Pharmacokinetics of coagulation factors: clinical relevance for patients with haemophilia.

Haemophilia is a recessively inherited coagulation disorder, in which an X-chromosome mutation causes a deficiency of either coagulation factor VIII (FVIII) in haemophilia A, or factor IX (FIX) in haemophilia B. Intravenous administration of FVIII or FIX can be used to control a bleeding episode, to provide haemostasis during surgery or for long term prophylaxis of bleeding. In special cases, activated factor VII (FVIIa) may be used instead of FVIII or FIX. The aim of this work is to review the pharmacokinetics of FVIII, FIX and FVIIa and to give an outline of the use of pharmacokinetics to optimise the treatment of patients with haemophilia. The pharmacokinetics of FVIII are well characterised. The systemic clearance (CL) of FVIII is largely determined by the plasma level of von Willebrand factor (vWF), which protects FVIII from degradation. Typical average CL in patients with normal vWF levels is 3 ml/h/kg, with an apparent volume of distribution at steady state (Vss) that slightly exceeds the plasma volume of the patient, and the average elimination half-life (t1/2) is around 14 hours. There are still some discrepancies in the literature on the pharmacokinetics of FIX. The average CL of plasma-derived FIX seems to be 4 ml/h/kg, the Vss is 3 to 4 times the plasma volume and the elimination t1/2 often exceeds 30 hours. FVIIa has a much higher CL (average of 33 ml/h/kg), and a short terminal t1/2 (at 2 to 3 hours). The Vss is 2 to 3 times the plasma volume. Since the therapeutic levels of coagulation factors are well defined in most clinical situations, applied pharmacokinetics is an excellent tool to optimise therapy. Individual tailoring of administration in prophylaxis has been shown to considerably increase the cost effectiveness of the treatment. Dosage regimens for the treatment of bleeding episodes or for haemostasis during surgery are also designed using pharmacokinetic data, and the advantages of using a constant infusion instead of repeated bolus doses have been explored. The influence of antibodies (inhibitors) on the pharmacokinetics of FVIII and FIX is in part understood, and the doses of coagulation factor needed to treat a patient can tentatively be calculated from the antibody titre. In conclusion, therapeutic monitoring of coagulation factor levels and the use of clinical pharmacokinetics to aid therapy are well established in the treatment of patients with haemophilia.     

Therapeutic approaches for haemophilia

The life-long episodic bleeding associated with inherited deficiencies of blood coagulation Factor VIII (FVIII) or Factor IX (FIX) can be well controlled with periodic iv. injections of FVIII or FIX concentrates. Either concentrate can be isolated from large human pools (i.e., plasma-derived FVIII or FIX concentrate) or from culture supernatants of recombinant cells engineered to secrete FVIII or FIX. The validated viral inactivation strategies used by manufacturers of FVIII and FIX concentrates have essentially eliminated the transmission of hepatitis B, hepatitis C and HIV viruses. The low yields and inherent instability of FVIII (and FVIIIa in particular) and the additional costs of viral inactivation methods make the annual cost/patient for prophylaxis and treatment of haemophilia very expensive. Several strategies have been adopted and proposed to improve yields of FVIII. These include: deletion of portions of FVIII which are not associated with function; mutations to prevent inactivation of FVIII by protease degradation; and synthesis of FVIII fragments to replace portions deleted in some FVIII deficient patients. An approach to improve FIX replacement involves the production of more coagulatively active FIX mutants. Another promising approach in both FVIII and FIX replacement is gene therapy. Two major issues that will have to be critically addressed before gene therapy for haemophilia can become widespread are whether the procedures will be well-tolerated in patients with significant liver impairment (due to previous exposure to hepatitis viruses) and whether consistent long-term delivery of the transgenes can be achieved.     

Safety of recombinant coagulation factors in treating hemophilia

Introduction: During the last decade, new FVIII/IX concentrates have been developed for the treatment of patients affected by hemophilia A/B. Significant progress has been achieved regarding their half-life, but the old issue of immunogenicity and new concerns about safety need to be addressed.

