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: Abstract 1113 In this study, we investigated the molecular basis of upregulation of factor VII (FVII) gene expression by ribavirin, and found that intracellular GTP depletion induced by ribavirin activated FVII gene transcription and modulated transcription elongation. In 2006, Yamamoto et al. reported that anti-hepatitis C virus (HCV) agent ribavirin elevated the activity of FVII in HCV-infected hemophilia patients; however, the precise mechanisms were still unknown. In addition, the anti-HCV mechanisms of ribavirin were not yet fully elucidated, although the extended studies have been done. We investigated the effects of ribavirin in vitro and confirmed the approximately 4-fold upregulation of FVII mRNA by ribavirin treatment in HepG2 cells. FVII mRNA was increased in a dose-dependent manner up to 100μg/mL of ribavirin at a lower concentration than therapeutic concentration of 150μg/mL. FVII mRNA induction by ribavirin was also observed in a time-dependent manner from 24 h to 72 h after treatment. Ribavirin metabolite ribavirin 5'-monophosphate is one of the IMP dehydrogenase (IMPDH) inhibitors, and the other IMPDH inhibitors mycophenolic acid (MPA) and 6-mercaptupurine (6-MP) also induced FVII upregulation. It is well known that inhibition of IMPDH causes intracellular GTP depletion, and guanosine supplementation to salvage GTP could reverse FVII mRNA increase in ribavirin-treated cells. These results indicated that cellular GTP reduction associated with FVII gene upregulation. The mechanisms of gene upregulation by GTP depletion were not elucidated. The promoter activities and mRNA stability of FVII were analyzed under ribavirin treatment. The FVII gene promoter activity was enhanced up to 1.5-fold by ribavirin treatment; however the activation did not reach 4-fold induction of FVII mRNA increase. There was no significant change of FVII mRNA half-life in ribavirin-treated cells. Since the promoter activation might display transcription initiation capacity, the contribution of transcription elongation stage was further investigated. Transcription elongation was regulated by phosphorylation of carbo-terminal domain (CTD) of RNA polymerase II (PolII). Transcription elongation factor P-TEFb (positive-transcription elongation factor b), which consists as a complex of CDK9 and cyclin T, phosphorylates Ser of PolII CTD. The kinase activity of P-TEFb could be inhibited by 5,6-dichlorobenzimidazole 1-b-D-ribofuranoside (DRB). In FVII gene upregulation, DRB completely canceled ribavirin-induced FVII mRNA increase. We also performed nuclear run-on assay to verify the potential transcription elongation capacity of paused PolII, and observed a dramatic increase of FVII mRNA in ribavirin-treated cells. These results suggested that ribavirin-induced FVII gene upregulation was caused not only by transcription initiation but also by accelerated transcription elongation rate. There are various transcription factor associated with transcription elongation in addition to P-TEFb, such as elongin, ELL (eleven nineteen-lysine rich leukemia). We found that ELL3, a member of ELL family protein, was upregulated by ribavirin treatment. A ELL3 mRNA increase occurred prior to FVII mRNA upregulation, and the ELL3 upregulation was also canceled by guanosine supplementation. These results indicated ELL3 induction by ribavirin was also a response to cellular GTP depletion. To confirm the contribution of ELL3 protein to FVII gene transcription elongation, we used siRNAs specific to ELL3 and as expected, knockdown of ELL3 resulted in diminished FVII upregulation. A chromatin immunoprecipitation (ChIP) revealed ELL3 recruitment to the FVII gene, and the recruitments of PolII and CDK9 were also enhanced by ribavirin treatment. Taken together, FVII gene upregulation by ribavirin was associated with intracellular GTP depletion. The GTP reduction mainly modulates transcription elongation rate rather than transcription initiation, though the relationships between cellular GTP depletion and enhanced transcription elongation must be investigated. This study uncovered candidate mechanisms of ribavirin and the other IMPDH inhibitors and highlights a development of novel pharmaceutical therapies for hemophilia. Disclosures: No relevant conflicts of interest to declare.