Non-Compartment and compartmental pharmacokinetics, efficacy, and safety of Kedrion FIX concentrate.

BACKGROUND: An open-label phase II, multicenter clinical trial was conducted at 11  Haemophilia Centres in  Italy, Romania, and Turkey, to evaluate the pharmacokinetics (PK), efficacy, and safety of high purity, plasma-derived, double virus inactivated and double nano-filtered factor IX (pd-FIX) concentrate (Kedrion FIX), EudraCT Number: 2005-006186-14. MATERIAL AND METHODS: 16 previously treated patients (PTPs) with severe or moderately severe haemophilia B were enrolled in the study. At enrolment, 14 underwent the first PK assessment (PK I), and the second PK (PK II) assessment was performed after six months of treatment (5 on-demand and nine prophylaxis) at the end of the study. PK parameters were evaluated by Non-Compartmental Analysis (NCA), One-Compartment model (OCM), and Two-Compartment Model (TCM). Efficacy of Kedrion FIX in all 16 patients was evaluated by the number of bleeding events, and clinical response following the infusions. Periodic FIX inhibitor assays and thrombogenicity tests were scheduled throughout the study to assess the safety of the drug. RESULTS: As compared to the published data on PK of pdFIX, Kedrion FIX displayed a longer half-life (22.37-55.73 hrs), reduced clearance, and regular volume of distribution at PK I by both NCA and OCM. The comparison of outcomes of PK II with those of PK I by OCM,  also showed significant changes, particularly in patients on prophylaxis, who showed some improved parameters of PK. Due to two outlier values at the end of the trial, the NCA parameters of PK I were not compared to those of PK II. Breakthrough bleeds were successfully treated with 1 or 2 infusions. No significant adverse events were observed during the study. DISCUSSION: During the six-month clinical study period, the use of Kedrion FIX resulted in a safe and effective pd-FIX concentrate with excellent PK characteristics.

Multiplex ligation-dependent probe amplification as first mutation screening for large deletions and duplications in haemophilia.

INTRODUCTION: Molecular characterization has shown a wide mutational spectrum underlying haemophilia A (HA) and haemophilia B (HB). Different molecular assays have allowed laboratories to perform genetic testing for F8 and F9 mutations. AIM: Recently, multiplex ligation-dependent probe amplification (MLPA), a simple technique for relative quantitation of targeted genomic regions, has been introduced in HA and HB for detection of large deletions and duplications. We want to verify if MLPA might be used at the beginning of the molecular investigation. METHODS: We used it to test 22 patients with suspected large deletions, nine patients negative for mutation detection by other methods and finally, 45 new patients as their first screening test. RESULTS: Carrier status was also established in 28 related females and gross rearrangements were also searched for by MLPA in 19 females with reduced FVIII or FIX levels. All suspected deletions were confirmed apart from two patients. In patients with a negative screening test, MLPA revealed one large duplication, while in two patients where MLPA was used as the first screening step, an exon duplication was detected. In females with reduced FVIII or FIX, no large deletions or duplications were found. CONCLUSIONS: Owing to its simplicity, MLPA seems useful at the beginning of the molecular investigation, saving all the following steps, where positive. Single exon deletion diagnosis requires caution due to the risk of misdiagnosis, but benefits of MLPA appear to overcome the pitfalls.

Correlation between FIX genotype and pharmacokinetics of Nonacog alpha according to a multicentre Italian study.

INTRODUCTION: Pharmacokinetic (PK) studies on recombinant FIX concentrate, Nonacog alpha, were conducted with different sampling time designs which gave rise to not complete and homogenous outcomes. In addition, patient's FIX genotype/PK relationship has never been investigated. AIM: Investigate how different sampling times may affect PK parameters and try to find a FIX genotype/PK relationship. PATIENTS AND METHODS: A cohort pharmacokinetic, Nonacog Alpha single-dose, open-label, non-comparative study was conducted in eight Comprehensive Care Haemophilia Centres in Italy. Seventeen previously treated moderate or severe haemophilia B patients were enrolled. Factors IX:C one-stage clotting assay, FIX genotype and PK analysis were centralized. RESULTS: The evaluation of PK outcomes showed a quite long half-life, smaller clearance and volume of distribution of Nonacog Alpha in comparison with the results from previously reported studies, where blood sampling was stopped too early. The relationship between PK outcomes and FIX genotype showed that small deletions displayed the higher clearance and shorter half-life, the nonsense mutations (the lower and the longer respectively), and missense mutations were in between. CONCLUSIONS: It is evident that area under the curve (AUC) and other PK parameters depend from the sampling time design. In order to have a complete evaluation of clotting factors in vivo decay, blood samples must be collected until the baseline factor concentration has been achieved again. Due to the relationship between FIX genotype and clearance, tailored prophylaxis of HB patients could be partially predicted by genotyping.

