H. H. Brackmann

Immune Tolerance for the Treatment of Factor VIII Inhibitors ‐ Twenty Years' ‘Bonn Protocol’

Around 20 years ago, a mother admitted her hemophilic son, 1 % years of age, knowing that he had an inhibitor, to our center by ambulance. He showed severe bleeding episodes into his right shoulder, right upper arm and right chest. The inhibitor titer at that time was :>500 Bethesda units (BU). Some months earlier, in 1974, we had heard about a publication by Kurczinsky and Penner [ I ] about the successful treatment of bleeding episodes in patients with an inhibitor with activated prothrombin complex concentrates. When calling a manufacturer (Immuno), asking for a similar product, we were told that this was still in development, and not available at the moment. In this serious situation, we decided to combine high dosages of factor VIII with a regular prothrombin concentrate. This regimen was given twice a day to the patient, and the bleeding could be controlled. After 3 weeks the patient recovered completely, and he could be dismissed from the hospital. Astonishingly, during this treatment the inhibitor titer decreased to slightly more than 40 BU. Therefore, it appeared to be reasonable to evaluate this procedure also in other hemophilic patients with an inhibitor suffering from acute bleeding episodes. We observed that in some patients there was a booster effect on the inhibitor, and in some not, but the inhibitor titer decreased in all cases after some weeks of treatment. We decided not to stop the treatment, but to continue this procedure, and realized that the inhibitor finally disappeared. Subsequently, also the half-life of factor VIIl normalized. However, it took around 2 years to develop the dosage schedule that we are using today. Methods

Haemophilia A: from mutation analysis to new therapies

Haemophilia is caused by hundreds of different mutations and manifests itself in clinical conditions of varying severity. Despite being inherited in monogenic form, the clinical features of haemophilia can be influenced by other genetic factors, thereby confounding the boundary between monogenic and multifactorial disease. Unlike sufferers of other genetic diseases, haemophiliacs can be treated successfully by intravenous substitution of coagulation factors. Haemophilia is also the most attractive model for developing gene-therapy protocols, as the normal life expectancy of haemophiliacs allows the side effects of gene therapy, as well as its efficiency, to be monitored over long periods.

Induction of immune tolerance in haemophilia A inhibitor patients by the 'Bonn Protocol': predictive parameter for therapy duration and outcome.

The treatment of inhibitors is one of the most challenging fields in haemophilia care. The present study reports the results of 60 haemophilia A inhibitor patients treated according to the ‘Bonn Protocol’ and evaluates predictors for the duration and outcome of therapy. Successful immune tolerance could be achieved in 52 patients (86.7%) while the therapy failed in eight patients (13.3%). The immune tolerance achieved was longlasting in all 52 patients, with no inhibitor relapse in up to 20–years follow–up. The course of ITT was influenced by several factors. Interruptions of treatment during the ITT course led to a substantial prolongation of ITT duration (median 39.9 months vs 14.1 months in continuously treated patients). Infections of intravenous central lines appeared to be frequently coincided with ITT prolongation and sometimes even ITT failure. Further negative predictors towards the ITT duration were high inhibitor titres at enrolment or during ITT. There was also a tendency towards longer ITT duration in patients exhibiting the prevalent intron 22 inversion. As a consequence of our data treatment interruptions and infections of intravenous central lines should be avoided during the course of ITT. Furthermore our data suggest, that ITT should be started at low inhibitor titres preferably with a high factor VIII dosage protocol.

Missense mutations at ALA-10 in the factor IX propeptide: an insignificant variant in normal life but a decisive cause of bleeding during oral anticoagulant therapy.

