Spontaneous Bleeding Episodes Research Articles

Hemophilia A in the third millennium

Hemophilia A is an X-linked hereditary bleeding disorder due to the deficiency of coagulation factor VIII (FVIII). According to the degree of FVIII deficiency, mild, moderate or severe forms are recognized. Although patients with mild hemophilia A usually bleed excessively only after trauma or surgery, those with severe hemophilia experience frequent episodes of spontaneous or excessive bleeding after minor trauma, particularly into joints and muscles. The modern management of hemophilia began in the 1970s and is actually based upon several plasma-derived or recombinant FVIII products. In addition, the synthetic drug desmopressin can be used to prevent or treat bleeding episodes in patients with mild hemophilia A. Long-term and continuous substitution therapy (prophylaxis), the recommended treatment in severe hemophilia, prevents bleeding and the resultant joint damage. In the last twenty years the high standard of hemophilia care has greatly improved the quality of life of patients and their life expectancy has reached that of the non-hemophilic male population, at least in high-income countries. The most serious and challenging complication of treatment of hemophilia A is the development of inhibitors, which renders FVIII concentrate infusion ineffective and exposes patients to an increased risk of morbidity and mortality. In this narrative review, the actual knowledge on the clinical features and management of patients with hemophilia A is summarized.

Kisspeptin, Leptin, and Retinol-Binding Protein 4 in Women with Polycystic Ovary Syndrome

Objectives: To compare plasma kisspeptin, serum leptin, and serum retinol-binding protein 4 (RBP4) levels in women with and without polycystic ovary syndrome (PCOS) and to correlate these among each other and with clinical, hormonal, and metabolic parameters. Methods: Ninety women, including 54 women with PCOS and 36 without PCOS, participated in this study. For all patients, history and physical examinations were performed and blood samples were collected between days 3 and 8 of a spontaneous bleeding episode in the PCOS group and during normal menses of controls. Plasma kisspeptin, serum leptin, and serum RBP4 levels were measured using specific commercial assays. Results: Kisspeptin, leptin, and RBP4 levels were significantly higher in the PCOS group than in controls. Kisspeptin and RBP4 levels were significantly higher among obese PCOS patients than controls. Leptin levels were higher among obese PCOS patients than non-obese PCOS patients or controls. Kisspeptin and leptin levels of PCOS patients were significantly correlated with RBP4 levels. When only obese PCOS patients were analyzed, kisspeptin levels correlated with only the free androgen index. Conclusions: These findings suggest that kisspeptin, leptin, and RBP4 are associated with metabolic disturbances in women with PCOS.

Corifact™/Fibrogammin® P in the prophylactic treatment of hereditary factor XIII deficiency: results of a prospective, multicenter, open-label study

Factor XIII (FXIII) deficiency is an extremely rare congenital condition that is associated with a high risk of potentially life-threatening intracranial hemorrhage, poor wound healing, spontaneous abortion, and a life-long tendency towards spontaneous bleeding and severe bleeding after trauma or surgery. Routine prophylaxis with FXIII concentrate is recommended in all individuals with FXIII levels <1 IU/dL from the time of diagnosis, and in some severely affected patients with FXIII levels of 1–4 IU/dL. Fibrogammin® P is a highly purified, pasteurized, plasma-derived concentrate that has been available in Europe and other countries since 1993 and has recently been approved as Corifact™ in the USA. To support the US registration of Corifact™, a 52-week, prospective, multicenter, open-label study was conducted in 41 patients (mean age 19 years; range <1–42 years) with congenital FXIII deficiency. Corifact™/Fibrogammin® P was administered intravenously at an initial dose of 40 IU/kg every 4 weeks, with dosing adjusted to maintain a trough FXIII activity level of 5–20%. No spontaneous bleeding episodes requiring FXIII treatment were reported during the study (primary endpoint). Preoperative use of Corifact™/Fibrogammin® P successfully prevented postoperative bleeding in two surgeries. Corifact™/Fibrogammin® P was well tolerated during a total exposure of ∼455 subject-months. No patient withdrew from treatment, and there were no reports of virus transmission or thromboembolism-related events. This study adds to the wealth of data gained from clinical trials and almost 20 years of clinical use confirming that Corifact™/Fibrogammin® P is an effective and well-tolerated prophylactic treatment for congenital FXIII deficiency.

Efficacy, PK and Safety Results of a Phase I/II Clinical Trial of Recombinant Fusion Protein Linking Coagulation Factor IX with Albumin (rIX-FP) in Previously Treated Patients with Haemophilia B (PROLONG - 9FP)

