Albertini A, Lenfant CL, Mannucci PM, Sixma JJ (eds): Biotechnology of Plasma Proteins. Curr Stud Hematol Blood Transf. Basel, Karger, 1991, No 58, pp 63-68

Experiences with Recombinant Factor VIIa in Haemophiliacs Ulla Hedner

Treatment of haemophiliacs with antibodies against F VIII/F IX is a matter of major concern in haemophilia care. Although a number of sophisticated methods for removal of antibodies have been designed, such procedures are suitable only in life-threatening situations since they are complicated and expensive [ 1]. Much effort has been focused on finding an agent capable of activating the final common pathway of the coagulation cascade inducing haemostasis independent of the presence of F VIII: C/F IX : C. Such an agent should be haemostatically active but also safe with regard to thromboembolic complications. Currently used prothrombin complex concentrates (PCC) or activated PCC (APCC) seem only to be active in about 50-60% of the bleeds [2]. Furthermore, thromboembolic complications are being reported following the use of PCC or APCC [3]. F VIIa should be an attractive candidate since it is not proteolytically active until after having formed a complex with tissue factor or phospholipids thereby minimizing the risk of inducing a systemic activation of the coagulation system. Plasma-derived F Vlla (pF Vila) was found to be haemostatically active in a few haemophilia A patients treated in association with muscle or joint bleeds as well as extraction of primary molars [4, 5]. The first patients treated got doses of pF VIla of 2-5 kg/kg resulting in plasma levels of F VIIa of 1.4-2.0 U/ml, a slight if any shortening of the APTT and a shortening of the prothrombin time of at most 2 s [4]. Later it was shown that higher doses (up to 10-15 µg/kg) of pF VIIa given at shorter intervals also were effective in major joint bleeds [5]. Following these doses the plasma levels of F VII rose to 6-7 U/ml. In parallel, a transient shortening of the APTT was seen as well as a prothrombin time shortening. As encouraging as these results were it seemed obvious that a large-scale production of a pure plasma-derived F VIIa would meet with substantial difficulties.

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Recombinant F Vila was cloned and expressed in BHE cells [6]. Recombinant F VIIa (rF VIIa) produced on a large scale in mammalian cells was purified and characterized with regard to amino acid backbone structure, carbohydrate and y-carboxylatíon [7]. Α spontaneous conversion of F VII into F VIIa occurred during the purification procedure [8]. The amino acid sequence was identical with that found in pF VIIa and the carbohydrate structure was very similar. Of the 10 y-carboxylated glutamic acid residues of pF VIIa, 9 were fully and 1 partially y-carboxylated in the rF VIIa. The rF VIIa normalized the prolonged APTT of haemophilic plasma, both Α and Β, in vitro, provided it was added in amounts of 3.8 µg/ml plasma roughly corresponding to a dose of 150 jig/kg [9]. The cuticle bleeding time was normalized in haemophilia Α as well as in haemophilia Β dogs [ 10]. The safety of F VIIa was addressed in a number of ways. The plasma-derived F VIIa was given to normal dogs and any coagulation changes previously described to occur on the administration of PCC/APCC [11] were followed. No signs of a generalized consumption coagulopathy were observed [4]. The safety, especially with regard to the induction of a generalized coagulopathy, was also checked in rabbits whose tissue factor is known to form proteolytically active complexes with human F Vlla. In doses of rF VIIa up to 5,000 U/kg (roughly corresponding to a dose of 150 µg/kg) no coagulation changes indicating a generalized proteolytic activity in the circulating blood were observed [9]. Any enhancing activity by F VIIa on the development of a systemic coagulopathy induced by endotoxin was checked in a rabbit model. The rabbits got two different doses of endotoxin (0.03 or 0.30 jig/kg/h). Each group of animals was divided into two subgroups obtaining rF VIIa in a dose of either 100 or 300 jig/kg in addition to the endotoxin infusion. A negative control group received saline and two positive controls groups receiving only endotoxin were included. No enhancing effect of rF VIla in any of the doses used was found as measured with the leucocyte and platelet counts, the fibrinogen level and the ethanol gelation test as a measure of soluble fibrin and thereby free thrombin activity [Diness et al., to be pub1.]. Clinical experience with rF VIIa: In total 22 patients have been given rF VIIa (Novostase, NovoSeven) on a compassionate use basis or corresponding authorization from health authorities in other countries than the USA (table 1). Of these, 13 have been treated in association with lifethreatening bleeds with no alternative treatment after the administration of PCC/APCC had been proved not to be efficient. One patient had haemophilia Β and inhibitors against F IX : C, one had acquired antibodies against F VIII: C and the rest (11 patients) had haemophilia A with

