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Publications and source records attributed to M Morfini.
In this article, we provide a summary of the generally accepted approaches to the design and analysis of studies examining the pharmacokinetic (PK) profile of an infused coagulation factor in patients with a deficiency of one or more of these factors. Furthermore, we briefly review the known PK results for various commercially available coagulation factor preparations under single and continuous infusion.
We report the case of a patient with mild haemophilia A, due to a Tyr2105Cys mutation in exon 22 of the C1 domain, who developed a high-titre factor VIII inhibitor (maximum titre 1600 BU) with recurrent severe haemorrhages and fatal intracranial bleeding. Based on published data, it appears that although this mutation occurs rarely in patients with mild or moderate haemophilia A, it is frequently associated with the development of high-titre inhibitors.
BACKGROUND: the basic prerequisite of Factor VIII clotting assay (FVIII:C) by One-Stage Method is that all other than FVIII clotting factors are present in constant concentration in each dilution of both standard reference and patient's plasma curves. On the contrary, the plasma content of each dilution is decreasing as the dilution factor increases. OBJECTIVES AND METHODS: to keep exactly constant the plasma content in each mixture, we performed all dilutions of both standard reference and patient's plasma with FVIII deficient plasma and further with a fixed amount of buffer (method B). To show the discrepancies between this method and regular method A, using buffer to make dilutions, a comparative study was conducted on FVIII: C assay on samples at known FVIII concentration and in patients' plasma. Imidazole or Owren's buffers and five different aPTT reagents were employed, both in method A and B. RESULTS: a discrepancy between FVIII: C assays obtained by method A and B was observed, mainly when Pathrontin SL and Imidazole buffer were used. The assays derived from method B always better fit with the expected, calculated, values of FVIII:C concentrations. Furthermore, FVIII: C was assayed in 60 patients: the outcome of method A was always higher than values of method B. The discrepancy between the two methods was higher at FVIII concentrations below 50 U/dL but null at 100 U/dL. The A slope was steeper than B slope and the difference was statistically significant starting from the 1/10 dilution. Accordingly, FVIII: C of patients' plasma obtained by method A was always higher that those obtained by method B, even 2 or 3 times for FVIII level < or = 10 U/dL or 1.4-1.6 times for FVIII levels between 10 and 25 U/dL. CONCLUSIONS: only method B is able to give FVIII: C assays in agreement with the expected values. The dilution of reference standards and samples with FVIII deficient plasma is crucial to accurately evaluate the post-infusion FVIII concentrations in pharmacokinetic studies or the trough level during prophylactic therapy and to investigate the discrepancy among different FVIII: C assays. In addition, the assessment of severity and classification of hemophilia should be reviewed.
BACKGROUND: The results of a number of studies in pigs and mice suggest that absence of von Willebrand factor (vWF) protects against the development of atherosclerosis. We studied whether patients with a complete deficiency of vWF (type 3 von Willebrand disease [vWD]) develop fewer atherosclerotic vessel wall changes than healthy controls. METHODS AND RESULTS: This study included 47 individuals with type 3 vWD and 84 healthy controls. Early atherosclerotic changes were assessed by measuring the thickness of the intima-media in the carotid and femoral arteries by B-mode ultrasonography. Advanced atherosclerotic changes were quantified by summing the maximal thickness of atherosclerotic plaques in the carotid and femoral arteries and were expressed as a plaque score. Established risk factors were determined to adjust for possible differences between the groups. We found no substantial difference in intima-media thickness between vWD patients and controls (adjusted difference for carotid artery 0.007 mm, 95% CI -0.022 to 0.036 mm; femoral artery 0.069 mm, 95% CI -0.056 to 0.19 mm). Similar proportions of patients and controls had atherosclerotic plaques (19% and 17%, respectively). No difference was found in the plaque score between groups (adjusted difference -0.22 mm, 95% CI -0.69 to 0.26). Among vWD patients, we found no effect of treatment with vWF concentrates on intima-media thickness or plaque score. CONCLUSIONS: The results of this study indicate that vWF does not play a substantial role in human atherogenesis.