Mortality and causes of death in Italian persons with haemophilia, 1990-2007.

Although a number of studies have analysed so far the causes of death and the life expectancy in haemophilic populations, no investigations have been conducted among Italian haemophilia centres. Thus, the aim of this study was to investigate mortality, causes of deaths, life expectancy and co-morbidities in Italian persons with haemophilia (PWH). Data pertaining to a total of 443 PWH who died between 1980 and 2007 were retrospectively collected in the 30 centres who are members of the Italian Association of Haemophilia Centres that chose to participate. The mortality rate ratio standardized to the male Italian population (SMR) was reduced during the periods 1990-1999 and 2000-2007 such that during the latter, death rate overlapped that of the general population (SMR 1990-1999: 1.98 95% CI 1.54-2.51; SMR 2000-2007: 1.08 95% CI 0.83-1.40). Similarly, life expectancy in the whole haemophilic population increased in the same period (71.2 years in 2000-2007 vs. 64.0 in 1990-1999), approaching that of the general male population. While human immunodeficiency virus infection was the main cause of death (45%), 13% of deaths were caused by hepatitis C-associated complications. The results of this retrospective study show that in Italian PWH improvements in the quality of treatment and global medical care provided by specialized haemophilia centres resulted in a significantly increased life expectancy.

Effects of secondary prophylaxis started in adolescent and adult haemophiliacs.

While primary prophylaxis is a well-established and recommended method of care delivery for children with severe haemophilia, fewer studies have documented the benefits of secondary prophylaxis started in adolescence or adulthood. To evaluate the role of secondary prophylaxis started in adolescent and adult severe haemophiliacs, a retrospective observational cohort study was conducted in 10 Italian Centres that investigated 84 haemophiliacs who had bled frequently and had thus switched from on-demand to prophylactic treatment during adolescence (n = 30) or adulthood (n = 54). The consumption of clotting factor concentrates, the orthopaedic and radiological scores, quality of life and disease-related morbidity were compared before and after starting secondary prophylaxis. Prophylaxis reduced the mean annual number of total and joint bleeds (35.8 vs. 4.2 and 32.4 vs. 3.3; P < 0.01) and of days lost from work/school (34.6 vs. 3.0, P < 0.01). A statistically significant reduction in the orthopaedic score was observed during prophylaxis in adolescents, but not in the whole cohort. Patients used more factor concentrates with corresponding higher costs on prophylaxis, but experienced a better quality of life. With respect to on-demand treatment, higher factor consumption and cost of secondary prophylaxis were balanced by marked clinical benefits and greater well-being in this cohort of adolescent/adult haemophiliacs.

F8 gene mutation profile and ITT response in a cohort of Italian haemophilia A patients with inhibitors.

Anti factor VIII (FVIII) antibodies represent the main complication of replacement therapy in severe cases of haemophilia and most patients with inhibitor have gross gene rearrangements or point mutations that hamper the production of normal circulating FVIII. In this study we have investigated 82 haemophilia A patients with inhibitors. Seventy six were severe, three were moderate and three were mild. We screened the patients for the causative mutations using long range PCR for the recurrent intron 22 inversion (invint22), multiplex PCR for intron 1 inversion (invint1) and conformation sensitive gel electrophoresis followed by DNA sequencing for all other mutation types in the F8 gene. Diverse genetic defects were detected in the severe cases (with a predominance of severe molecular defects): F8 gene inversions, large deletions and non-sense mutations account for 71% of the mutations. Only missense and splicing mutations were identified in the non-severe patients and we confirmed that the presence of inhibitors correlates well with the presence of severe mutations, but a proportion of severe patients develops inhibitors despite the presence of diverse less severe mutations. When we have analysed the subgroup of patients who underwent immunetolerance, we have found that F8 gene large deletions are likely to be a high risk factor also for immunetolerance therapy unresponsiveness, while no clear evidence has been demonstrated for other mutation types.

