Mapping the binding site of C1-inhibitor for polyanion cofactors.

The serpin, C1-inhibitor (also known as SERPING1), plays a vital anti-inflammatory role in the body by controlling pro-inflammatory pathways such as complement and coagulation. The inhibitor's action is enhanced in the presence of polyanionic cofactors, such as heparin and polyphosphate, by increasing the rate of association with key enzymes such as C1s of the classical pathway of complement. The cofactor binding site of the serpin has never been mapped. Here we show that residues Lys284, Lys285 and Arg287 of C1-inhibitor play key roles in binding heparin and delivering the rate enhancement seen in the presence of polyanions and thus most likely represent the key cofactor binding residues for the serpin. We also show that simultaneous binding of the anion binding site of C1s by the polyanion is required to deliver the rate enhancement. Finally, we have shown that it is unlikely that the two positively charged zones of C1-inhibitor and C1s interact in the encounter complex between molecules as ablation of the charged zones did not in itself deliver a rate enhancement as might have been expected if the zones interacted. These insights provide crucial information as to the mechanism of action of this key serpin in the presence and absence of cofactor molecules.

Manufacturing of plasma-derived C1-inhibitor concentrate for treatment of patients with hereditary angioedema.

Background: Replacement therapy with plasma-derived C1-inhibitor (C1-INH) has been used for decades to treat patients with hereditary angioedema (HAE) with C1-INH deficiency. Objective: This article reviewed the rationale for using C1-INH replacement therapy in patients with HAE and the process of manufacturing plasma-derived C1-INH. Methods: The manufacture of C1-INH is an involved and carefully monitored process that includes screening and selection of prospective donors, the collection of source plasma, and purification with dedicated pathogen reduction steps. Donor eligibility is determined by restrictive criteria established and monitored by regulatory agencies as well as voluntary standards implemented by plasma collection centers that exceed government regulations. Individual and pooled donations are tested for transfusion-transmissible infections, including hepatitis B virus, hepatitis C virus, human immunodeficiency virus, parvovirus B19, and hepatitis A virus, by using enzyme-linked immunosorbent assays or nucleic acid amplification technologies. Frozen plasma that is cleared for manufacturing undergoes controlled thawing and centrifugation, and the resulting supernatant (i.e., cryoprecipitate-depleted plasma) is used to manufacture several plasma-derived therapies, including C1-INH. In addition to chromatography steps, the manufacturing process consists of dedicated and effective pathogen reduction steps, including pasteurization, hydrophobic interaction chromatography or polyethylene glycol precipitation, and virus filtration. Manufacturers continuously monitor the safety profile of C1-INH products by robust pharmacovigilance processes that enable systematic collection and evaluation of all suspected adverse drug reaction reports as well as evaluation of safety information from all other sources. Results and Conclusion: These procedures used in donor screening, donation and manufacturing pool testing, manufacturing, and pharmacovigilance ensure that plasma-derived C1-INH products have the safety, quality, identity, potency, and purity that is necessary to provide the intended therapeutic effect.

Costs and effects of on-demand treatment of hereditary angioedema in Italy: a prospective cohort study of 167 patients.

OBJECTIVES: To explore treatment behaviours in a cohort of Italian patients with hereditary angioedema due to complement C1-inhibitor deficiency (C1-INH-HAE), and to estimate how effects and costs of treating attacks in routine practice differed across available on-demand treatments. DESIGN: Cost analyses and survival analyses using attack-level data collected prospectively for 1 year. SETTING: National reference centre for C1-INH-HAE. PARTICIPANTS: 167 patients with proved diagnosis of C1-INH-HAE, who reported data on angioedema attacks, including severity, localisation and duration, treatment received, and use of other healthcare services. INTERVENTIONS: Attacks were treated with either icatibant, plasma-derived C1-INH (pdC1-INH) or just supportive care. MAIN OUTCOME MEASURES: Treatment efficacy in reducing attack duration and the direct costs of acute attacks. RESULTS: Overall, 133 of 167 patients (79.6%) reported 1508 attacks during the study period, with mean incidence of 11 attacks per patient per year. Only 78.9% of attacks were treated in contrast to current guidelines. Both icatibant and pdC1-INH significantly reduced attack duration compared with no treatment (median times from onset 7, 10 and 47 hours, respectively), but remission rates with icatibant were 31% faster compared with pdC1-INH (HR 1.31, 95% CI 1.14 to 1.51). However, observed treatment behaviours suggest patterns of suboptimal dosing for pdC1-INH. The average cost per attack was €1183 (SD €789) resulting in €1.58 million healthcare costs during the observation period (€11 912 per patient per year). Icatibant was 54% more expensive than pdC1-INH, whereas age, sex and prophylactic treatment were not associated to higher or lower costs. CONCLUSIONS: Both icatibant and pdC1-INH significantly reduced attack duration compared with no treatment, however, icatibant was more effective but also more expensive. Treatment behaviours and suboptimal dosing of pdC1-INH may account for the differences, but further research is needed to define their role.