Bleeding complications are the most common and unwanted side‐effect of oral anticoagulant therapy. We report three patients in whom mutations in the factor IX (FIX) propeptide were found to cause severe bleeding during coumarin therapy. Strikingly, the bleeding occurred within the therapeutic ranges of the prothrombin time (PT) and international normalized ratio (INR). In all three patients coumarin therapy caused an unusually selective decrease of FIX activity (FIX:C) to levels below 1–3%. Upon withdrawal of coumarin, FIX:C increased to subnormal or normal values of 55%, 85% and 125%, respectively. Analysis of the FIX gene revealed two different missense mutations affecting the Ala‐10 residue in the propeptide coding region: Ala[GCC] to Val[GTC] in two patients and Ala[GCC] to Thr[ACC] in one patient. No further mutation was detected by screening 195 random blood donors for mutations at Ala‐10, thus excluding a frequent polymorphism at this position. The mutation in the FIX propeptide at a position which is essential for the carboxylase recognition site causes a reduced affinity of the carboxylase enzyme to the propeptide. This effect leads to an impaired carboxylase epoxidase reaction which is decisively triggered by the vitamin K concentration. Determination of FIX and APTT in addition to PT and INR is therefore recommended in coumarin‐treated patients with an uncommon bleeding pattern.

Pain status of patients with severe haemophilic arthropathy

Patients with severe haemophilia A growing up before the establishment of prophylactic treatment frequently developed significant haemarthropathies. The goal of the following study was to clarify the role of haemarthropathic pain for haemophilic patients. Furthermore, we aimed to determine to what degree daily activities are influenced by the impairment and which therapeutic modalities are used in pain management. Using a questionnaire we consulted 71 haemophiliacs concerning their complaints and how they were treated in 1999 (average age 43 years; range 21–63 years). The pain in the large joints and spine and the effect of specific treatment was estimated by a visual analogue scale. On average, there were four joints with major pain and 0.5 with minor pain. The most frequent sources of pain were the ankle joints (45%), followed by the knee (39%), spine (14%) and elbow (7%). Fifty percent of all patients complained of pain throughout the day if no treatment was applied. In 29% of patients, pain persisted after application of factor VIII (FVIII), while 12% claimed that pain still remained after use of FVIII and painkillers. Restriction in activities of daily life was reported by 89% of the group and 85% reported on an impact of pain on their mood. Patients primarily used FVIII to decrease pain, followed in frequency by use of anti‐inflammatory drugs, orthopaedic footwear, liniments and bandages. Haemophilic patients with haemarthropathy are chronic pain patients. By means of the questionnaire, it is possible to reveal the ‘silent’ sufferers. Sufficient pain treatment is essential so as to increase the patient’s quality of life and avoid inadvertent abnormal postures possibly resulting in increased loading of joints and subsequent bleeding episodes.

SMALL DELETION/INSERTION MUTATIONS WITHIN POLY-A RUNS OF THE FACTOR VIII GENE MITIGATE THE SEVERE HAEMOPHILIA A PHENOTYPE

Recently Young et al. [1] reported a haemophilia A family in whom a single base deletion within an A nm of exon 14 of the factor VIII (FVIII) gene did not lead to the severe phenotype that is typically expected from frame shift mutations. We wish to point out that mitigation of the severe haemophilia A phenotype in patients with small deletion/insertion mutations of exon 14 may not be rare.

Contamination of coagulation factor concentrates with human parvovirus B19 genotype 1 and 2

Human parvovirus B19 (B19) DNA has frequently been detected in plasma-derived coagulation factor concentrates. Furthermore, transmission of B19 infection was observed, indicating presence of the infectious virus despite routine viral inactivation/removal procedures during the manufacturing process. Recently, human parvovirus DNA isolates, variant from B19, have been identified resulting in classification of B19 virus into three distinct genotypes, with all viruses previously classified as B19 belonging to genotype 1. So far, there is no information available on contamination of clotting factor concentrates with genotype 2. Therefore, we analysed 202 different factor concentrate lots for genotype 1 and 2 DNA by PCR. Analysis of one hundred eighty-one lots representing 13 different products, administered over the last three years, was compared to 21 lots (8 products) used until the early 1980s which had not been treated by viral inactivation procedures. Genotype 1 DNA was detected in 77/181 (42.5%) currently administered lots, and 17/21 (81%) previously used lots. The level of genotype 1 DNA contamination was similar in currently and previously administered concentrates. Genotype 2 DNA was found in 5/202 (2.5%) lots, all of which were co-contaminated with genotype 1 DNA. DNA sequence analysis showed that the PCR-double positive concentrates contained typical genotype 1 and genotype 2 DNA. Because genotype 2 appears to cause a similar spectrum of diseases as genotype 1, simultaneous detection of genotype 2 by nucleic acid amplification testing (NAT), now widely applied to plasma pools for genotype 1, would give an added level of safety to blood products.