AbstractAbstract 1121Factor IX (FIX) replacement therapy is the standard of care for patients with haemophilia B. FIX products currently available have a relatively short half-life, requiring 2–3 times a week intravenous prophylactic treatment to achieve a significant bleeding reduction. A recombinant FIX albumin fusion protein (rIX-FP) was generated by genetic fusion of human recombinant albumin to rFIX to extend the half life of FIX. A Phase I/II open-label, multicenter, clinical study of rIX-FP has been completed in previously treated patients with severe hemophilia B (FIX ≤ 2%) as part of the PROLONG - 9FP clinical developmental program. The objectives of the study were to evaluate the prevention of bleeding episodes during once weekly prophylaxis and to assess the hemostatic efficacy of rIX-FP for the treatment of bleeding, in addition to safety and pharmacokinetic (PK) assessments.The study consisted of a 10 to 14 day PK evaluation period, and a 3 to 12 month safety and efficacy evaluation period, during which subjects received either on-demand or prophylaxis treatment. Subjects receiving weekly prophylactic treatment were initially treated with a dose based upon the subject's PK profile, bleeding phenotype, and physical activity level. The dose could be adjusted based on clinical outcome, while maintaining a 7 day treatment interval. Subjects receiving on-demand treatment were treated with a dose based upon the subject's PK profile and WFH recommendations for treatment of bleeding episodes.Seventeen study subjects from hemophilia treatment centers in Israel and Bulgaria participated in the study, 13 of whom received weekly prophylaxis treatment and 4 of whom received on-demand treatment. Following a single infusion of 25 IU/kg rIX-FP (n=13), the PK parameters (t1/2 = 94 hrs, AUC0-inf= 3414 hr*IU/dL) were comparable to those previously reported from the Phase I study (Blood prepublished Aug 2, 2012). In addition, rIX-FP maintained a baseline-corrected mean trough level of 3.8% and 2.7% at Day 7 and Day 14, respectively, after 25 IU/kg rIX-FP administration. There were no AEs considered as possibly related to rIX-FP. There were no allergic reactions, inhibitors to FIX or antibodies to rIX-FP reported. All 10 prophylaxis subjects who were previously receiving routine prophylaxis with FIX were maintained successfully on weekly treatment with rIX-FP for the entire study. Furthermore, three prophylaxis subjects who were previously treated on-demand were maintained successfully on weekly prophylaxis treatment with rIX-FP with at least 85% reduction in the annualized spontaneous and total bleeding rate compared to the annualized bleeding rate prior to study entry. All of the bleeding events were treated successfully, including approximately 90% of the events with a single infusion of rIX-FP. The mean weekly product consumption of rIX-FP (IUs) was reduced compared to the consumption of the previous FIX product (IUs). No subject was withdrawn from the study due to safety concerns or lack of hemostatic efficacy. This Phase I/II study has demonstrated the clinical efficacy of rIX-FP for once weekly routine prophylaxis to prevent spontaneous bleeding episodes and treatment of bleeding episodes, in addition to the excellent safety characteristics and improved PK profile. A detailed analysis of the efficacy of weekly routine prophylaxis and treatment of bleeding episodes, improved PK and safety properties of rIX-FP will be presented. Disclosures:Martinowitz:CSL Behring: Honoraria, Investigator for CSL clinical study of rIX-FP Other. Lubetsky:CSL Behring: Investigator for CSL clinical trial of rIX-FP Other. Santagostino:CSL Behring: Honoraria, Investigator for CSL Behring clinical trial of rIX-FP Other, Research Funding, Speakers Bureau. Jotov:CSL Behring: sub-investigator for CSL clinical trial of rIX-FP Other. Barazani-Brutman:CSL Behring: study coordinator for CSL Behring clinical trial of rIX-FP Other. Voigt:CSL Behring: Employment. Moises:CSL Behring: Employment. Jacobs:CSL Behring: Employment. Lissitchkov:CSL Behring: Investigator for CSL Behgring clinical trial of rIX-FP Other.

The modern treatment of haemophilia: a narrative review.

Haemophilia A and B are X chromosome-linked bleeding disorders that are included among the rare diseases and are caused by mutations in the genes for factor VIII (FVIII) and factor IX (FIX)1. Both these clotting factors are part of the intrinsic pathway of blood coagulation. Individual with haemophilia may have severe, moderate or mild forms of the diseases, defined by factor plasma levels of 1% or less, 2 to 5% and 6 to 30%, respectively. The prevalence of haemophilia A is 1 case in 5,000 male live births, while that of haemophilia B is 1 case in 30,0002,3. Although patients with mild haemophilia usually bleed excessively only after trauma or surgery, those with severe haemophilia experience frequent episodes of spontaneous or excessive bleeding, particularly into joints and muscles, after minor trauma3. The modern management of haemophilia began in the 1970s and currently includes several plasma-derived and recombinant factor VIII products4. This article reviews the recent history, current knowledge, the most important progress and expected improvements in haemophilia care.

Malignant disease in the haemophilic population: moving towards a management consensus?

The Malignancy in Haemophilia Workshop Group convened a consensus working group of haematologists and oncologists to review topics related to malignancy in haemophilia. The treatment of malignant disease in this population is increasingly relevant as both outcome and lifespan continue to improve. Although adequate guidance exists for control of spontaneous bleeding episodes and of haemostasis in general surgery, information for management of haemostasis in patients with various malignancies is sparse. To date, no clinical guidelines exist for management of complex bleeding problems, diagnosis, therapy and follow-up of malignancies in haemophilia. Furthermore, it remains unclear whether or not morbidity and mortality outcomes associated with malignancies are affected by haemophilia or by its treatment. Through presentation of five malignancies - prostate cancer, colorectal cancer, acute leukaemia, bladder cancer and hepatocellular carcinoma - important issues are highlighted, such as risk from bleeding as a symptom of malignancy; risks from invasive screenings and how these should be handled in haemophilic individuals; the implications of chemotherapy and treatment schedules, bone marrow suppression, radiotherapy, or surgery; and the likelihood of an interaction between treatment for haemophilia and malignancy outcomes. Ultimately, the aim is to establish consensus guidelines to direct and harmonize future treatment policy for malignant disease in the haemophilic population.

Laparoscopy-Assisted Small Bowel Resection for Treatment of Adult Small Bowel Intussusception in a Patient with Factor VII Deficiency

Factor VII deficiency is a rare congenital bleeding disorder characterized by episodes of spontaneous bleeding in severely affected individuals. It is rare intussusception due to submucosal hematoma in coagulation factor deficiency patient. We recently experienced an adult small bowel intussusception in a patient with factor VII deficiency. A 36-yr old female patient with coagulation factor VII deficiency who was referred to our hospital underwent emergency surgery for treatment of the small bowel intussusceptions. Emergency laparoscopy-assisted small bowel resection was performed for treatment of small bowel intussusception caused by submucosal hematoma. The patient was successfully treated with close laboratory monitoring and laparoscopy-assisted small bowel resection.