To establish the pharmacokinetic profile of activated recombinant coagulation factor VII (rFVIIa; NovoSeven in children with haemophilia A, and to compare it with the pharmacokinetic profile in adults with haemophilia A. Twelve children (2-12 years) received one single dose of rFVIIa 90 and 180 micrograms kg(-1) in randomized order separated by a washout period of 48 h to 1 month. Six adults (18-55 years) received a single dose of rFVIIa 90 micrograms kg(-1). The pharmacokinetic analyses were based on a non-compartmental method. In children, the plasma level of FVII increased proportionally with the dose. The total body clearance normalized for body weight was significantly faster in children than in adults (FVII:C, 58 vs. 39 mL kg(-1) h(-1) and FVIIa, 78 vs. 53 mL kg(-1) h(-1), P < 0.05). A trend towards a larger volume of distribution at steady-state in children than in adults was observed (P > 0.05). Dose proportionality was established for plasma concentrations of FVII in children with haemophilia A at the dose levels investigated (90 and 180 micrograms kg(-1) rFVIIa). Following administration of rFVIIa 90 micrograms kg(-1), significantly faster clearance was observed in children compared with adults, suggesting that higher doses of rFVIIa may be needed to achieve the same plasma levels as in adults.
BACKGROUND: Antibodies to glycoprotein (GP) IIb-IIIa and/or HLA may render platelet transfusions ineffective to stop bleeding or to cover surgery in patients with Glanzmann's thrombasthenia (GT). Anecdotal reports suggest recombinant factor (rF)VIIa might be a therapeutic alternative in these situations. OBJECTIVES: An international survey was conducted to evaluate further the efficacy and safety of rFVIIa in GT patients. PATIENTS: We analyzed the use of rFVIIa during 34 surgical/invasive procedures and 108 bleeding episodes in 59 GT patients including 29 with current or previous antiplatelet antibodies, and 23 with a history of refractoriness to platelet transfusion. RESULTS: rFVIIa was effective in 29 of the 31 evaluable procedures, and in 77 of the 103 evaluable bleeding episodes of which eight had a recurrence. A significantly higher success rate was observed in severe bleeding episodes when an arbitrarily defined 'optimal regimen' derived from the Canadian pilot study results (> or = 80 micro g kg(-1) rFVIIa/injection, dosing interval < or = 2.5 h, three or more doses before failure declaration) was used compared with other regimens (77%; 24/31 vs. 48%, 19/40; chi(2), P = 0.010). Patients given maintenance doses had significantly fewer recurrences within 48 h of bleed cessation compared with those not given any (Fisher's exact test, P = 0.022). One thromboembolic event and one blood clot in the ureter occurring in surgical patients following prolonged continuous infusion of high-dose rFVIIa and antifibrinolytic drug use have been previously reported. CONCLUSION: rFVIIa seems a potential alternative to platelet transfusion in GT patients, particularly in those with antiplatelet antibodies and/or platelet refractoriness.
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Participants in an international conference on prophylactic therapy for severe haemophilia developed a consensus summary of the findings and conclusions of the conference. In the consensus, participants agreed upon revised definitions for primary and secondary prophylaxis and also made recommendations concerning the need for an international system of pharmacovigilance. Considerations on starting prophylaxis, monitoring outcomes, and individualizing treatment regimens were discussed. Several research questions were identified as needing further investigation, including when to start and when to stop prophylaxis, optimal dosing and dose interval, and methods for assessment of long-term treatment effects. Such studies should include carefully defined cohorts, validated orthopaedic and quality-of-life assessment instruments, and cost-benefit analyses.
A survey of principal pharmacokinetic (PK) studies on factor VIII (FVIII) and factor IX (FIX) plasma- and rDNA-derived concentrates, analysed by means of the PKRD program, has been performed. Notwithstanding the accurate definition of the study design, released in 1991 by the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis (SSC-ISTH), a large variability of PK parameters has been pointed out. In the majority of the PK studies, the size of the population is small. In this situation, a careful individualization of haemophilia therapy is strongly recommended. The tailored prediction of loading and maintenance dosages and the need for strict control of trough FVIII/IX levels are mandatory not only to decrease the risk of bleeds but also to spare financial resources. Recently, the old problem of FVIII assay standardization has again become a concern among physicians, especially after the introduction of B-domain deleted rFVIII concentrate. The discrepancies between the widely used one-stage clotting assay and the chromogenic substrate assay seem to be solved by the introduction of a product-specific laboratory standard.
