A review of the two major regulatory pathways for non-proprietary low-molecular-weight heparins.

With the expiry or pending expiry of originator low-molecular-weight heparin (LMWH) patents, pharmaceutical companies have invested in developing non-proprietary versions of LMWHs. LMWHs are manufactured by depolymerising highly purified unfractionated heparin. In contrast to traditional synthetic drugs with well-defined chemical structures, LMWHs contain complex oligosaccharide mixtures and the different manufacturing processes for LMWHs add to the heterogeneity in their physicochemical properties such that the European Medicines Agency (EMA) and the US Food and Drug Administration (FDA) consider existing originator LMWHs to be distinct medicinal entities that are not clinically interchangeable. The FDA views LMWHs as drugs and has approved two non-proprietary (generic) LMWHs, using the Abbreviated New Drug Application pathway. In contrast, the World Health Organization and the EMA view LMWHs as biological medicines. Therefore, the EMA and also the Scientific and Standardization Subcommittee on Anticoagulation of the International Society on Thrombosis and Haemostasis and the South Asian Society of Atherosclerosis and Thrombosis have all published specific guidelines for assessing non-proprietary (biosimilar) LMWHs. This manuscript reviews why there are two distinct pathways for approving non-proprietary LMWHs. Available literature on non-proprietary LMWHs approved in some jurisdictions is also reviewed in order to assess whether they satisfy the requirements for LMWHs in the three guidance documents. The review also highlights some of the significant difficulties the two pathways pose for manufacturers and an urgent need to develop a consensus governing the manufacture and regulation of non-proprietary LMWHs to make them more widely available.

Immune responses associated with perioperative exposure and reexposure to topical bovine thrombin do not impair hemostasis.

Topical bovine thrombin has been associated with immune responses and anecdotal reports of coagulopathy. This open-label study assessed the impact on clinical hemostasis of human antibodies to bovine thrombin (aBT) or factor V/Va (aBV/Va) in response to topical bovine thrombin (THROMBIN-JMI) in patients both with and without preexisting anti-bovine antibodies. Noninferiority analysis assessed primary endpoint for mean shift from baseline activated partial thromboplastin time (aPTT) at 48 hours postsurgery; secondary endpoints included changes from baseline antibodies/titers and coagulation parameters through 8 weeks postsurgery. A total of 550 patients underwent surgery with THROMBIN-JMI utilized at investigator's discretion. Adjusted mean aPTT change in (+)aBT/(+)THROMBIN-JMI cohort was greater than (-)aBT/(-)THROMBIN-JMI cohort; 4.67-second upper confidence bound exceeded 4.5-second margin (based on assumed mean aPTT of 30 seconds) and noninferiority was not met. Post hoc analysis indicated noninferiority would have been met had noninferiority margin been set prior at relative 15% of actual baseline aPTT. Antibodies/titers were unchanged by THROMBIN-JMI exposure 48 hours postsurgery and unrelated to postsurgical changes in coagulation. Thus, THROMBIN-JMI exposure in patients with/without preexisting aBT or aBV/Va does not alter hemostasis.

The United States Food and Drugs Administration approves a generic enoxaparin.

The Food and Drug Administration (FDA) approved on July 23, 2010, the abbreviated new drug application (ANDA) by Sandoz Inc for a generic enoxaparin (Lovenox) as the FDA is satisfied with the active ingredient ''sameness'' and the interchangeability of Lovenox with this generic version. Regulatory authorities that consider low-molecular-weight heparins (LMWHs) as drugs approve generic LMWHs via an ANDA pathway, whereas EMEA views them as biological medicines and will therefore regulate copies of LMWHs via a biosimilar pathway because only similar copies can be manufactured. Furthermore, European Medicines Agency (EMEA) requires appropriately powered clinical trails to establish comparable efficacy and safety profiles for biosimilar and their branded LMWHs counterparts. The safety issues that have led to withdrawal of some generic LMWHs from some Asian and South American countries mandate the need for a broad consensus defining the minimum attributes copies of LMWHs required for efficacy and safety.

Safety and efficacy of thrombin-JMI: a multidisciplinary expert group consensus.