Management of a child with von Willebrand disease (type 2A) and extensive burns: A case report

Von Willebrand disease (VWD) is a common inherited bleeding disorder. Its diagnosis is based on the association of bleeding symptoms and specific biological data [1,2]. The cornerstone of its treatment is the replacement of the deficient protein during acute spontaneous bleeding episodes or before invasive procedures. It may induce haemorrhagic morbidities in severe burn since the coagulation homeostasis is frequently impaired by itself in that setting and since excision and closure of burn wound are regularly required in such patients. However, the specific management of patients with both severe burn and type 2A VWD has not yet been described. We therefore report a paediatric case of type 2A VWD admitted for severe burn.

Efficacy of Baxter's Longer Acting rFVIII (BAX 855) in Mouse Models of Hemophilia A

Abstract 4350Factor VIII (FVIII) is a critical component of the intrinsic coagulation pathway. FVIII concentrates are used in patients with hemophilia A to provide a hemostatic FVIII level sufficient to treat and prevent bleeding episodes. Multiple prophylactic administrations per week are necessary to maintain a FVIII level of at least 1% of normal to effectively prevent or reduce spontaneous bleeding episodes. A longer acting FVIII concentrate would reduce the frequency of infusions. Baxter and Nektar have developed a longer-acting PEGylated variant (BAX 855) of Baxter's recombinant FVIII (ADVATE process). The aim of the presented studies was to evaluate the efficacy of BAX 855 in two primary pharmacodynamics in hemophilia A mice: a tail-tip bleeding model (TTBM) and a carotid occlusion model (COM).BAX 855 was tested at a dose of 200 IU/kg rFVIII. ADVATE™ served as the active reference item, and formulation buffer as the negative control item. Mice received intravenous treatment with either BAX 855 or ADVATE™ 12–40h prior to the experiment. Buffer was administered 5–15min prior to the experiment.Animals were anesthetized using ketamine and xylazine. In the TTBM (n=16) the tip of the tail was cut off and total blood loss [mg] was assessed over 60 minutes. In the COM study (n=10) the left carotid artery was exposed and the endothelium was denuded by topical application of FeCl3. Time to occlusion [min] was assessed.Buffer-treated animals had a median blood loss of 951 mg in the TTBM and did not show vessel occlusion within 30 min in the COM. In the TTBM, a prophylactic effect of ADVATE™ could be observed for up to 18 h, leading to a reduction in blood loss to 121 mg after this treatment interval. Prophylactic efficacy after treatment with BAX 855 could be observed for up to 40 h with a median blood loss of 73 mg after 30 h and 436 mg after 40 h. In the COM, treatment with ADVATE™ 12 h before the experiment shortened the time to occlusion to 3.8 min. With BAX 855, a similar efficacy could be observed for up to 24 h after administration (5.2 min).In summary, the results of our studies show that BAX 855 is efficacious in these two mouse models of hemophilia A and that the efficacy of BAX 855 is prolonged compared with an unmodified rFVIII. Furthermore, the intravenous injection of BAX 855 was well tolerated in all animals across all treatment groups without any signs of acute toxicity. Disclosures:Schiviz:Baxter Innovations GmbH: Employment. Barbara:Baxter Innovations GmbH: Employment. Lawo:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment. Turecek:Baxter Innovations GmbH: Employment. Schwarz:Baxter Innovations GmbH: Employment. Muchitsch:Baxter Innovations GmbH: Employment. Hoellriegl:Baxter Innovations GmbH: Employment.

Management of the Sponataneous Bleeding Episodes in Factor VII Deficiency. A Prospective Evaluation of the STER,