Plasma-derived C1-INH for managing hereditary angioedema in pediatric patients: A systematic review.

Presently, medications approved for children with Hereditary Angioedema (HAE) are extremely limited. This is especially the case for children under 12 years of age. For this reason we reviewed and summarized the data on treatment of children with HAE. Available data indicate that plasma derived C1-inhibitor is a safe, effective treatment option for HAE in pediatric patients, including those below 12 years of age. Other therapies are also appear safe for the under 12 year of age, but less data are available. Importantly, home-based treatment of HAE in this age group appears to be safe and effective and can improve quality of life. These findings support current HAE consensus guidelines which strongly recommend the use of plasma derived C1-inhibitor as a first-line treatment in children and encourage home and self-treatment.

Pathogen safety of human C1 esterase inhibitor concentrate.

BACKGROUND: Human plasma-derived products--such as C1 esterase inhibitor (C1-INH) concentrate, used to treat hereditary angioedema--carry with them the risk of transmitting blood-borne viruses and, theoretically, prion proteins. To minimize this risk, three complementary approaches are implemented: selection and testing of plasma donations for the absence of pathogenic blood-borne viruses, similarly testing and releasing the plasma pool for fractionation, and ensuring that the manufacturing process includes validated steps for pathogen inactivation and removal. STUDY DESIGN AND METHODS: This article describes the selection of plasma for the production of C1-INH and the studies used to confirm the pathogen reduction capacity of the manufacturing process: three independent virus reduction steps--pasteurization, hydrophobic interaction chromatography (HIC), and virus filtration--and two prion reduction steps. Samples of product intermediates from the manufacturing steps were spiked with a panel of enveloped and nonenveloped viruses and two prion preparations and subjected to a valid scaled-down version of the respective manufacturing steps resulting in the quantification of the pathogen reduction factors. RESULTS: Validation studies demonstrated overall virus reduction factors for all viruses of more than 15 log, considerably exceeding the potential amount of virus present in a plasma pool for fractionation. Prion proteins were also efficiently removed by the manufacturing process, as currently determined in evaluating the prion removal capacity of the ammonium sulfate precipitation and HIC steps. CONCLUSION: The pathogen reduction capacity demonstrated here indicates that the manufacturing process of the C1-INH Berinert is highly effective for reducing enveloped and nonenveloped viruses and prion proteins.

Micrurus snake venoms activate human complement system and generate anaphylatoxins.

BACKGROUND: The genus Micrurus, coral snakes (Serpentes, Elapidae), comprises more than 120 species and subspecies distributed from the south United States to the south of South America. Micrurus snake bites can cause death by muscle paralysis and further respiratory arrest within a few hours after envenomation. Clinical observations show mainly neurotoxic symptoms, although other biological activities have also been experimentally observed, including cardiotoxicity, hemolysis, edema and myotoxicity. RESULTS: In the present study we have investigated the action of venoms from seven species of snakes from the genus Micrurus on the complement system in in vitro studies. Several of the Micrurus species could consume the classical and/or the lectin pathways, but not the alternative pathway, and C3a, C4a and C5a were generated in sera treated with the venoms as result of this complement activation. Micrurus venoms were also able to directly cleave the α chain of the component C3, but not of the C4, which was inhibited by 1,10 Phenanthroline, suggesting the presence of a C3α chain specific metalloprotease in Micrurus spp venoms. Furthermore, complement activation was in part associated with the cleavage of C1-Inhibitor by protease(s) present in the venoms, which disrupts complement activation control. CONCLUSION: Micrurus venoms can activate the complement system, generating a significant amount of anaphylatoxins, which may assist due to their vasodilatory effects, to enhance the spreading of other venom components during the envenomation process.