Areas covered: After the implementation of virucidal methods, both plasma-derived and recombinant clotting factor concentrates achieved a very safe profile. The development of anti-FVIII antibodies is the major adverse event of replacement therapy with both FVIII concentrates. Furthermore, the new extended half-life concentrates, protein fused or pegylated, raised some concerns about their side effects.

Expert Opinion: The treatment of hemophilia A with inhibitors by induction of immunotolerance and using by-passing concentrates, improved the quality of life of patients but did not allow them to have a life expectancy like that of patients without inhibitors. The new humanized monoclonal antibody (MAb) ACE910, mimicking FVIII function, seems to be able to reduce the bleedings of hemophilia A patients with inhibitors. The post-marketing surveillance will clarify if the adverse events observed during the phase III clinical trials and compassionate use were due to the association with a Prothrombin activated complex concentrate or to the prothrombotic effect of the drug itself.     

Modulators of the coagulation cascade: focus and recent advances in inhibitors of tissue factor, factor VIIa and their complex

Recent developments in the field of haemostasis and thrombosis highlighted the crucial role of the tissue factor/factor VIIa complex (TF/FVIIa) in the initiation of coagulation processes. Nowadays, anticoagulant therapies involving heparin or coumarin derivatives, thrombin or factor Xa inhibitors are generally associated with side effects such as bleeding and thrombocytopenia. In this context, the inhibition of TF, FVIIa and their complex by efficient antithrombotic drugs represents a new strategy to reduce this bleeding and to prevent thrombosis events. Moreover, TF/FVIIa inhibition is shown to be useful in the treatment of biological processes independent of the clotting cascade such as angiogenesis and cancer. Among the natural and genetically engineered TF/FVIIa inhibitors, injections of the recombinant protein rNAPc2 show clinical improvements, such as reduced bleeding and thromboembolism, over classical drugs used in the therapy of coronary angioplasty and hip or knee replacement surgery. The knowledge of the 3D-structure of TF/FVIIa complex and examination of co-crystal data of some drugs bound to this complex led to the design and synthesis of numerous TF/FVIIa inhibitors. Among them, the p-amidinophenylurea 18 (Ki = 0.027 microM), the pyrimidinones PHA-927 (30, IC50 = 0.016 microM) and PHA-798 (31, IC50 = 0.014 microM) and the pyridinone 37 (IC50 = 0.052 microM) are highly potent inhibitors of the TF/FVIIa complex, deprived of activity towards thrombin (IC50 > 30-100 microM) and factor Xa (IC50 > 10-100 microM), other proteases involved in the coagulation cascade. Both pyrimidinones prevent arterial thrombosis in non-human primate models of thrombosis and represent a safe approach to anti-thrombotic therapy in patients with cardiovascular risk factors.     

Design,parallel synthesis,and crystal structures of pyrazinone antithrombotics as selective inhibitors of the tissue factor VIIa complex

Structure-based drug design (SBDD) and polymer-assisted solution-phase (PASP) library synthesis were used to develop a series of pyrazinone inhibitors of the Tissue Factor/Factor VIIa (TF/VIIa) complex. The crystal structure of a tripeptide-alpha-ketothiazole complexed with TF/VIIa was utilized in a docking experiment to identify the pyrazinone core as a starting scaffold. The pyrazinone core could orient the substituents in the correct spatial arrangement to probe the S1, S2, and S3 pockets of the enzyme. A multistep PASP library synthesis was designed to prepare the substituted pyrazinones varying the P1, P2, and P3 moieties. Hundreds of pyrazinone TF/VIIa inhibitors were prepared and tested in several serine protease enzyme assays involved in the coagulation cascade. The inhibitors exhibited modest activity on TF/VIIa with excellent selectivity over thrombin (IIa) and Factor Xa. The structure-activity relationship of the pyrazinone inhibitors will be discussed and X-ray crystal structures of selected compounds complexed with the TF/VIIa enzyme will be described. This study ultimately led to the synthesis of compound 34, which exhibited 16 nM (IC50) activity on TF/VIIa with >6250 x selectivity vs Factor Xa and thrombin. This potent and highly selective inhibitor of TF/VIIa was chosen for preclinical, intravenous proof-of-concept studies to demonstrate the separation between antithrombotic efficacy and bleeding side effects in a nonhuman primate model of electrolytic-induced arterial thrombosis.     