BACKGROUND: The use of bovine thrombin has been an effective approach to aiding hemostasis during surgery for over 60 years. Its use has a reported association with the development of antibodies to coagulation factors with limited evidence to the clinical significance. METHODS: The Collaborative Delphi survey methodology was used to develop a consensus on specified topic areas from a panel of 12 surgeons/scientists who have had experience with topical thrombins; it consisted of 2 rounds of a Web-based survey and a final live discussion. RESULTS: Some key issues that reached consensus included: bovine, human plasma-derived and recombinant human thrombin are equally effective hemostatic agents with similar adverse event rates, and immunogenicity to a topical protein rarely translate into adverse events. CONCLUSIONS: Although a risk of immunogenicity is associated with all topical thrombins, no conclusive clinical evidence is available that these antibodies have any significant effect on short- and long-term clinical consequences.

A safety review of topical bovine thrombin-induced generation of antibodies to bovine proteins.

BACKGROUND: Topical bovine thrombin has been used to accelerate attainment of hemostasis in the surgical setting for >60 years, and its immunogenicity has been widely reported. Although the development of antibodies is inherent in the introduction of any non-self-therapeutic protein such as bovine-sourced thrombin, there are questions about the relationship between the presence of antibodies to constituents of the therapeutic protein preparation and the occurrence of clinically relevant adverse events (AEs). OBJECTIVE: This review examines the proposed mechanisms for the immunogenicity of topical bovine thrombin preparations and summarizes available evidence from randomized clinical trials, observational studies, and case reports to explore possible relationships between the reported immunogenicity of topical bovine thrombin and the occurrence of AEs. METHODS: A search of MEDLINE (1966-August 2008) for studies published in English was conducted using the Medical Subject Heading terms surgery, antibodies, and hemorrhage, as well as equivalent key words for bovine, adverse events, and thrombin. For inclusion in the review, studies had to report clinical or laboratory safety data for patients exposed to topical bovine thrombin during surgery. RESULTS: The evidence suggests that patients with repeated perioperative exposure to topical bovine thrombin have a 3- to 10-fold greater risk for development of antibodies to topical bovine thrombin than do patients with no history of surgery-related exposure to this agent. Early case reports associated the development of anti-bovine protein antibodies with bleeding and/or thrombotic complications. However, in one prospective, randomized controlled trial comparing topical bovine thrombin with topical recombinant human thrombin, 99.5% of patients in each treatment arm developed postoperative AEs. In an-other, 54% and 55% of patients in the respective treatment arms developed postoperative AEs. In a prospective, randomized controlled trial that compared topical bovine thrombin and plasma-derived human thrombin, 95.5% of patients in each treatment arm developed postoperative AEs. CONCLUSIONS: Repeated perioperative exposure to topical bovine thrombin may increase both the prevalence and titers of antibodies to >or=1 protein contained in nonhomogeneous topical bovine thrombin preparations. However, the evidence reviewed does not support a definitive association between preoperative or postoperative generation of anti-bovine protein antibodies and an increased risk of AEs in surgical patients treated with topical bovine thrombin.

Early intraplatelet signaling enhances the release of human platelet PAR-1 and -4 amino-terminal peptides in response to thrombin.

Activation of washed human platelets initiated with alpha-thrombin, SFLLRN, or AYPGKF invariably results in the generation of PAR-1-(1-41) and PAR-4-(1-47). PAR-1-(1-41) and PAR-4-(1-47) are amino-terminal peptides generated when PAR-1 and -4 are cleaved in their first extracellular domains after R(41) and R(47), respectively, to expose the tethered ligand domains of PAR-1 and -4. Since soybean trypsin inhibitor decreases generation of PAR-1-(1-41) and PAR-4-(1-47) and other platelet aggregation-related responses to these three agonists, but does not inactivate alpha-thrombin, a platelet trypsin-like proteinase apparently activates PAR-1 and -4 to propagate PAR-dependent platelet responses. This study identified the signaling pathways implicated in the generation of the platelet proteinase that in turn produces PAR-1-(1-41) and PAR-4-(1-47), to thereby drive the subsequent PAR-dependent platelet aggregation-related responses to alpha-thrombin, SFLLRN, or AYPGKF. Only inhibitors of signaling enzymes that prevented ATP release (forskolin, PGE(1), or BIMI-1) prevented or delayed the generation of PAR-1-(1-41) and PAR-4-(1-47) in response to all three agonists. SBTI prevented platelet aggregation initiated by alpha-thrombin, SFLLRN, or AYPGKF but did so less effectively when it was added 10 s after each agonist. Thus, the platelet-derived proteinase acts within 10 s of each agonist addition to generate PAR-1-(1-41) and PAR-4-(1-47). Furthermore, alpha-thrombin may not effectively catalyze PAR-1-(1-41) and PAR-4-(1-47) generation. We propose that unidentified ATP-dependent phosphorylation reactions catalyzed by PKC help to generate the platelet-derived proteinase that propagates human platelet PAR-1 and -4 activation by the three agonists.