NovoSeven in immune tolerance therapy.

&NA; The development of inhibitors in haemophilia patients is one of the most serious challenges to effective treatment. The effect of factor (F) concentrate and treatment regimen on titre levels was studied in nine patients with haemophilia A or B and inhibitors. Patients with haemophilia A were subdivided into those treated with recombinant activated factor VII (rFVIIa) on demand or those treated prophylactically with activated prothrombin complex concentrate (aPCC) and rFVIIa. A third group comprised haemophilia B patients receiving rFVIIa prophylaxis and on‐demand aPCC or rFVIIa. On‐demand therapy with rFVIIa proved to be effective and safe treatment for acute bleeding episodes in haemophilic patients. In group 1, inhibitor titres decreased markedly 0.5‐21 months prior to immune tolerance therapy (ITT) and remained low for a further 1‐19 months. However, use of rFVIIa instead of aPCC as prophylactic treatment in group 2 patients undergoing ITT failed to show favourable results for rFVIIa. This may be due to the short half‐life of rFVIIa (2.3‐2.9 h). The data presented suggest that exclusive use of rFVIIa in acute bleeding episodes prior to commencing ITT is an effective method of decreasing inhibitor titre, thereby optimizing conditions for ITT.

Clinical and radiographic scores in haemophilic arthropathies: how well do these correlate to subjective pain status and daily activities?

Summary. Haemophilic patients who reached adulthood before the establishment of prophylactic treatment frequently show multiple and substantial arthropathies. The aim of this study was to determine to what extent haemophiliacs subjective impairment due to arthropathies correlates with objective clinical and radiographic parameters. By means of a questionnaire and a visual analogue scale, we consulted 79 haemophiliacs concerning their joint‐pain status, how these were treated and to what extent their daily activities had been affected. Using a scoring system suggested by the Advisory Committee of the World Federation of Haemophilia, clinical evaluation was performed. Radiographs of 60 patients were assessed by means of the Petterson scale. The results were statistically compared. We found a significant correlation between pain intensity and clinical pathology as well as between pain intensity and radiographic joint damage for both knees and for the right ankle. The number of painful joints correlated well with the number of clinically/radiographically affected joints. The more pronounced the objective damage to joints, the more frequently patients claimed to have constant pain, depressive episodes and a dependency on pain‐relieving medication. The more pronounced the objectively assessed damage to the knee and ankle joint, the higher the likelihood that the patient suffers from severe joint pain and reduction of activity. Treatment of painful symptoms from arthropathies is often insufficient. Scores and questionnaires may help to define the haemophiliacs pain status more clearly, thereby offering a possibility of assessment and long‐term observation.

European data of a clinical trial with a sucrose formulated recombinant factor VIII in previously treated haemophilia A patients.

Summary.  To increase the safety of antihaemophilic treatment, the production process of full‐length recombinant factor VIII (FVIII) KOGENATE® Bayer (Kogenate®FS)has been modified. Human albumin is no longer added as stabilizer during purification and in final formulation. Instead, the new KOGENATE® Bayer production process uses sucrose as a stabilizer in the formulation and adds solvent/detergent virus inactivation step. An European clinical trial was carried out in Germany and France in previously treated patients with severe haemophilia A who had more than 100 exposure days to exogenous FVIII. Pharmacokinetic data was analysed according to one‐stage and chromogenic assays. Efficacy and safety during home therapy and in surgical procedures were evaluated; inhibitor formation was carefully monitored. Safety and efficacy were evaluated in 33 European patients for 24 months. Patients received more than 13 million IU KOGENATE® Bayer. Over 75% of patients accrued more than 100 exposure days with the new product. Of 875 bleeding episodes, 90.7% were treated with 1 or 2 infusions and 75.8% of responses to treatment were rated as ‘excellent’ or ‘good’. Prophylactic treatment was the most common mode of therapy (60.7% of infusions). The product was well‐tolerated and FVIII recovery studies were consistent throughout the study period. Only 0.26% of adverse events were reported to be drug related. No evidence of de novo inhibitor formation was observed. Overall, KOGENATE® Bayer was efficacious, safe and well‐tolerated for the treatment of haemophilia A in multitransfused patients.