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Table 1. Patients given rF VIIa (Novostase, NovoSeven)

inhibitors. Two patients died, one having a retroperitoneal bleed complicated with a renal insufficiency. In this latter patient haemostasis was achieved and the effect of rF VIIa treatment was described as astoundingly good. However, insufficient amounts of rF VIIa were available at the time so the treatment could not be completed. The other patient, who died, was the elderly man (76 years old) with an acquired inhibitor against F VIII. He developed a bleed in the tongue extending into a nasopharyngeal bleed threatening to suffocate him. He was subjected to an emergency tracheotomy from which he was then bleeding profusely. His bleed was controlled by rF VIIa given in doses of 75-90 jig/kg every 3rd to 4th hour. Unfortunately, he developed a profuse gastrointestinal bleeding that killed him. The steroid therapy he had been on for some time was considered as having contributed to the development of a haemorrhagic gastritis and the fatal bleeding. One patient with haemophilia A and inhibitors was a boy with a profuse epistaxis and a destroyed nasal septum as a result of repeated episodes of severe epistaxis in the past. Novostase (NovoSeven) in a dose of 52-75 sg/kg every 3-4 h for 7 days controlled the bleeding initially but the patient started to rebleed. In order to control this heavy bleeding FEIBA and F IX concentrates had to be added. No side effects of the 11 days of rF VIIa treatment were observed [ 12]. One of the very first patients given rF VIIa was treated in association with a massive laryngeal bleed threatening to suffocate the patient. It was successfully reversed by rF VIIa and the results were published [ 13]. Among the following all through successful treatments there was one boy with a cerebellar bleed, one with a posttraumatic spinal bleed with neurological symptoms, and one with a severe mesenteric-retroperitoneal bleed. In all these patients the rF VIIa controlled the bleeding efficiently. The one haemophilia B patient treated was 11 years old and given rF VIIa because of a profuse bleeding with hypotension and dropping haemoglobin following surgical fasciotomy because of a compartment syndrome in his left forearm. He had in the past developed serious anaphylatic reactions on the administration of F IX

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9 patients subjected to surgery 13 patients on compassionate use 8 hemophilia Α + inhibitors 11 hemophilia Α + inhibitors 1 F XI deficiency + inhibitors against F XI 1 hemophilia B + inhibitors 1 acquired inhibitors against F VIII :C