Abstract 3368 IntroductionPatients with an inherited factor VII (FVII) deficiency may display a wide range of clinical phenotypes, from an asymptomatic condition to serious hemorrhagic episodes such as fatal central nervous system (CNS) or gastrointestinal (GI) bleeds (Mariani G et al. Thromb Haemost 2005; 93: 481–7). Symptomatic patients can be divided into two major categories: those with mild-to-moderate bleeding tendency and individuals with a severe bleeding tendency which may be more severe than that in hemophilia. The former group mainly experience mucosal bleeding, a clinical picture that mimics that of a platelet disorder and often does not call for treatment. In contrast, for the most severely affected patients, characterized by life- or limb-threatening hemorrhages, more aggressive Replacement Therapy (RT) as long or short-term prophylxis may be required. AimWithin the frame of the Seven Treatment Evaluation Registry we have prospectively collected large numbers of treatments for spontaneous bleeding episodes and evaluted schedules and side-effects. Clinicaltrials.gov identifier: NCT01269138. ResultsAs of May 2011, 64 evaluable spontaneous bleedings (managed in 16 haemophilia treatment centres, 11 countries), were observed in 40 FVII-deficient subjects (25 females and 15 males) with residual FVII coagulant activity (FVIIc) levels between <1% and 20% (90% of patients had FVIIc below 5% of normal). Bleeding episodes were grouped into the following categories: 1. CNS + GI (n=2+4), 2. Hemarthroses (n=23), 3. Muscle hematomas (n=6), Epistaxis (n=4), Gum bleeding (n=7), Menorrhagia (n=11), Other (n=7) (hemorrhoidal n=2, subcutaneous hematomas n=2, easy bruising n=1, hematuria n=1, hemoperitoneum n=1). Recombinant FVIIa was given to treat 47 episodes, plasma derived FVII (pdFVII) concentrates 7, Prothrombin Complex Concentrate (PCC) 3 and Fresh Frozen Plasma (FFP) 7 (Table 1). No thrombosis episodes were reported during the thirty-day follow up.Table 1Type of Bleeding, Replacement Therapy (RT) schedules and outcomeBleedsnRTRT daysn. of dosesTotal dose range (μg/Kg IU/Kg ml/Kg)OutcomeCNS & GI6rFVIIa 31–41–8rFVIIa 160-240median 160Excellent 1 P.Effective 2pdFVII 2pdFVII 15-60–P.Effective 1 Effective 1FFP 1FFP 2–Effective 1Hemarthrosis23rFVIIa 221–21–7rFVIIa 18 – 110060Excellent 19 Effective 3PCC 1PCC 38–Excellent 1Hematomas (muscle & subcut.)8rFVIIa 41–61–12rFVIIa 54- 8066Excellent 3 Effective 1pdFVII 1pdFVII 56–Excellent 1PCC 2PCC 30–Excellent 1 n.e. 1FFP 1FFP 72–Effective 1Menorrhagia11rFVII 101–61–8rFVII 20 – 12041Excellent 5 Effective 5pdFVII 1pdFVII 135Effective 1Epistaxis & Gum bleeding12rFVIIa 511–3rFVIIa 27- 18071Excellent 4 Effective 1pdFVII 4pdFVII 21-5644Excellent 3 Effective 1FFP 3FFP 1.8-122.6Effective 3Other5rFVIIa 31-111-52rFVIIa 33-1716†33Excellent 1 Effective 2pdFVII 1pdFVII 18–Excellent 1FFP 1FFP 31–P.Effective 1*patient treated for severe hemoperitoneum; P.Effective:Partly Effective; n.e.: not evaluable ConclusionTreatment schedules and outcomes depend on the clinical setting. As for the severest bleeds (CNS and GI), on demand RT is probably not the best choice and prophylaxis should be considered as first line option especially in babies and children. Hemarthroses can be successfully treated for one day only; the number of doses used vary (from 1 to 7) as variable is the total dose of rFVIIa (from 35 to 210 μg/Kg/bw, mean 73.7, median 60) but an average intermediate dose of 60 μg/Kg/bw, possibly split in two doses, seems appropriate. The same holds for muscle and subcutaneous hematomas, always with one-day treatment schedule. RT protocols for menorrhagia varied either in terms of duration or rFVIIa total dose (1 to 6 days, total rFVIIa dosing ranging from 20 to 135 μg/Kg/bw, mean 88, median 40); treatment duration, however, did not appear to influence the outcome as one day RT showed the same efficacy as multiple days schedules. Successful RT for gum bleeding and epistaxis required treatment for one day only with doses of rFVIIa ranging from 27 to 180 μg/Kg/bw (mean 87.8, median 71) and of pdFVII ranging from 21 to 56 IU/Kg/bw (mean 41.5, median 44.5). Others bleed management were reported (hemorrhoidal, hematuria, easy bruising, peritoneal bleeding) but numbers do not allow to draw any conclusion as is the case for treatments carried out with FFP and PCCs. Disclosures:Knudsen:Novonordisk: Employment.

Immune tolerance induction therapy for patients with hemophilia A and FVIII inhibitors particularly using low‐dose regimens

Inhibitory antibodies against infused clotting factor VIII concentrates (FVIII) developed in 20-30% of patients with hemophilia A. Bypass therapy may control the bleeds in patients with FVIII inhibitors, however, immune tolerance induction (ITI) therapy is the only proven modality for eradicating FVIII inhibitors. Since the cost of high-dose (200 IU/kg) ITI is extremely expansive, we conducted this study to identify whether low-dose ITI can be an alternative strategy besides high-dose ITI or bypass therapy. Patients with hemophilia A and FVIII inhibitors treated by ITI in Kaohsiung Medical University Hospital from January 2000 to January 2010 were enrolled. Regimens of ITI therapy included high-dose (100 IU/kg) and low-dose (30-50 IU/kg). High-dose ITI therapy for two high responders (HRs) and low-dose ITI therapy for three HRs and all low responders (LRs) were performed. Complete tolerance was achieved in 2 HRs with high-dose regimen, and in one HR and 19 LRs with low-dose regimens. We administered low-dose ITI combined with immune suppressants treatment for one of the patient with extremely high FVIII inhibitor titers and the inhibitor level markedly declined and no spontaneous bleeding episode was noticed during the treatment period. The outcome of ITI in our study was satisfactory without clinically significant complications. Low-dose ITI regimens can effectively treat patients with high responder inhibitors, including one patient with extremely high inhibitor levels over 700 BU. Low-dose ITI may be an alternative modality for FVIII inhibitors management, especially in countries with limited resources.

Experience with Ulcerated, Bleeding Autologous Dialysis Fistulas

The incidence of ulcerated, bleeding, autologous, hemodialysis fistulas has been felt to be increasing in recent years. This review was undertaken to examine our experience with patients who presented with episodes of spontaneous bleeding in the presence of an ulcerated lesion over their autologous dialysis accesses. A database of hemodialysis access procedures was reviewed for individuals with spontaneous bleeding from ulcerations. Twenty-four patients were identified with 28 ulcerating lesions over a ten-year period from 2001 to 2011. Each had at least a single episode of spontaneous bleeding. Treatment methods were reviewed including five techniques of surgical intervention. Two of 24 patients expired from major hemorrhagic events before obtaining surgical consultation (8.3%). Twenty-one patients (87.5%) underwent 28 surgical procedures for correction of the ulcers. One patient (4.1%) with simultaneous ulcers healed under antibiotic therapy during close observation in hospital. Simple suturing of the ulcer was found to be inconsistent in effectively maintaining hemostasis. Surgical excision of the ulcer with primary closure, vein patching of the fistula, or end-end anastomosis were equally effective in definitively correcting the problem. The ulcerated autologous dialysis fistula is a life-threatening lesion and requires prompt surgical intervention to reduce mortality. The frequency of this problem appears to be increasing. Simple suturing of the ulcers was not consistently effective in remedying the problem and should be utilized as a temporizing measure until surgical correction can be undertaken. Fistulas can be uniformly salvaged with surgical intervention enabling uninterrupted dialysis treatments.