BACKGROUND: Hemophilia A patients with inhibitors are generally treated with preparations containing activated coagulation factors to achieve hemostasis by bypassing factor (F)VIII. OBJECTIVES: We developed an assay for monitoring the kinetic of thrombin generation in human FVIII inhibitor plasma reconstituted in vitro with activated prothrombin complex concentrate, FEIBA, and in plasma samples from hemophilia A patients taken after FEIBA treatment. PATIENTS AND METHODS: For pharmacokinetic studies three patients with severe hemophilia A and with a high-titer inhibitor received a single dose of FEIBA. Repeated FEIBA treatment was monitored in one patient with acquired hemophilia A. Coagulation was triggered in citrated plasma by adding a low concentration of tissue factor/phospholipid complex and CaCl2 in the presence of a fluorogenic thrombin substrate. The intensity of the fluorescence signal (FU) was continuously monitored, and the rate of increase in the fluorescence signal for every time point, which reflects the actual thrombin concentrations, was calculated. RESULTS: The maximum rate of substrate conversion, which indicates the highest thrombin concentration, was approximately 1900 FU min(-1) in a normal plasma pool. Practically no thrombin generation was observed in the FVIII inhibitor plasma, but when it was spiked with FEIBA, the rate and the peak of thrombin generation increased dose-dependently to close to normal. Plasma samples from FVIII inhibitor patients treated with a single dose of FEIBA had an improved thrombin maximum within an hour after treatment, which gradually returned to baseline values with a half-life of 4-7 h. Changes in the characteristic parameters of thrombin generation coincided with the repeated administration of FEIBA in a patient with acquired hemophilia A. CONCLUSIONS: This assay enables the pharmacodynamic and pharmacokinetic properties of bypassing therapies to be monitored, thus helping to optimize treatment.
When the one-stage clotting assay is used in comparison with the chromogenic and immunological assays, plasma levels of factor (F)VIII are underestimated by 40-50% after infusion of B-domain deleted recombinant FVIII (BDD-rFVIII) in patients with hemophilia. A possible way to counteract the underestimation of FVIII levels by the one-stage assay is the adoption of a recombinant FVIII reference standard instead of a plasma standard. To evaluate the usefulness of such a standard [ReFacto Laboratory Standard (RLS)], the pharmacokinetic parameters of a single dose of BDD-rFVIII (25 U kg(-1)) were evaluated in a multicenter study carried out in 18 patients with severe hemophilia A. The very low in vivo recovery, obtained with the combination of the one-stage assay and plasma reference standard, was increased up to the values obtained by the chromogenic assay when the results were expressed in terms of RLS. When the plasma standard was used, the one-stage/chromogenic ratio was 0.82 +/- 0.12 for FVIII levels above 25 U dL(-1) and 1.42 +/- 0.99 for FVIII levels below 25 U dL(-1). Using the RLS, the one-stage/chromogenic ratio increased to 1.01 +/- 0.19 at FVIII levels above 25 U dL(-1), as a consequence of a complete overlap of the two decays; however, at FVIII levels below 25 U dL(-1), the one-stage/chromogenic ratio was still 1.6 +/- 0.85. After the twelfth hour, FVIII concentrations obtained by chromogenic assay were always lower than those resulting from the one-stage clotting assay, independently of the standard used. Results obtained by chromogenic assay were not affected by the type of standard used. Compared with those obtained by the one-stage assay, higher values of clearance, lower volume of distribution area and shorter plasma half-life or mean residence time were obtained by chromogenic assay because of a shape change of the decay curve due to a shift to higher values in the first part (time interval 0-12 h) and to lower values in the second part of the decay curve (time interval 12-48 h). As a consequence, the slope of the decay curve obtained by means of chromogenic assay was steeper. In conclusion, the more homogeneous results of in vivo recovery and pharmacokinetic analysis, due to the decrease of discrepancy between the two methods when RLS was used, make the cheaper and more widely used one-stage assay preferable to the more expensive chromogenic assay, on condition that the ReFacto specific standard has used.