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concentrates. Novostase (NovoSeven) was given in doses of 60-90 µg/kg every 3rd hour for 3 weeks, whereafter the dose intervals were increased to 4 h. Antifibrinolytic treatment was instituted 4 days after the start of the treatment and given intravenously (0.01 g/kg) every 6 h. The bleeding was controlled during the first week of rF VIIa treatment. Three weeks after the start of treatment a skin grafting was performed under the cover of rF Vlla and tranexamic acid. The estimated blood loss during the operation was 200 ml. During the treatment he had a resistant Staphylococcus aureus growing from wounds as well as Escherichia coli from the central line. No complications occurred. At most this patient received 0.7 mg/kg/24 h of rF VIla without any side effects. He has later been treated with rF Vila for joint bleeds on a few occasions also with good effect. In total, 9 patients have been subjected to surgery under the cover of rF Vlla as the sole coagulation factor. The very first patient given rF VIla was subjected to synovectomy [14]. He was given a dose of 54 µ g/kg every 4 h for the first 2 days and then at every 6 h for 10 days. No abnormal per- or postoperative bleeding occurred. Three extensive tooth extractions (dental surgery) were performed and 1 patient, 18 months old, was treated in association with a posttraumatic mouth bleed that was not controlled by repeated F VIII infusions and tranexamic acid. He had a loose front tooth extracted, was bleeding from several venopunctures including one in the jugular vein and had a haemoglobin of 49 g/1 when an inhibitor against F VIII : C was diagnosed. He was started on Novostase (NovoSeven) at a dose of 60 g/kg that was increased to 90 µg/kg every 3 h for 12 h after which the dose intervals were prolonged and the dose reduced. His bleeding was immediately controlled. One patient was subjected to an extensive herniotomy on a dose of rF Vlla of 75 µg/kg. No peroperative bleeding occurred. His second dose (75 jig/kg) was given 2 h later and he was then intended to be on the same dose every 4 h. However, he started oozing from the wound right before the next dose so the interval between doses was shortened to 3 h. At 26 h after surgery overt bleeding from the wound started, stopped at the administration of extra rF VIIa but relapsed on day 3 (45 h postoperatively). In spite of tranexamic acid added at this point and extra rF Vlla given by continuous infusion (38 jig/h) the bleeding was not fully contrilled and porcine F VIII had to be given. Another of the patients was 21 years old and had a spontaneously ruptured absess of the left thigh that made drainage of the area imperative. He was subjected to explorative surgery under the cover of rF VIIa (90 /4g/kg) and extensive myonecrotic tissue was removed. He was infected by salmonella. No bleeding occurred initially but towards the latter part of surgery an episode of

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profuse bleeding developed. Low levels of fibrinogen (0.8 g/1), az-antiρlasmin (46%) as well as AT III (24%) and high levels of D dimers were found. The bleeding slowed down on addition of cryoprecipitate, freshfrozen plasma, and red cells. He was kept on an unchanged dose of rF VIIa (70 µg/kg) every 3rd h that was increased to 90 µg/kg. The same series of events was repeated 2 days later with further surgical resection of necrotic tissue. Also this time the bleeding episode slowed down on the same therapy. The patient was continuously kept on rF VIIa in a dose of 90 µg/kg every 3rd hour. Unfortunately, he later started to bleed excessively from the surgical wound and a surgical bleeding was suspected. He became hypovolemic, developed bradycardia and finally died. The interpretation of the treatment by the physician in charge was that the rF VIIa successfully allowed the surgery to be performed and to maintain haemostasis. The data did not suggest that the rF VIIa caused consumption in this patient with a substantial bacteraemia during the surgical manipulation in the area of extensive tissue necrosis. Both these latter circumstances may have contributed to the problem. Furthermore, a patient, 66 years old, having a F XI deficiency with substantial bleeding problems in the past and an inhibitor against F XI was subjected to orchiectomy because of a massive bleeding from his left testis, under the cover of F VIla (60 jig/kg) initially and then every 3rd hour. No bleeding occurred. At 16 h after surgery the patient developed substernal chest pain similar to what he had experienced once before several years ago. No ischaemic changes were found in ECG and the pain was relieved on nitroglycerine. Monitoring: Peak plasma F VII levels of 30-40 U/ml seem to be adequate for haemostasis in major bleeds including major surgery. Through levels of ? 10 U/ml seem to be advisable in major bleeds. The prothrombin time shortens and seems to be around 9-10 s during a haemostatically effective treatment. Also the APTT shortens. It is, however, hard to decide on any preferred levels at present, expecially since the results obviously vary with regard to reagents used. Further studies are being carried out on this. Summarizing: The rF VIIa has been found to be haemostatically active also in extremely complicated excessively bleeding haemophilia patients with inhibitors against F VIII : C provided it is given in doses of 70-90 µg/kg at short intervals (3-4 h) at least initially. There is some evidence that the addition of antifibrinolytic therapy in adequate doses may allow a lower dose of rF VIIa for sufficient haemostasis. No side effects have been observed. All patients are being followed with regard to any development of antibodies against F VII. So far, no signs of such antibodies have been seen.