抗血栓药物的临床应用研究进展

非正常凝血可导致血栓形成、动脉粥样硬化等疾病,抗血栓药物能有效预防血栓的形成.该文依据抗血栓药物的作用机制和临床研究,对抗血小板聚集、抗凝血和溶栓类抗血栓药物进行综述.     

治疗性人源化单克隆抗体研究进展

目前正在研究中的生物技术药物有1/4以上是单克隆抗体药物,临床治疗中人抗鼠抗体反应(HAMA)的出现使鼠源性单克隆抗体的应用受到极大限制.因此,对于疗程长、需反复给药的单克隆抗体药物,人源化是其重要而必然的发展方向.现阶段,已有30余个通过细胞工程和基因工程制备的人源化抗体应用于临床,适应证包括变态反应性疾病、恶性肿瘤、器官移植排斥反应、心血管疾病、病毒感染等难治性疾病.此外尚有近百个人源化抗体药物正处在临床前或临床研究阶段.预计它们将在人类疾病的治疗中发挥重要作用.     

The safety of pharmacologic options for the treatment of persons with hemophilia

ABSTRACT

Introduction: The mainstay of treatment of hemophilia A and B is the replacement of the congenitally deficient coagulation factor through the intravenous infusion of specific concentrates (factor VIII, FVIII, in hemophilia A; factor IX, FIX, in hemophilia B). Several commercial brands of FVIII or FIX products extracted from human plasma or engineered using recombinant DNA technology are available.

Areas covered: We analyze the safety aspects of plasma-derived and recombinant FVIII and FIX products licensed in Europe, focusing on their pathogen safety and inhibitor and thrombosis risks. The safety aspects of bypassing agents (i.e., activated prothrombin complex concentrates and recombinant activated factor VII) used for treatment of bleeding episodes in inhibitor patients will be also briefly discussed.

Expert opinion: The analysis of the published literature documents the high degree of safety from pathogen risk for both plasma-derived and recombinant products available for hemophilia treatment. The main threat to factor concentrate safety is represented by the development of neutralizing alloantibodies against the infused coagulation factor, which in hemophilia A seem to occur more frequently following the administration of recombinant than plasma-derived FVIII products. Great expectations are placed on newer products, particularly on those based upon mechanisms of action other than FVIII replacement.     

抗肿瘤人源化抗体药物的研究进展

随着单克隆抗体技术的发展,治疗性抗体药物逐渐占据全球市场,主要应用于肿瘤治疗、抗移植排斥、自身免疫性疾病的治疗等。抗体药物可根据人源化程度不同,分为鼠源抗体、嵌合抗体、人源化抗体和全人源抗体。本文着眼于目前治疗性抗体药物研究热点,聚焦于抗肿瘤人源化抗体药物,结合已上市热点抗肿瘤人源化抗体药物实例,从抗体人源化技术以及人源化抗体生产的角度来阐述目前研究状况。同时,立足于国内外抗肿瘤人源化抗体药物的现状,分析未来发展趋势。     

Factor X inhibitors

Factor X plays a central role in coagulation, being the point of convergence of the extrinsic and intrinsic pathways of blood clotting. It may also act as one of the links between the coagulation and inflammatory pathways. These findings suggest that factor X may represent an attractive target for a new antithrombotic drug. Indeed, a factor X inhibitor, fondaparinux, has already been approved for clinical use to prevent post-operative deep vein thrombosis. Factor X inhibitors are also being evaluated for use in the treatment of the acute coronary syndromes, pulmonary embolism and deep vein thrombosis. Oral factor X inhibitors are also being developed, which may be of use in the outpatient prevention and/or treatment of stroke and thromboembolism.     