Coordinate activation of human platelet protease-activated receptor-1 and -4 in response to subnanomolar alpha-thrombin.

We previously demonstrated that human platelets activated with SFLLRN release PAR-1 activation peptide, PAR-1-(1-41), even in the presence of hirudin. This observation suggests that during their activation, platelets generate a protease that activates PAR-1. In this study, PAR-1 and -4 activation peptides were detected 10 s after

Plasma-derived biological medicines used to promote haemostasis.

Several biological medicines derived from human and animal plasmas can effectively improve haemostasis in individuals with inherited or acquired defects in haemostasis. Factor VIII and factor VIII/vWF and factor IX concentrates are used to treat haemophilia A, von Willebrand disease and hemophilia B respectively. Cryoprecipitates are used to treat hypofibrinogenemia and von Willebrand disease where desmopressin (DDAVP) is ineffective or when plasma-derived factor VIII/vWF concentrates are unavailable. Thrombin-containing topical haemostatic agents and fibrin sealants are used to control perioperative bleeding. Intravenous immunoglobulin has several uses, including management of patients with autoimmune thrombocytopenias and patients with acquired factor VIII deficiency. Similar to most protein-based biological medicines, all the above products can elicit some level of antibody response, with clinical consequences that vary from mild anaphylaxis to loss of product efficacy. An ongoing potential safety concern with any biological medicine derived from blood/plasma is transmission of blood-borne pathogens. This safety concern has lessened significantly in the past decade as a result of the institution of more effective pre- and post-donation screening that tests for potential pathogens, and institution of pathogen reduction strategies to which many plasma-derived biological medicines are now routinely subjected. This article considers the manufacture, standardization, clinical efficacy and adverse event profiles of the plasma-derived biological medicines currently used to promote haemostasis in patients with inherited or acquired functional defects in haemostasis. It also considers approaches employed to minimize infectivity of biological medicines derived from human and animal plasmas and to manage patients who develop antibodies (inhibitors) to clotting factor concentrate infusions.

Factors that contribute to the immmunogenicity of therapeutic recombinant human proteins.

Use of recombinant human proteins has revolutionized medicine by providing over 200 highly purified hormones and proteins that effectively treat many inherited and acquired peptide hormone and protein deficiencies. With the exception of therapeutic monoclonal antibodies, these biological medicines are synthesized by cultured cells using DNA sequences that would yield proteins with identical amino acid sequences as endogenous human proteins. Therefore, there was the broad expectation that recombinant human biological medicines would be non-immunogenic in patients capable of synthesizing even sub-optimal levels of these therapeutic proteins to which they are innately tolerant. However, the widespread clinical use of recombinant human proteins has demonstrated that nearly all of them are immunogenic. This observation suggests that factors additional to differences in amino acid sequences of endogenous and biotherapeutic proteins contribute to the immunogenicity of therapeutic proteins. The main aim of this review is to summarize some of the factors that are known to contribute to the immunogenicity of recombinant therapeutic proteins.

Encapsulated human primary myoblasts deliver functional hFIX in hemophilic mice.