[Safety and efficacy of purified human C1-esterase inhibitor in the treatment of patients with sepsis].

One of the new experimental trends in pharmacotherapy of sepsis is the use of C1-esterase inhibitor (C1I) from the group of immunobiological agents influencing the complement system. An open prospective study on the safety and efficacy of C1I revealed its positive effect on the death rate among the patients with sepsis. In a dose of 12000 IU, C1I had a significant inhibitory action on the complement system activity, as well as an antiinflammatory effect by blocking the complement-dependent link in the systemic inflammation. The adverse and significant adverse events were not associated with the use of C1I.

Hereditary angioedema: a current state-of-the-art review, VIII: current status of emerging therapies.

OBJECTIVE: To provide an overview on the current status of emerging therapies for hereditary angioedema (HAE) in the United States. DATA SOURCES: Summary statements were obtained from each pharmaceutical company regarding their agent. STUDY SELECTION: Each agent is undergoing or has completed phase 3, double-blind, placebo-controlled trials. RESULTS: Berinert P, a purified, virus-inactivated, human plasma-derived C1 inhibitor (C1-INH) concentrate, is being investigated in 2 international, multicenter, prospective trials. Experience with this agent in Europe and Canada indicates it is effective and safe. Cinryze is a nanofiltered C1-INH replacement therapy demonstrated to be effective and safe in acute and prophylactic arms of a phase 3, double-blind, placebo-controlled study. Rhucin, a recombinant human C1-INH replacement therapy from transgenic rabbits, has been shown to be effective and safe in phase 2 and phase 2/3 studies, with an additional phase 3 study ongoing. DX-88 or ecallantide, a potent and specific inhibitor of plasma kallikrein, achieved all primary and secondary efficacy end points in a placebo-controlled, double-blind, phase 3 study, with a second phase 3 study ongoing. Icatibant, a potent and specific peptidomimetic bradykinin 2 receptor antagonist, was studied in 2 phase 3 trials: FAST 1 (For Angioedema Subcutaneous Treatment) did not achieve statistical significance for the primary end point but did so for secondary end points, whereas FAST 2 achieved statistical significance for primary and secondary end points. CONCLUSIONS: The future treatment of HAE in the United States appears promising based on progress being made in drug development for this orphan disease.

Self-administration of C1-inhibitor concentrate in patients with hereditary or acquired angioedema caused by C1-inhibitor deficiency.

BACKGROUND: Administration of C1-inhibitor concentrate is effective for prophylaxis and treatment of severe angioedema attacks caused by C1-inhibitor deficiency. The concentrate should be administered intravenously and hence needs to be administered by health care professionals, which might cause considerable delay in treatment and inconvenience for patients. OBJECTIVE: The aim of this study was to investigate the feasibility, efficacy, and safety of on-demand and prophylactic self-administration of C1-inhibitor concentrate in patients with frequent attacks of angioedema. METHODS: Patients with hereditary or acquired C1-inhibitor deficiency who had very frequent angioedema attacks were trained to self-administer C1-inhibitor concentrate. The study consisted of 31 patients using on-demand treatment and 12 patients using prophylaxis with C1-inhibitor concentrate. Mean follow-up was 3.5 years. RESULTS: All patients were capable of self-administering the concentrate, with technical failure rates of self-injection being less than 2%. Times between the onset of the attack and the initiation of relief or complete resolution of symptoms in the on-demand group were significantly shortened (2.2 hours and 7.9 hours, respectively) compared with the situation before the start of self-administration. In the prophylaxis group self-administration of C1-inhibitor concentrate decreased the angioedema attack rate from 4.0 to 0.3 attacks per month. CONCLUSION: Intravenous self-administration of C1-inhibitor concentrate is a feasible and safe option and results in more rapid and more effective treatment or prevention of severe angioedema attacks in patients with C1-inhibitor deficiency. CLINICAL IMPLICATIONS: Self-administration of C1-inhibitor concentrate could be a valuable and convenient treatment modality to prevent or treat angioedema attacks in patients with C1-inhibitor deficiency.