Hepatitis C virus quasispecies in the natural course of HCV-related disease in patients with haemophilia.

Patients with haemophilia show high prevalence of hepatitis C infection but low rate of progressive liver disease when they are not co-infected with HIV. The balance between host immune system and hepatitis C virus (HCV) variability seems to play a major role in the evolution of the HCV-related disease. To address this point we have studied, in a group of selected patients with haemophilia, the composition and in some cases the evolution, of the highest variable envelope gene within the hypervariable region 1 (HVR1) of the HCV, which is the region more directly exposed to the host immune response. Five of 12 patients show a very high homogeneity of the HVR1 and four of those had severe progressive liver disease. These results seem to confirm the major role of the immunity in driving the variability of the HCV rather than the high degree of different HCV strains to which haemophiliacs have been in touch with, during their long-term replacement therapy. Our results seem in keeping with other studies on different type of patients, where a low degree of quasispecies variability has been demonstrated in relationship with the progression and the severity of their liver disease.

Intra-articular rifamycin in haemophilic arthropathy.

Synoviorthesis is the intra-articular injection of chemical or radioactive substances able to produce fibrosis of hypertrophied synovium, which has proved effective in the treatment of chronic haemophilic synovitis. Between September 1999 and October 2001 we treated 28 outpatients (25 with haemophilia A, three with haemophilia B). Our treatment was focused on pain and functional limitation of joints. A schedule was adopted to treat each joint using intra-articular rifamycin once a week, repeated five times. Patients were covered with factor replacement on demand. Oral analgesia was offered as required because of acute but transient painful inflammatory reaction. Their median age was 34 years (range 15-60 years). The indication for synoviorthesis was chronic synovitis characterized by recurrent haemarthroses, persistent pain and limited range of motion (ROM). Thirty-five joints were treated with a total of 169 injections, including six joints (20%) in patients with inhibitors. In five patients two joints were treated in the same session. Thirty procedures were completed: 24 (80%) were considered effective (as excellent or good), while six were considered insufficient (20%). Pain was reduced in 96% of cases and in 70% the ROM was improved. In our experience intra-articular infiltration with rifamycin appears to be effective in reducing joint pain and in improving the ROM. The procedure presents a low risk of bleeding, can be used for patients with inhibitors and multiple joints can be treated without any additional cost.

Concurrent total hip and knee replacements in a patient with haemophilia with inhibitors using recombinant factor VIIa by continuous infusion.

The introduction of activated recombinant factor VIIa (rFVIIa) has allowed elective surgery to be safely performed in haemophiliacs with inhibitors. The main problems associated with its use are the short half-life, necessitating frequent intravenous injections, and its very high cost. Here we describe, for the first time, the performance of total hip and knee replacements in a haemophiliac with inhibitors at the same operation. The amount of rFVIIa concentrate used (8.57 mg) was similar to that normally used for a single joint replacement. The use of continuous infusion allowed for easier administration and further contributed to the reduction in cost as it avoids the peak levels associated with bolus injections.

Large FVIII gene deletion confers very high risk of inhibitor development in three related severe haemophiliacs.

Haemophilia A displays a broad heterogeneity of genetic defects and of clinical severity. Inhibitor development is the main complication of replacement therapy in severe cases and most patients with inhibitors have gross gene rearrangement or point mutations, which hamper the production of normal circulating factor VIII (FVIII). We have investigated three related severe haemophilia A patients, all of whom have high titre inhibitors. By using long-range polymerase chain reaction (PCR) for FVIII gene inversion, we observed an unusual pattern in these patients. We therefore decided to screen the whole FVIII gene by conformation-sensitive gel electrophoresis. A large FVIII gene deletion spanning exon 2 to exon 25 was identified and we were able to obtain a 18.5 kb PCR product, which is specific for this mutation and useful for determining the carrier state in this family. All three haemophiliacs carrying this very large gene deletion show similar clinical history and very high-titre inhibitors, supporting the observation that inhibitor development seems to be an inherited characteristic. On the basis of our observations we think that this subgroup of patients at very high risk of inhibitor development should be identified by mutation analysis whenever possible, before the beginning of replacement therapy.