OBJECTIVE: To evaluate the coagulative/fibrinolytic cascade and the circulating markers of the endothelial injury in systemic sclerosis (SSc). METHOD: Plasma was obtained from 29 patients with SSc and tested for thrombin-antithrombin (TAT), fragments 1+2 (F1+2), dermatansulphate (DS), thrombomodulin (TM), lipoprotein (a) [Lp(a)], von Willebrand factor (vWF), tissue type plasminogen activator (tPA), plasminogen activator inhibitor (PAI), D-dimers, intercellular adhesion molecole-1 (ICAM-1), vascular cell adhesion molecule (VCAM), and E-selectin. The data were correlated with lung (forced vital capacity, diffusing lung capacity for carbon monoxide, vital capacity) and skin (skin score) involvement. RESULTS: Coagulation was significantly activated (increase in F1+2, P <.001; TAT, P <.01; and Lp(a), P <.05). TM was not significantly different from controls. vWF was significantly increased (P <.01), and its supranormal multimers increased in more than 50% of patients. DS was significantly increased in diffuse cutaneous SSc (P <.01). Fibrinolysis was impaired as shown by reduced D-dimers (P <.01) and decreased levels of PAI (P < 0.01). The markers of endothelial injury were also significantly elevated. DS correlated significantly with forced vital capacity (P <.01) and forced vital capacity ratio (P <.01). CONCLUSION: Injury to the endothelium reduces endothelial function, as suggested by impairment of fibrinolysis and activation of the coagulative pathway. The loss of the balance between fibrinolysis and coagulation contributes to vessel engulfment with fibrin and breakdown of vessel patency. The increase of circulating DS suggests that this factor may be a new marker of endothelial injury.
Factor VIII (FVIII)-bypassing agents have complex modes of action but all control bleeding in inhibitor patients by triggering the generation of thrombin. No routine test is available for monitoring this therapy in patients with inhibitors against FVIII. We present an assay that records FEIBA- or FVIIa-mediated changes in thrombin generation (TG) in FVIII inhibitor plasma samples. In plasma samples spiked with FEIBA TG was normalized above 0.4 U/ml, while for recombinant FVIIa (rFVIIa) more than 12.5 microg/ml were required to induce TG in the absence of tissue factor (TF). Addition of TF increased the TG potential of rFVIIa in vitro. This assay seems suitable for monitoring the pharmacokinetics of inhibitor bypassing agents during treatment and possibly for predicting responses to treatment.
The general rules of pharmacokinetics have been applied to the study of the behaviour of clotting factor concentrates in patients with haemophilia. Since 1980, the continuous development of innovative plasma- and rDNA-derived concentrates and the implementation of new virucidal methods in manufacturing processes has prompted us to define a standard approach to this issue. Model-based methods, based upon one or two open compartment models, were available when this work was initiated. Unfortunately, these methods are supported by very little biological data and are profoundly affected by the goodness-of-fit of the data. In contrast, the model-independent method, which is not affected by errors in fitting, provides reproducible and reliable estimates of the behaviour of clotting factor concentrates in patients with haemophilia. Further, the calculations required for the model-independent method are quite simple and can be computed using a pocket minicomputer. The need for an accurate standardization has been recognized by the Factor VIII/IX Sub-Committee which, in 1991, issued the first recommendations on the pharmacokinetic evaluation of Factor VIII/IX concentrates. A recent revision of the recommendations has been made available on the web site of the International Society on Thrombosis and Haemostasis. The most crucial changes - sample size, study design, dosages in single-dose studies, potency assessment, need for well-defined standards, optimal number of points, and most important outcomes - are discussed in this report. In addition, the model-independent and compartmental methods are described.