Factor X inhibitors

Factor X plays a central role in coagulation, being the point of convergence of the extrinsic and intrinsic pathways of blood clotting. It may also act as one of the links between the coagulation and inflammatory pathways. These findings suggest that factor X may represent an attractive target for a new antithrombotic drug. Indeed, a factor X inhibitor, fondaparinux, has already been approved for clinical use to prevent post-operative deep vein thrombosis. Factor X inhibitors are also being evaluated for use in the treatment of the acute coronary syndromes, pulmonary embolism and deep vein thrombosis. Oral factor X inhibitors are also being developed, which may be of use in the outpatient prevention and/or treatment of stroke and thromboembolism.     

Downregulation of CD40 signal and induction of TGF-β by phosphatidylinositol mediates reduction in immunogenicity against recombinant human Factor VIII

Factor VIII (FVIII) is an important coagulation cofactor and its deficiency causes Hemophilia A, a bleeding disorder. Replacement therapy using recombinant FVIII is currently the first line of therapy for Hemophilia A, but the development of neutralizing antibody is a major clinical complication for this therapy. Recently, it has been shown that FVIII associated with phosphatidylinositol (PI)-containing lipidic nanoparticles reduced development of neutralizing antibodies in Hemophilia A mice (Peng A, Straubinger RM, Balu-Iyer SV. 2010. AAPS J 12(3):473-481). Here, we investigated the underlying mechanism of this reduction in antibody response in culturing conditions. In vitro, PI interfered with the processing of FVIII by cultured dendritic cells (DC), resulting in a reduction in the upregulation of phenotypic costimulatory signal CD40. Furthermore, PI increased secretion of regulatory cytokines Transforming Growth Factor β1 and Interleukin 10 (IL-10) but reduced the secretion of proinflammatory cytokines IL-6 and IL-17. The data suggest that PI reduces immunogenicity of FVIII by modulating DC maturation and inducing secretion of regulatory cytokines.     

Contribution of in vivo models of thrombosis to the discovery and development of novel antithrombotic agents

Cardiovascular and cerebrovascular diseases continue to be the leading cause of death throughout the world. Over the past two decades, great advances have been made in the pharmacological treatment and prevention of thrombotic disorders (e.g., tissue plasminogen activators, platelet GPIIb/IIIa antagonists, ADP receptor antagonists such as clopidogrel, low-molecular weight heparins, and direct thrombin inhibitors). New research is leading to the next generation of antithrombotic compounds such as direct coagulation FVIIa inhibitors, tissue factor pathway inhibitors, gene therapy, and orally active direct thrombin inhibitors and coagulation Factor Xa (FXa) inhibitors. Animal models of thrombosis have played a crucial role in discovering and validiting novel drug targets, selecting new agents for clinical evaluation, and providing dosing and safety information for clinical trials. In addition, these models have provided valuable information regarding the mechanisms of these new agents and the interactions between antithrombotic agents that work by different mechanisms. This review briefly presents the pivitol preclinical studies that led to the development of drugs that have proven to be effective clinicallly. The role that animal models of thrombosis are playing in the discovery and development of novel antithrombotic agents is also described, with specific emphasis on FXa inhibitors. The major issues regarding the use of animal models of thrombosis, such as the use of positive controls, appropriate pharmacodynamic markers of activity, safety evaluation, species-specificity, and pharmacokinetics, are highlighted. Finally, the use of genetic models in thrombosis/hemostasis research and pharmacology is presented using gene-therapy for hemophilia as an example of how animal models have aided in the development of these therapies that are now being evaluated clinically. In summary, animal models have contributed greatly to the discovery of currently available antithrombotic agents and will play a primary role in the discovery and characterization of the novel antithrombotic agents that will provide safe and effective pharmacological treatment for life-threatening thrombotic diseases.     