BACKGROUND: Hemophilia B is a bleeding disorder caused by defective factor IX (FIX), currently treated by regular infusions of plasma-derived or recombinant FIX. We propose a gene therapy strategy based on the implantation of cells secreting FIX enclosed in alginate microcapsules as a highly desirable alternative treatment. We have reported sustained delivery of human factor IX (hFIX) in immunocompetent mice implanted with encapsulated primary mouse myoblasts engineered to secrete hFIX. As a step towards the treatment of human patients, in this study we report the implantation of encapsulated human primary myoblasts secreting hFIX in hemophilia B mice. METHODS: Human primary myoblasts were transfected with plasmids pKL4M-hFIX, pLNM-betaIXL, pMFG-hFIX, and transduced with retrovirus MFG-hFIX. Two human primary myoblast clones secreting approximately 1 microg hFIX/10(6) cells/day were enclosed in biocompatible alginate microcapsules and implanted intraperitoneally into SCID and hemophilic mice. RESULTS: Circulating hFIX (peak of approximately 120 ng/ml) was detected in hemophilia B mice on day 1 after implantation. Human FIX delivery was transient, however, becoming undetectable on day 14. Concurrently, anti-hFIX antibodies were detected. At the same time, activated partial thromboplastin time (APTT) was reduced from 94 s before treatment to 78-80 s. Tail bleeding time decreased from 15 min to 1.5-7 min after treatment, some mice being normalised. These findings indicate that the delivered hFIX is biologically active. Similarly treated NOD/SCID mice had circulating hFIX levels of 170 ng/ml on day 1 that remained detectable for 1 month, albeit at low levels. Cell viability of microcapsules retrieved on day 60 was below 5%. CONCLUSIONS: Our findings indicate that encapsulated human primary myoblasts secrete functional hFIX. Furthermore, implantation of encapsulated human primary myoblasts can partially correct the phenotype of hemophilia B mice, supporting the feasibility of this gene therapy approach for hemophilia B. However, the long-term viability of the encapsulated human myoblasts must first be improved.

Review: Laboratory markers quantifying prothrombin activation and actions of thrombin in venous and arterial thrombosis do not accurately assess disease severity or the effectiveness of treatment.

Thrombin is normally produced for hemostasis and physiological wound healing. Increased thrombin production in vivo, cell activation and inflammation mediated in part by thrombin are hallmarks of both arterial and venous thrombosis. Thrombin generates (pro) coagulant, mitogenic, inflammatory and anticoagulant responses by interacting with a variety of cells in vivo. Both direct and indirect thrombin inhibitors are effective drugs for preventing and treating the consequences of arterial and venous thrombosis. For these reasons, measurements of the production and activities of thrombin in vivo have the potential for gauging the extent of thromboembolism and the responses of patients to anticoagulant, antiplatelet and anti-inflammatory drugs. However, a critical review of published information suggests that measurement of thrombin production and activity in patients at risk for and in patients with significant thrombosis generally does not provide information useful for clinical decision-making. This lack of clinical utility of levels of thrombin production in vivo may arise from two causes: the inability of the measurement to differentiate between physiological (hemostatic) and disease-related (pathological) sources and/or causes of thrombin productio n in vivo, and the inability of antithrombotic treatment modalities to permanently eliminate the stimuli that cause increased thrombin production evident in venous and arterial thrombosis.

Sustained and therapeutic levels of human factor IX in hemophilia B mice implanted with microcapsules: key role of encapsulated cells.

BACKGROUND: A gene therapy delivery system based on microcapsules enclosing recombinant cells engineered to secrete a therapeutic protein was explored in this study. In order to prevent immune rejection of the delivered cells, they were enclosed in non-antigenic biocompatible alginate microcapsules prior to being implanted intraperitoneally into mice. We have shown that encapsulated C2C12 myoblasts can temporarily deliver therapeutic levels of factor IX (FIX) in mice, but the C2C12 myoblasts elicited an immune response to FIX. In this study we report the use of mouse fetal G8 myoblasts secreting hFIX in hemophilia mice. METHODS: Mouse G8 myoblasts were transduced with MFG-FIX vector. A pool of recombinant G8 myoblasts secreting approximately 1500 ng hFIX/10(6) cells/24 h in vitro were enclosed in biocompatible alginate microcapsules and implanted intraperitoneally into immunocompetent C57BL/6 and hemophilic mice. RESULTS: Circulating levels of hFIX in treated mice reached approximately 400 ng/ml for at least 120 days (end of experiment). Interestingly, mice treated with encapsulated G8 myoblasts did not develop anti-hFIX antibodies. Activated partial thromboplastin time (APTT) of plasmas obtained from treated hemophilic mice was reduced from 107 to 82 sec on day 60 post-treatment, and whole blood clotting time (WBCT) was also corrected from 7-9 min before treatment to 3-5 min following microcapsule implantation. Further, mice were protected against bleeding following major trauma. Thus, the FIX delivery in vivo was biologically active. CONCLUSIONS: Our findings suggest that the type of cells encapsulated play a key role in the generation of immune responses against the transgene. Further, a judicious selection of encapsulated cells is critical for achieving sustained gene expression. Our findings support the feasibility of encapsulated G8 myoblasts as a gene therapy approach for hemophilia B.