The objective was to establish the pharmacokinetic properties of porcine factor VIII (pFVIII) at three different doses in a single patient. The patient, born in 1952, had severe haemophilia A and developed an inhibitor to human FVIII (hFVIII) in 1966 aged 14 years. He was first treated with pFVIII in 1980. Apart from a short period of treatment with hFVIII in 1998 which resulted in the reappearance of the inhibitor, pFVIII has been constantly used since 1984. No inhibitor against human or porcine FVIII had been recordable over the 2-year period prior to the study. Three separate pharmacokinetic studies were performed using a washout period of 72 h and doses of 10 U kg (-1), 25 U kg (-1), or 50 U kg (-1), respectively, with sampling at preinfusion, then at 15 and 30 min and 1, 3, 6, 9, 12 and 24 h postinfusion. The FVIII levels were measured using both plasma derived one stage APTT based assay and chromogenic assay. The results were computed using a model-independent analysis and a model-dependent analysis. The respective clearances (mL h(-1)kg(-1)) at doses 50 U, 25 U or 10 U kg (-1) were 1.32, 1.33 1.8 (bioassay) and 2.54, 2.93, 9.43 (chromogenic). The respective half-lives (h) at doses 50 U, 25 U, and 10 U kg (-1) were 23.71, 16.54, 25.17 (bioassay) and 15.71, 17.39, and 10.66 (chromogenic). The respective recoveries (u dL(-1)/u kg(-1)) at doses 50 U, 25 U, and 10 U kg (-1) were 2.32, 2.44, 2.7 (bioassay) and 1.42, 1.16 and 0.9 (chromogenic). It was found that the two compartment model best fitted the curves of the bioassay and a one compartmental model best fitted the curves of the chromogenic assay. The pharmacokinetic studies are the first to be performed at different dose levels and using different assay methods for pFVIII. Using the bioassay, they show a long half-life and high recovery compared to hFVIII. The differences between the bioassay and the chromogenic assay reflect their different biological basis and are of relevance when potency labelling is performed using chromogenic assay (European Pharmacopeia).
We characterized a symptomatic CRMred factor X (FX) deficiency produced by the Glu19Ala mutation in the gamma-carboxyglutamic-rich domain. FX activity levels in plasma were markedly reduced in prothrombin time assays (< 1-5%), whereas in activated partial thromboplastin assays (16%) and in RVV assays (17%) the reduction in activity mirrored that in antigen levels (17%). Activation of recombinant 19Ala-FX by factor IXa/factor VIIIa or RVV, and the activity in thrombin generation assays, were comparable to those of wild-type FX. Differently, complete activation of recombinant 19Ala-FX required a factor VIIa/TF concentration 30-fold higher than that of wild-type FX. The recombinant FVIIa significantly reduced PT values in 19Ala-FX reconstituted plasma, thus suggesting an alternative approach for treatment of FX deficiencies characterized by defective FX activation. The study of this FX deficiency provides an "in vivo" and "in vitro" model for the investigation of Gla domain interactions.
Recombinant factor VIII and factor IX concentrates, human-plasma-derived albumin, and samples from previously untreated patients with hemophilia were examined for the presence of TT virus (TTV) by using polymerase chain reaction testing. Blood samples from the patients were obtained prospectively before and every 3 to 6 months after therapy was begun. TTV was detected in 23.5% of the recombinant-product lots and 55.5% of the albumin lots tested. Only first-generation factor VIII recombinant concentrates stabilized with human albumin were positive for TTV, whereas all second-generation (human protein-free) concentrates were negative for the virus. In 59% of patients treated with either first- or second-generation recombinant factor concentrates, TTV infection developed at some point after the initial infusion. Infection with TTV in these patients before and after treatment did not appear to be clinically important. Thus, first-generation recombinant factor VIII concentrates may contain TTV and the source of the viral contamination may be human albumin.
Recombinant factor VIIa (rFVIIa; NovoSeven, Novo Nordisk, Bagsvaerd, Denmark) appears effective and relatively safe for the treatment of bleeding and for surgical prophylaxis in patients with Glanzmann thrombasthenia as reported to the International Registry on rFVIIa and Congenital Platelet Disorders. One of the shortcomings of the Registry data is the heterogeneity of treatment protocol, including dosage, number of doses used, duration of treatment before declaration of failure, and mode of rFVIIa administration (bolus v continuous infusion). The data are not yet sufficient to define optimal regimens for various indications such as the type of bleeding or the type of procedures. The place of this drug compared to platelet transfusion in the overall management of patients with Glanzmann thrombasthenia will need to be determined in relationship to a number of challenges and unresolved issues in the clinical care of these patients. These issues include: how to improve local measures for patients with mucosal bleeds, optimal management of young women during menarche, optimal platelet transfusion regimens for various indications, the relationship between antiplatelet antibodies detected by monoclonal antibody-specific immobilization of platelet antigens (MAIPA) and effectiveness of platelet transfusion, whether there are other biological tests that may correlate with effectiveness of platelet transfusion, and management of pregnancy and delivery regarding antiplatelet immunization.