Recombinant human C1-inhibitor (Ruconest®) has been granted market authorisation in Europe for the treatment of acute angioedema attacks in patients with hereditary angioedema due to C1-inhibitor deficiency. Recombinant human C1-inhibitor is made from the milk of transgenic rabbits and is an alternative to C1-inhibitor products manufactured from human blood. Here we review the background to the therapeutic use of recombinant C1-inhibitor in hereditary angioedema, and summarise mechanism of action, manufacturing process, pharmacology, pharmacokinetics, and clinical efficacy and safety, as well as cost effectiveness.
C Erik Hack MD PhD
Department of Immunology, Dermatology/Allergology and Rheumatology, University Medical Center Utrecht, The Netherlands
Key words: Hereditary angioedema; recombinant C1-inhibitor; acute attack; efficacy; safety
Declaration of interest
The author has received consultancy fees from Pharming Technologies.
C1-inhibitor (C1INH) is a blood protein that belongs to the superfamily of the so-called serpins (or serine protease inhibitors).(1) Since the 1960s, it has been recognised that a deficiency of C1INH leads to the clinical disease hereditary angioedema (HAE).(2) Recombinant human C1INH (rhC1INH; Ruconest®; Pharming Technologies, Leiden, The Netherlands; marketed by Swedish Orphan Biovitrum, Stockholm, Sweden) has been developed for the treatment of HAE.
HAE results from a genetic deficiency of the blood protein C1INH and has an estimated prevalence of 1 in 50,000.(3,4) The deficiency is heterozygous. Thus, patients with HAE have one normal and one mutated C1INH gene, and synthesise normal endogenous C1INH, albeit at lower levels than healthy individuals. The disease becomes clinically manifest in childhood or adolescence as episodic, recurrent acute attacks of soft tissue swelling, which can either occur without any known trigger or be precipitated by physical trauma or medications, notably angiotensin-converting enzyme (ACE) inhibitors.(4,5) Attacks can occur in the submucosal tissues of the gastrointestinal tract (abdominal attacks), the larynx (laryngeal attacks), the oropharynx and of the urogenital region, or in the subcutaneous tissues of the face, trunk, arms or legs. Attacks may occur at a single or multiple locations. For example, we reported multiple locations in more than 50% of HAE patients with a severe peripheral attack.(6)
The frequency of acute angioedema attacks across HAE patients varies widely, from more than one attack per week to fewer than one attack per year, and may also vary in individual patients over time. The frequency of attacks is not linked to any genetic or biochemical parameter. The clinical severity of acute attacks is also variable. It is generally accepted that abdominal attacks need immediate treatment because they are extremely painful,(7) as do attacks in the upper airways because of the risk of asphyxiation.(8) A recent study shows that the medical need for treatment of attacks in extremities is generally underestimated, as many patients with these attacks have severe pain and dysfunction of the involved extremity.(6)
C1INH is the major inhibitor of several complement proteases and contact-system proteases. Low functional C1INH levels lead to poor control of either pathway, which become more readily activated. This results in the episodic generation of vasoactive peptides such as bradykinin, which, by enhancing vasopermeability, mediate angioedema.(4,5)
Without treatment, HAE attacks worsen over the first 24 hours to subside over the next 48–72 hours.(4,5) With the rationale that restoration of homeostatic control of contact and complement systems might be a good approach to treat and prevent angioedema attacks in HAE patients as a cause for HAE, various C1INH products manufactured from pooled human plasma –plasma-derived C1INH (pdC1INH) – have been developed as a treatment of HAE patients with acute attacks. Two of these products are currently approved for the treatment of acute attacks of HAE.(9,10) In addition, a recombinant inhibitor of kallikrein, a key enzyme of the contact system called ecallantide, produced in Pichia pastoris, and a peptide antagonist of the bradykinin-receptor 2, icatibant, have been developed as a therapy for symptomatic HAE.(11,12)
Recombinant human C1-inhibitor (rhC1INH; International nonproprietary name: conestat alfa; marketed as Ruconest®) is the recombinant analogue of human C1INH, and is manufactured from the milk of rabbits expressing the gene encoding human C1INH. A major advantage of the transgenic platform is a guaranteed continued supply of rhC1INH, whereas pdC1INH products for manufacture are dependent upon the availability of human plasma. In addition, rhC1INH is devoid of risk for transmission of human blood-borne pathogenic viruses and prions. Thus, rhC1INH offers a safer, more widely available therapeutic alternative to the existing pdC1INH products used as a replacement therapy for acute HAE attacks. RhC1INH is the topic of this drug evaluation.