Prophylaxis In Congenital Factor VII Deficiency, Indications, Efficacy and Safety: Results of the STER

AbstractAbstract 665 IntroductionProphylaxis is considered a difficult endeavour in FVII deficiency, especially because of the very short FVII zymogen and FVIIa half-lives, mainly in childhood. The Seven Treatment Evaluation Registry (STER, www.targetseven.org) is a prospective observational, multi-centre, web-based registry concerned with the evaluation of treatments for spontaneous bleeding episodes, surgery and prophylaxis in patients with FVII deficiency. As regards prophylaxis, STER provides the frame for a structured and detailed data capture aiming at: a) identifying patients in whom prophylaxis is advisable, b) defining clinical settings in which prophylaxis is necessary and c) describing effective and safe dosing schedules. Materials and MethodsTogether with basic clinical and laboratory data related to the patient, the following information has been collected for each prophylaxis treatment course: i. Type (primary or secondary, short- and long-term), ii. Indication iii. Start/stop date iv. Schedule, v. Concomitant medications and illness vi. Bleeding episodes, vii. Other complications. The prophylactic regimen adopted reflected the decision of each enrolling physician as was the type of replacement therapy. FVIIc assays were performed at each centre lab, whereas inhibitor assays were centralized. ResultsNineteen patients with FVII deficiency (12 females, 7 males, age ranging from new born to 71 y.) were enrolled as a long-term prophylaxis was deemed necessary by the treating physician. FVIIc levels ranged from <1% to 4.9 of normal, median 1.5). Patients' disease severity was characterized by a striking prevalence of the severest symptoms (CNS and GI bleeding bleeding, hemarthrosis) as disease hallmark. Twelve subjects received recombinant FVIIa (rFVIIa), six received plasma-derived (pd) products (pdFVII concentrates [n=3], Fresh Frozen Plasma (FFP) [n=3]); 1 subject was given both pdFVII and an APCC. The 12 patients who were prophylaxed with rFVIIa (7 for a primary and 5 for a secondary course) were treated for the following reasons: recurrent hemarthrosis (n=4), CNS bleeding (3), menorrhagia (2), other (3). The 3 patients treated with pdFVII were reported for secondary prophylaxis courses after hemarthrosis (n=2) and hematuria, and the 3 patients treated with FFP were reported for courses after CNS bleeding (n=1), GI (1) and menorrhagia (1). Reported treatment schedules were quite various: the commonest frequency of rFVIIa administrations was “3 times a week” (n=5) ranging from daily (n=1) to once weekly (n=2); for menorrhagia, one dose on the 1st & 2nd days of the cycle were administered. The single dose of rFVIIa ranged from 14 to 80 μg/kg. Clinical efficacy of the rFVIIa prophylaxis regimens was excellent (n=10) with small intervals and medium or high dosing (14-80μg/kg). The regimens based on a dose of 30μg/kg once/week or on a dose of 16.5μg/kg 3 times/week, were considered partially effective. Prophylaxis with pdFVII was performed at a mean dose of 17 IU/kg (range: 9–30 U) 2 to 3 times /weekly: only the outcomes of the 3 times weekly regimen were reported as excellent. FFP was given at a mean dose of 15 ml/Kg twice a week but was only effective in preventing provoked bleeds. Long term prophylaxis courses carried out in 2 patients who had previously developed an inhibitor to FVII (both following multiple treatments) deserve a special mention: one received a long-term rFVIIa prophylaxis course with excellent results, while the other was given pdFVII and the APCC in association with satisfactory results. In none of the described prophylaxis courses spontaneous or catheter-related thromboses were reported. ConclusionsWe have identified a subset of patients with a FVII deficiency who may need a continuous, long-term prophylaxis treatment; these patients have a severe disease characterized by CNS or GI bleeding, hemarthrosis (mostly as presenting symptoms) or menorrhagia associated with low to very low FVIIc levels. Reported dosing and schedules varied widely so it is difficult to ascertain which is the optimal prophylaxis schedule but the 3 dose weekly regimens associated with medium to high replacement therapy doses appear the most efficacious. We have also shown that long-term prophylaxis is feasible in patients with an inhibitor to FVII. Disclosures:Knudsen:Novonordisk: Employment.

Peripheral transvenular delivery of adeno-associated viral vectors to skeletal muscle as a novel therapy for hemophilia B

AbstractMuscle represents an important tissue target for adeno-associated viral (AAV) vector-mediated gene transfer of the factor IX (FIX) gene in hemophilia B (HB) subjects with advanced liver disease. Previous studies of direct intramuscular administration of an AAV-FIX vector in humans showed limited efficacy. Here we adapted an intravascular delivery system of AAV vectors encoding the FIX transgene to skeletal muscle of HB dogs. The procedure, performed under transient immunosuppression (IS), resulted in widespread transduction of muscle and sustained, dose-dependent therapeutic levels of canine FIX transgene up to 10-fold higher than those obtained by intramuscular delivery. Correction of bleeding time correlated clinically with a dramatic reduction of spontaneous bleeding episodes. None of the dogs (n = 14) receiving the AAV vector under transient IS developed inhibitory antibodies to canine FIX; transient inhibitor was detected after vector delivery without IS. The use of AAV serotypes with high tropism for muscle and low susceptibility to anti-AAV2 antibodies allowed for efficient vector administration in naive dogs and in the presence of low- but not high-titer anti-AAV2 antibodies. Collectively, these results demonstrate the feasibility of this approach for treatment of HB and highlight the importance of IS to prevent immune responses to the FIX transgene product.