Persistence of parvovirus B19 DNA in synovium of patients with haemophilic arthritis.

A progressive arthropathy develops commonly in haemophiliacs and its pathogenesis is not fully understood. Human parvovirus B19 has been associated with several diseases including acute and chronic arthropathy and some studies suggest its implication in chronic inflammatory diseases of the joints such as rheumatoid arthritis. In haemophiliacs parvovirus B19 infection occurs very frequently because of its transmission with plasma derivatives. In order to assess a role of B19 virus in haemophilic arthritis, synovial tissue samples from patients with haemophilia with arthritis and from patients, nonhaemophiliacs, with arthrosis or with joint trauma were examined for B19 DNA by nested PCR. In addition, the prevalence of antibody to parvovirus B19 NS1 protein as a possible serological marker of persistent B19 infection was tested and the association of the outcome of parvovirus infection with genetic diversity of B19 P6 promoter sequences was investigated. B19 DNA was detected in the synovial tissue of 31% of haemophiliacs with progressive arthropathy and of 5% of control patients. Fourteen out of 17 patients (82%) with haemophilic arthritis and with B19 DNA in their synovial membranes had IgG antibodies against the nonstructural protein NS1 of parvovirus B19. On the other hand, 19% of patients with haemophilia with B19 PCR negative synovial tissue and 21% of controls showed anti-NS1 antibodies. The P6 promoter presented specific sites of point mutations shared frequently by isolates from patients with haemophilia and arthritis. These results indicate that B19 DNA can persist in the synovial membranes of patients with haemophilic arthritis significantly more frequently in comparison to control individuals with arthrosis or joint trauma and show a correlation between anti- NS1 antibody presence and B19 DNA persistence in the synovial tissue.

Recombinant FVIIa (NovoSeven) continuous infusion and total hip replacement in patients with haemophilia and high titre of inhibitors to FVIII: experience of two cases.

In this report we describe our experience of total hip replacement in two patients with severe haemophilia A and high titres of inhibitors to FVIII. We used rFVIIa replacement therapy by continuous infusion to perform the surgery. The total amount of rFVIIa used in these two patients was very similar but the manner of administration was quite different. In our experience, it is an advantage to use a higher dose for shorter periods than a lower dose for a longer treatment period. Tranexamic acid by continuous infusion, and parallel saline infusion were useful for good haemostasis and avoided local thrombophlebitis in the side of rFVIIa infusion.

Detection of B-cell monoclonality in fine needle aspiration by PCR analysis.

Cytologic examination of fine-needle aspiration (FNA) sometimes fails to diagnose the malignant nature of B-cell proliferations. In this study we analyzed the Ig gene rearrangement of 49 FNA samples by polymerase chain reaction (PCR) in order to evaluate whether molecular analyses can improve the accuracy of FNA. Twenty-six patients had non-Hodgkin's lymphoma, 11 had reactive lymphoid diseases, 5 had chronic inflammation and 7 had carcinoma. A semi-nested PCR was performed using an oligoprimer specific for consensus sequences of the V regions (FR3A) and two oligoprimers derived from conserved sequences of the J regions (LJH and VLJH). Histologic examination always followed the molecular and cytologic analysis. The sensitivity of PCR and FNA morphological examination in detecting a neoplastic pattern was 92% and 78%, respectively. When samples were considered inadequate for cytologic examination, PCR always reached a diagnosis consistent with the histologic features. Our results demonstrate that PCR analysis of FNA specimens is a reliable and sensitive method capable of enhancing the diagnostic accuracy of cytologic examination.

Myasthenic syndrome and oligoclonal lymphocytosis: evolution into chronic lymphocytic leukemia.

We report a patient with myasthenic syndrome who, 2 years after diagnosis, developed an oligoclonal lymphocytosis. This disorder was sustained by both kappa+ and lambda+ CD5+ B-cell clones; over the following year, the white blood cell count increased and phenotypic characterization revealed a clear imbalance in the immunoglobulin light chain ratio (84% kappa+). Accordingly, persistence of a kappa+ B-cell clone was disclosed by molecular analysis of immunoglobulin heavy chain gene rearrangements. Our results may suggest that prolonged immune system stimulation due to an autoimmune disease can drive a benign lymphoproliferation into a B-cell neoplastic process.