of measurement. These suggested that CT+CFT based on NATEM is a suitable parameter for the monitoring of emicizumab. In order to develop a NATEM-based hemostatic scale convertible to FVIII:C, we secondly measured CT+CFT in the samples (n = 81) from PWHA with various FVIII:C levels in our hospital. The samples were classified based on FVIII:C into three groups, such as Tertile (T)1 with undetectable FVIII:C (