Increased plasma metastin levels in adolescent women with polycystic ovary syndrome

ObjectiveThis study was designed to:(1)measure metastin levels in women with polycystic ovary syndrome (PCOS) and in adolescent controls;(2)investigate the possible correlations between metastin and PCOS-related reproductive and metabolic disturbances. Study designThe study was a clinical study. Nineteen adolescent women with PCOS, twenty-three adult women with the syndrome, and twenty adolescent controls were selected. Blood samples were collected between day 1 and day 5 of a spontaneous bleeding episode in the PCOS groups and of a menstrual cycle of the controls at 9 a.m. after an overnight fast. Circulating levels of LH, FSH, prolactin, testosterone (T), free testosterone, DHEAS, sex hormone-binding globulin, insulin, glucose and metastin were measured. Result(s)Plasma metastin levels are increased in adolescent women with PCOS compared to adolescent controls. Plasma metastin levels were positively correlated with LH levels, 2-h glucose levels and T levels. Conclusion(s)These results indicate that metastin is increased in adolescent PCOS women. The increased metastin levels were positively correlated with LH and T levels, and may affect the development of PCOS in adolescents.

Clinical and molecular characterization of a re-established line of sheep exhibiting hemophilia A.

Large animal models that accurately mimic human hemophilia A (HA) are in great demand for developing and testing novel therapies to treat HA. To re-establish a line of sheep exhibiting a spontaneous bleeding disorder closely mimicking severe human HA, fully characterize their clinical presentation, and define the molecular basis for disease. Sequential reproductive manipulations were performed with cryopreserved semen from a deceased affected ram. The resultant animals were examined for hematologic parameters, clinical symptoms, and responsiveness to human FVIII (hFVIII). The full coding region of sheep FVIII mRNA was sequenced to identify the genetic lesion. The combined reproductive technologies yielded 36 carriers and 8 affected animals. The latter had almost non-existent levels of FVIII:C and extremely prolonged aPTT, with otherwise normal hematologic parameters. These animals exhibited bleeding from the umbilical cord, prolonged tail and nail cuticle bleeding time, and multiple episodes of severe spontaneous bleeding, including hemarthroses, muscle hematomas and hematuria, all of which responded to hFVIII. Inhibitors of hFVIII were detected in four treated animals, further establishing the preclinical value of this model. Sequencing identified a premature stop codon and frame-shift in exon 14, providing a molecular explanation for HA. Given the decades of experience using sheep to study both normal physiology and a wide array of diseases and the high homology between human and sheep FVIII, this new model will enable a better understanding of HA and facilitate the development and testing of novel treatments that can directly translate to HA patients.

Successful Long Term Therapeutic Expression of Factor VIII in Hemophilia A Dogs After Administration of AAV-cFVIII Using a Two-Chain or Single Chain Delivery Approach.

Abstract 546While adeno-associated virus (AAV) is a promising gene delivery vector, it has been challenging to deliver FVIII due to the large size of the FVIII cDNA and the high frequency of FVIII antibody formation in hemophilia A (HA) patients. We used two approaches to overcome the size limitation of AAV for FVIII: (1) two-chain delivery in which the canine FVIII (cFVIII) heavy chain (HC) is delivered in one AAV vector and the cFVIII light chain (LC) is delivered in a second AAV vector and (2) single chain delivery in which the B-domain deleted cFVIII cDNA with minimal regulatory elements is within one AAV vector. In the two-chain approach AAV-HC (4.0 Kb) and AAV-LC (3.9 Kb) with a liver specific promoter was co-injected at a dose of 6×1012 vector genomes/vector/kg or 1.25×1013vg/vector/kg using AAV8 or AAV9 via hepatic infusion. Five hemophilia A dogs treated with AAV-HC and AAV-LC expressed 0.5-11% cFVIII in a dose-dependent manner. The mean cFVIII activity based on Coatest assay for the low dose was 1.3% (>1220d)(Linus)(AAV8) and 0.6% (>1770d)(H19)(AAV9), while for the high dose it was 5.2% (800d)(F24)(AAV8) and 2.4% (>1270d)(Woodstock)(AAV9). One dog (J60) had a splenectomy due to a complication at the time of surgery and has maintained high levels of expression (mean 11.0%; >820d). The WBCT consistently remained at a mean of 17.6 min for low dose dogs and 13.7 min for high dose dogs compared to 8-12 min in normal dogs. Using novel reagents that we generated specific to cFVIII, we developed assays to detect cFVIII antigen levels and IgG antibodies. Despite receiving equal doses of each vector, at day 85 the cFVIII-LC antigen levels (71.7 ± 19.2 ng/ml) were >10-fold higher than would be predicted based on activity while the cFVIII-HC antigen levels (14.6 ± 9.2 ng/ml) were >3-fold higher than activity. Since functional FVIII synthesis relies on the co-transduction of AAV-HC and AAV-LC in the same cell, this suggests that only a portion of the vector co-transduces and expresses cFVIII in the same cell and that the light chain is secreted more efficiently than the HC. No IgG antibodies to cFVIII were detected at any time point in these dogs. Three dogs have maintained FVIII expression for >3.5 years and two dogs for >2 years with ongoing observation. No spontaneous bleeding episodes have been observed in these dogs for a cumulative observation of >16 years while >80 bleeding episodes would be expected during this time period. The second approach, the single chain delivery, overcomes the co-transduction requirement of the two-chain approach by ensuring that each transduced cell expresses functional FVIII. However, it is difficult to efficiently package the large 5.2 Kb single chain construct into an AAV vector. Since no significant differences were observed between AAV8 and AAV9 using the two-chain approach, we used AAV8 to deliver the single chain cFVIII by peripheral vein infusion at 2×1013vg/kg or 4×1013vg/kg. The mean cFVIII activity was 0.7% (>430d) for the low dose dog (L51) and 6.8% (>290d) and 2.2% (>110d) for the high dose dogs (M06, M50). cFVIII HC and LC ELISA showed that cFVIII antigen levels correlated with activity. WBCT was a mean of 19.1 min for L51, 15.3 min for M06 and 11.6 min for M50. No spontaneous bleeding episodes have been observed in these dogs. The high dose dogs had no IgG antibodies to FVIII. L51 had transient IgG antibodies to FVIII until d52 in the absence of a Bethesda titer. A rise in FVIII expression in L51 coincided with the disappearance of anti-cFVIII antibodies. Comparison of single chain and two-chain delivery of FVIII reveals that (1) long term therapeutic levels of cFVIII in a dose-dependent manner can be obtained with both delivery approaches; (2) circulating cFVIII antigen levels are >10-fold higher than activity in the two-chain delivery in contrast to single chain delivery in which antigen correlates with activity; and (3) high antigen levels may facilitate tolerance to FVIII in the setting of liver-directed gene transfer, since a transient non-inhibitory antibody was observed in only one dog with very low FVIII levels. Notably, no cellular toxicity due to continuous expression of various forms of FVIII was found in these animals based on long-term sustained FVIII expression levels and normal liver enzymes in all eight HA dogs. Further studies to characterize the immune responses to the transgene will define the optimal vector approach. These data will form the basis for clinical studies in humans with severe HA. Disclosures:No relevant conflicts of interest to declare.