Tissue factor - a therapeutic target for thrombotic disorders

Exposure of blood to tissue factor (TF) sets off the coagulation cascade. TF is a transmembrane protein that serves as an essential cofactor for activated coagulation factor VII (FVIIa). TF may be exposed locally by vascular injury (such as balloon angioplasty) or by spontaneous rupture of an atherosclerotic plaque. Expression of TF may also be induced on monocytes and endothelial cells in conditions like sepsis and cancer, causing a more generalised activation of clotting. TF may thus play a central role in thrombosis in a number of settings, and attention has turned to blocking TF as a means to prevent thrombosis. Inhibiting the inducible expression of TF by monocytes can be achieved by 'deactivating' cytokines, such as interleukin (IL)-4, -10 and -13, or by certain prostanoids; by drugs that modify signal transduction, such as pentoxifylline, retinoic acid or vitamin D(3), or by antisense oligonucleotides. Such approaches are for the most part at a preclinical stage. The function of TF can be blocked by antibodies that prevent the binding of FVIIa to TF; by active site-inhibited FVIIa, which competes with native FVIIa for binding; by antibodies or small molecules that block the function of the TF/FVIIa complex; and by molecules, such as TF pathway inhibitor or nematode anticoagulant peptide C2, which inhibit the active site of FVIIa in the TF/FVIIa complex after first binding to activated factor X. The latter two agents have entered Phase II clinical trials. Perhaps most intriguing is the use of anti-TF agents locally, which holds the promise of stopping thrombosis at a specific site of injury without the bleeding risk associated with systemic anticoagulation.     

Inhibitors of Tissue Factor. Factor VIIa for anticoagulant therapy

Factor VIIa (FVIIa) is a key serine protease involved in the initiation of the coagulation cascade. It is a glycosylated disulfide-linked heterodimer comprised of an amino-terminal gamma-carboxyglutamic acid-rich (Gla) domain and two epidermal growth factor (EGF)-like domains in the light chain, and a chymotrypsin-like serine protease domain in the heavy chain. FVIIa requires tissue factor (TF), a membrane bound protein, as an essential cofactor for maximal activity towards its biological substrates Factor X, Factor IX and Factor VII (FVII). Inhibition of TF.FVIIa activity may prevent the formation of fibrin clots and thus be useful in the management of thrombotic disease. The development of TF.FVIIa inhibitors to validate this target has been of great interest. A wide array of strategic approaches to inhibiting the biochemical and biological functions of the TF.FVIIa complex has been pursued. This has been greatly aided from our understanding of the structures for TF, FVII, FVIIa, and the TF.FVIIa complex. These approaches have resulted in inhibitors directed specifically towards either FVIIa or TF. Antagonists include active site inhibited FVIIa, TF mutants, anti-TF antibodies, anti-FVII/FVIIa antibodies, naturally-occurring protein inhibitors, peptide exosite inhibitors, and protein and small molecule active site inhibitors. These antagonists can inhibit catalysis directly at the active site as well as impair function by binding to exosites that may interfere with substrate, membrane, or cofactor binding. The rationale of TF.FVIIa as a target and the development, characteristics and biological uses of TF.FVIIa inhibitors are discussed.     

Recent research developments in the direct inhibition of coagulation proteinases--inhibitors of the initiation phase

Physiologic clotting is a defensive action. The new cell-based model of hemostasis proposes three steps--initiation, amplification and propagation--occurring on specific cell surfaces to generate a thrombus in a tightly regulated manner. The initiation phase relies on key players including tissue factor (TF), factor VIIa (fVIIa), platelets, Ca2+, phospholipids, and factor X/Xa (fX/fXa). Exposure of TF on sub-endothelial and other blood cells triggers a coagulation response, which may have to be inhibited to prevent a deleterious thrombotic effect. Inhibiting TF-initiated coagulation, akin to 'nipping coagulation in the bud', is predicted to have major advantages, including a more efficient separation of the antithrombotic and hemorrhagic responses. The availability of crystal structures of TF, fVIIa and TF-fVIIa complex makes structure-based drug design feasible. Although no initiation phase small molecule inhibitor has reached the clinic as yet, several molecules have displayed promise. We discuss recent results on the discovery of inhibitors of the initiation phase with special emphasis on peptides, peptidomimetics and organic small molecules.     