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References 1

Prof. Ulla Hedner, MD, Biopharmaceuticals Division Research, Novo Nordisk, DK-2880 Bagsværd (Denmark)

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Nilsson IM, Berntorp E, Zettervall O: Induction of immune tolerance in patients with hemophilia and antibodies to factor VIII by combined treatment with intravenous IgG, cyclophosphamide, and factor VIII. N Engl J Med 1988;318:947-950. 3 Sjamsoedin LIN, Heijnen L, Mauser- Bunschoten EP, van Geijlswijk JL, van Houwelingen Η, van Asten P, Sixma JJ: The effect of activated prothrombin-complex concentrate (FEIBA) on joint and muscle bleeding in patients with hemophilia A and antibodies to factor VIII. N Engl J Med 1981;305:717-758. 3 Sullivan DW, Purdy LJ, Billingham M, Glader BE: Fatal myocardial infarction following therapy with prothrombin complex concentrates, in a young man with hemophilia A. Pediatrics 1984;74:279-281. 4 Hedner U, Kisiel W: The use of human factor VIIa in the treatment of two hemophilia patients with high-titer inhibitors. J Clin Invest 1983;71:1836-1841. 5 Hedner U, Bjoern S, Bernvil SS, Tengborn L, Stigendahl L: Clinical experience with human plasma-derived factor VIIa in patients with hemophilia A and high-titer inhibitors. Haemostasis 1989;19:335-343. 6 Hagen FS, Gray CL, O'Hara P, Grand FJ, Saari GC, Woolbury, RG, Hart CE, Inselv M, Kisiel W, Kurschi K, Davie EW: Characterization of a cDNA coding for human factor VII. Proc Natl Acad Sci USA 1986;83:2412-2416. 7 Thim L, Bjoem S, Christensen M, Nicolaisen EM, Lund-Hansen T, Pedersen ΑΗ, Hedner U: Amino acid sequence and posttranslational modifications of human factor VIIa from plasma and transfected baby hamster kidney cells. Biochemistry 1988;27:7785-7793. 8 Bjoem S, Thim L: Activation of coagulation factor VII to VIIa. Res Disc' 1986;269:564-565. 9 Hedner U, Ljungberg J, Lund-Hansen T: Comparison of the effect of plasma-derved and recombinant human F VIIa in vitro and in a rabbit model. Blood coagulation and fib nolysis. Accepted, 1990. 10 Brinkhous KM, Hedner U, Garns JB, Diness V, Read MS: Effect of recombinant factor VIIa on the hemostatic defect in dogs with hemophilia, hemophilia B, and von Wíllebrand disease. Proc Natl Acad Sci USA 1989;86:1382-1386. 11 Hedner U, Nilsson IM, Bergentz SE: Various prothrombin complex concentrates and their effect on coagulation and fib nοlysis in vivo. Thromb Haemost 1976;35:386-395. 12 GruPp SA, Glazer S, Williams DA: Recalcitrant epistaxis in a hemophiliac with inhibitors: Experience with recombinant factor VII$. Blood 1989;74(suppl 1):390a(abstr 1489). 13 Macik BG, Hohneker J, Roberts HR, Griffin AM: The use of recombinant activated factor VII for treatment of a retropharyngeal hemorrhage in a hemophilic patient with a high titer inhibitor. Am J Med 1989;32:232-234. 14 Hedner U, Glazer S, Pingel S, Alberts KA, Blombäck M, Schulman S, Johnsson H: Successful use of recombinant factor VIIa in patients with severe haemophilia A during synovectomy. Lancet l988;ii: 1193.

Experiences with recombinant factor VIIa in haemophiliacs.

Albertini A, Lenfant CL, Mannucci PM, Sixma JJ (eds): Biotechnology of Plasma Proteins. Curr Stud Hematol Blood Transf. Basel, Karger, 1991, No 58, pp...
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