Successful eradication of acquired factor-VIII-inhibitor using single low-dose rituximab

It was with great interest that we read the recent recommendations regarding the treatment of acquired hemophilia A published in Haematologica1 and followed the subsequent discussion.2,3 The described treatment algorithm for this disease, which occurs at an incidence of 1.5/million/year,4 stresses the importance of early eradication of the acquired fVIII-inhibitor. Using prednisolone alone or in combination with cyclophosphamide, this approach will be successful in 30–50% of affected individuals.5 As Huth-Kuhne pointed out, the CD20-antibody rituximab may be an attractive alternative in non-responders or patients with contraindications to steroids.6 However, concerns have been raised because of rituximab side-effects including associations with progressive multifocal leukencephalopathy (PML).7 Furthermore, as Mannucci et al.2 emphasized, the use of this drug is hampered by its substantial costs. Restricting the administered dose to the lowest effective level, seems to be a reasonable strategy to minimize potential risks and costs of rituximab treatment. We report a 66-year old male AHA-patient successfully treated with single low-dose rituximab. The patient presented with multiple hematomas and severe anemia after recovering from a respiratory infection. Prolonged activated partial-thromboplastin-time (aPTT) in combination with undetectable fVIII-activity due to an fVIII-inhibitor was observed. There was no clinical history of any prior hemorrhages nor a family history of bleeding. Clinical and imaging examinations could not reveal any pathological findings suggesting an idiopathic cause of AHA. Prednisolone (1 mg/kg) was started immediately and 2 units of packed red blood cells had to be administered. Afterwards, bleeding ceased, correlating with an increase in fVIII-activity to 28%. Steroids were completely tapered over the next weeks and the patient was lost to follow-up. However, during the following year the patient experienced recurrent episodes of minor bleeding. Seventeen months after the first onset of AHA, he again presented at our institution for follow-up. He reported no spontaneous bleeding episodes; however, fVIII-activity was 15% translating into an fVIII-inhibitor of 133 BU (Bethesda Units). Two months later, severe limb-bleeding requiring surgical treatment occurred after a minor accident. FVIII-inhibitor could still be detected at high levels (79 BU). Because of impaired wound-healing, steroids were deemed contraindicated. Therefore, given our experience with patients with auto-immune diseases other than AHA,8 inhibitor-eradication treatment was started with a single flat dose of 100 mg rituximab. Treatment was well tolerated and three weeks later complete B-cell depletion could be demonstrated (Figure 1). There was no reoccurrence of significant bleeding and even an accident was tolerated without complications. At last presentation, nearly ten months after rituximab treatment, the patient had only a low residual fVIII-inhibitor activity, in spite of a normal B-cell count. FVIII-levels stabilized at a level of 15 to 20% and aPTT was never again found to be prolonged after rituximab treatment. Figure 1. Clinical course overview. Time since diagnosis in months (x-axis). fVIII-inhibitor titer in Bethesda Units (BU) (left y-axis). B-cell proportion (%) (right y-axis). R: time of rituximab application. The clinical course of our patient demonstrates that complete B-cell depletion can be achieved with low-dose rituximab in AHA. We are aware that given the persistently low fVIII-level, our patient does not meet the strict criteria for inhibitor eradication proposed by Huth-Kuhne. On the other hand, we have to acknowledge that he experienced no further bleeding and had inhibitor-titers near the detection limit. We, therefore, believe that future clinical trials should incorporate this low-dose regimen rather than the classical lymphoma-dose (e.g. 375 mg/m2) in the treatment of acquired hemophilia A.