Monoclonal Antibodies as Diagnostics; an Appraisal

Ever since the development of Hybridoma Technology in 1975 by Kohler and Milstein, our vision for antibodies as tools for research for prevention, detection and treatment of diseases, vaccine production, antigenic characterization of pathogens and in the study of genetic regulation of immune responses and disease susceptibility has been revolutionized. The monoclonal antibodies being directed against single epitopes are homogeneous, highly specific and can be produced in unlimited quantities. In animal disease diagnosis, they are very useful for identification and antigenic characterization of pathogens. Monoclonal antibodies have tremendous applications in the field of diagnostics, therapeutics and targeted drug delivery systems, not only for infectious diseases caused by bacteria, viruses and protozoa but also for cancer, metabolic and hormonal disorders. They are also used in the diagnosis of lymphoid and myeloid malignancies, tissue typing, enzyme linked immunosorbent assay, radio immunoassay, serotyping of microorganisms, immunological intervention with passive antibody, antiidiotype inhibition, or magic bullet therapy with cytotoxic agents coupled with anti mouse specific antibody. Recombinant deoxyribonucleic acid technology through genetic engineering has successfully led to the possibility of reconstruction of monoclonal antibodies viz. chimeric antibodies, humanized antibodies and complementarily determining region grafted antibodies and their enormous therapeutic use.     

Effect of Route of Administration of Human Recombinant Factor VIII on Its Immunogenicity in Hemophilia A Mice

Factor VIII is a multi-domain glycoprotein and is an essential cofactor in the blood coagulation cascade. Its deficiency or dysfunction causes Hemophilia A, a bleeding disorder. Replacement using exogenous recombinant Factor VIII (FVIII) is the first line of therapy for Hemophilia A. Immunogenicity, the development of binding (total) and neutralizing (inhibitory) antibody against administered protein is a clinical complication of the therapy. There are several product related factors such as presence of aggregates, route and frequency of administration and glycosylation have been shown to contribute to immunogenicity. The effect of route of administration of FVIII on antibody development in Hemophilia A is not completely understood. Here we investigated the effect of route of administration (s.c. or i.v.) on immunogenicity in Hemophilia A mice. The total and inhibitory titers were determined using ELISA and modified Bethesda Assay respectively. The results indicated that s.c. is more immunogenic compared to i.v. route in terms of total antibody titer development (binding antibodies) but no significant differences in inhibitory titer levels could be established. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:4480–4484, 2009     

单克隆抗体在疾病治疗中的应用

单克隆抗体(单抗)技术与分子生物学技术的结合使重组嵌合抗体、人源化抗体和全人抗体的技术得到迅速发展.由于这些单抗带有人抗体的Fc片段,因此,这些抗体不仅能中和抗原,更重要的是能介导细胞毒作用和激活补体系统.这些抗体已被用于治疗某些疾病,如癌症、免疫性疾病、感染性疾病和器官移植排斥.随着大规模CHO细胞生产工艺的发展,越来越多的单抗将应用于临床.     

单克隆抗体在疾病治疗中的应用

单克隆抗体(单抗)技术与分子生物学技术的结合使重组嵌合抗体、人源化抗体和全人抗体的技术得到迅速发展.由于这些单抗带有人抗体的Fc片段,因此,这些抗体不仅能中和抗原,更重要的是能介导细胞毒作用和激活补体系统.这些抗体已被用于治疗某些疾病,如癌症、免疫性疾病、感染性疾病和器官移植排斥.随着大规模CHO细胞生产工艺的发展,越来越多的单抗将应用于临床.