Plasma metastin in adolescent polycystic ovary syndrome.

This study was designed to investigate the correlationship between plasma metastin and pathogenesis of adolescent women with polycystic ovary syndrome (PCOS). From Jan. 2006 to Jun. 2006, 42 PCOS patients including 19 adolescent women and 23 adults with syndrome were treated in Second Affiliated Hospital of Sun Yat-Sen University. According to the range of age, those patients were divided into 19 cases in adolescent group (</= 19 years) and 23 cases in adult group (> 19 years). Meanwhile, 20 adolescent women were matched as controls. Blood samples were collected between day 1 and day 5 of a spontaneous bleeding episode in the PCOS groups and a menstrual cycle of the controls. The levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone (T), free T (FT), dehydroepiandrosterone sulfate (DHEAS), sex hormone-binding globulin (SHBG), insulin, glucose, and metastin were detected from day 1 to day 5 of spontaneous bleeding or withdrawal bleeding by progesterone. On the next day, oral glucose tolerance test (75 g) and insulin release test were performed on those above patients and controls. The area under curve (AUC), the ratio of fasting blood glucose to insulin (GIR) and homeostasis model assessment insulin resistance index (HOMA-IR) were calculated. (1) The level of hormone: the level of LH was in (12 +/- 7) U/L in adult group and (12 +/- 8) U/L in adolescent PCOS group, which were significantly higher than (6 +/- 4) U/L in controls (P < 0.05). The level of FT was (2.3 +/- 1.2) pmol/L in adult group, which was significantly higher than (1.3 +/- 0.8) pmol/L in adolescent group and (1.1 +/- 0.5) pmol/L in control group (P < 0.05). It was observed that the level of (3.1 +/- 2.7)micromol/L in adolescent group was significantly lower than (6.3 +/- 2.7) micromol/L in control group (P < 0.05). Meanwhile, the level of FAI of 5.6 +/- 4.1 in adult group was significantly higher than 3.0 +/- 1.3 in control group (P < 0.05). No significant difference in FSH, T and SHBG levels among three groups were observed (P > 0.05). (2) Metastin and metabolism: Both the levels of fasting blood insulin, 2-hour insulin and AUC of insulin were (13 +/- 7) mU/L, (88 +/- 59) mU/L and (133 +/- 80) mUxL(-1)xmin(-1) in adolescent group, which were significantly higher than (7 +/- 3) mU/L, (57 +/- 29) mU/L and (82 +/- 34) mUxL(-1)xmin(-1) in control group. The fasting blood insulin of (13 +/- 7) mU/L in adolescent group was significantly higher than (9 +/- 5) mU/L in adult group. The level of fasting blood glucose and 2-hour glucose were (5.01 +/- 0.44) mmol/L and (6.48 +/- 1.16) mmol/L in adult group, which were significantly higher than (4.68 +/- 0.29) mmol/L and (5.44 +/- 0.83) mmol/L in control group and (4.67 +/- 0.30) mmol/L and (5.93 +/- 1.44) mmol/L in adolescent group. The glucose AUC of (9.99 +/- 1.85) mmolxL(-1)xmin(-1) in adult group was significantly higher than (8.42 +/- 1.53) mmolxL(-1)xmin(-1) in control group (P < 0.05). HOMA-IR of 2.6 +/- 2.0 in adolescent group was significantly higher than 1.4 +/- 0.7 in control group. GIR of 10 +/- 8 in adolescent group was significantly lower than 16 +/- 10 in control group (P < 0.05). The metastin level of (0.25 +/- 0.19) pmol/L in adolescent group and (0.29 +/- 0.29) pmol/L in adult group were all significantly higher than (0.18 +/- 0.23) pmol/L in control group (PPh glucose were observed (r = 0.256, 0.286 and 0.267. P = 0.044, 0.025 and 0.043). The expression of metastin in adolescent PCOS women was significantly higher that of normal adolescent women. The increased level of metastin might be associated with pathogenesis of adolescent women with PCOS.

Directed Engineering of a High-expression Chimeric Transgene as a Strategy for Gene Therapy of Hemophilia A

Human coagulation factor VIII (fVIII) is inefficiently biosynthesized in vitro and has proven difficult to express at therapeutic levels using available clinical gene-transfer technologies. Recently, we showed that a porcine and certain hybrid human/porcine fVIII transgenes demonstrate up to 100-fold greater expression than human fVIII. In this study, we extend these results to describe the use of a humanized, high-expression, hybrid human/porcine fVIII transgene that is 89% identical to human fVIII and was delivered by lentiviral vectors (LVs) to hematopoietic stem cells for gene therapy of hemophilia A. Recombinant human immunodeficiency virus-based vectors encoding the fVIII chimera efficiently transduced human embryonic kidney (HEK)-293T cells. Cells transduced with hybrid human/porcine fVIII encoding vectors expressed fVIII at levels 6- to 100-fold greater than cells transduced with vectors encoding human fVIII. Transplantation of transduced hematopoietic stem and progenitor cells into hemophilia A mice resulted in long-term fVIII expression at therapeutic levels despite <5% genetically modified blood mononuclear cells. Furthermore, the simian immunodeficiency virus (SIV) -derived vector effectively transduced the human hematopoietic cell lines K562, EU1, U.937, and Jurkat as well as the nonhematopoietic cell lines, HEK-293T and HeLa. All cell lines expressed hybrid human/porcine fVIII, albeit at varying levels with the K562 cells expressing the highest level of the hematopoietic cell lines. From these studies, we conclude that humanized high-expression hybrid fVIII transgenes can be utilized in gene therapy applications for hemophilia A to significantly increase fVIII expression levels compared to what has been previously achieved.