Pharmacology, PK and PD
C1INH is synthesised in the liver as a single-chain plasma glycoprotein of 478 amino acids. Its molecular weight is approximately 78 kDa. C1INH has six N-linked and at least seven O-linked glycosylation sites; 28–30% of its molecular weight consists of carbohydrates. These carbohydrates are not necessary for its inhibitory activity. C1INH is a serpin and shares structural homology and a common inhibitory mechanism with other serpins, such as α1-antitrypsin, antithrombin III, α1-antichymotrypsin and α2-antiplasmin.1 C1INH is the only known inhibitor of activated C1s and C1r of the classical pathway of complement and of the mannan-binding lectin-associated serine proteases of the lectin pathway of complement, and the major inhibitor of activated factor XII, factor XI and kallikrein of the contact system of intrinsic coagulation. These proteases are designated target proteases for C1INH. Inhibition of target proteases by a serpin is achieved by proteolytical cleavage of a substrate-like peptide bond, designated P1–P1’, in the reactive centre of the inhibitor. Subsequently, covalent complexes between protease and C1INH are formed, in which the active site serine amino acid residue of the protease blocked by covalent linkage to the P1-residue of the inhibitor. These complexes are cleared from the circulation by the liver with an apparent half-life of clearance of 20–40 minutes. The kinetics of the interaction of rhC1INH with several target proteases has been studied in detail by the Pharming Group. Inhibition constants assessed for inhibition of activated factor XIIa, kallikrein, activated factor XIa and activated C1s by rhC1INH were found to be identical with those of pdC1INH.(13)
Levels of functional C1INH in plasma as well as doses of C1INH are often expressed as Units (U) per ml and U/kg, respectively. One U of C1INH, recombinant or plasma-derived, is equivalent to the amount of functional C1INH in 1ml of pooled plasma from healthy human donors. Normal levels of C1INH range from 0.7 to 1.3U/ml.
The pharmacokinetics of rhC1INH was initially studied in asymptomatic HAE patients who received two doses of 6.25–100U/kg body weight administered at a minimum interval of five weeks.(14) Asymptomatic HAE patients have low levels – often <0.2U/ml – of functional C1INH, and so the course of infused levels in time can be easily measured. Infusion of escalating doses of rhC1INH yielded dose-dependent increases in functional C1INH activity.
C1INH activity levels in the patients. Restoration of functional C1INH into the normal range (0.7U–1.3U/ml) was observed when 50 or 100U/kg rhC1INH was administered. Compared with pdC1INH, which has a half-life of at least 24 hours in humans, rhC1INH had a shorter half-life, which dose-proportionally increased from 28 minutes after infusion of 6.25 U/kg to 172 minutes following administration of 100U/kg of body weight.(14) These data suggested elimination of rhC1INH from the circulation via a saturable elimination mechanism. In preclinical studies, clearance of rhC1INH was prolonged by specific inhibitors of the asialoglycoprotein and the mannose receptors in the liver,(13) suggesting that the shorter half-life of rhC1INH results from incomplete capping of glycans with sialic acid residues and to the presence of oligomannose structures.(15) A pharmacodynamic effect was also observed in the asymptomatic HAE patients, as rhC1INH dose-dependently reduced the increased levels of activated C4, a complement protein.(14) Notably, normalisation of activated C4 was only achieved when C1INH activity levels were restored in the normal range.
Pharmacokinetics of rhC1INH was also measured in healthy volunteers, who received five administrations of rhC1INH at 100U/kg, once every three weeks. The volume of distribution was approximately 2.8l and elimination half-life was approximately two hours. No difference in pharmacokinetic profiles was observed following the first, third and fifth administrations of rhC1INH, suggesting neutralising antibodies against rhC1INH were not formed upon repeat administration.(13) Similar pharmacokinetic profiles were found in symptomatic HAE patients who received 100U/kg as a treatment of an acute angioedema attack.(16) Pharmacokinetic findings in these symptomatic patients were in line with those of the previous studies. In addition, a pharmacodynamic effect on C4 levels, which increased in the patients upon treatment with rhC1INH, was observed. The pharmacokinetic findings were consistent across the studies and rhC1INH distribution and elimination parameters were similar for healthy volunteers and for symptomatic and asymptomatic HAE patients.
Dosing and administration
Ruconest® is produced as a powder that is made up into a solution for injection. Once reconstituted in 14ml water for injections, it has a concentration of 150U/ml. The excipients used in the formulation are sodium citrate, sucrose and citric acid.13 Ruconest® must be injected intravenously. The marketing authorisation in Europe is for the treatment of an acute angioedema attack in HAE patients. The approved dose is 50U/kg for subjects below 84kg, and two vials of 2100U for subjects above 84kg. Subjects with a known rabbit allergy should not receive the drug, and it is recommended to test subjects for the presence of IgE against rabbit dander before exposure to Ruconest®. In the event of a positive test result, the subject should not receive the drug. Ruconest® has not been evaluated in children under 12 years of age.
Exploratory efficacy studies were conducted in 14 symptomatic HAE patients, who were treated with single doses of rhC1INH at 100U/kg per attack for a total of 21 acute angioedema attacks. A diagnosis of HAE was based on low levels of functional C1INH (<50% of normal) as well as low C4 levels, and absence of C1INH auto-antibodies (to exclude acquired angioedema). Patients were eligible for treatment when they presented at a clinical centre with a severe attack within eight hours (explorative studies) or five hours (randomised controlled and open label extension studies) after onset of symptoms. The dose selected for treatment was based on the pharmacokinetic data of the study in the asymptomatic patients and on the assumption that, for effective treatment of an acute angioedema attack, restoration of functional C1INH levels into the normal range was required. An interim report on nine patients who received 13 treatments with rhC1INH for an acute attack has been published.16 Main findings were: (i) the median time to onset of symptom relief, as measured with a visual analogue scale (VAS), was 30 minutes; (ii) the response rate – the percentage of patients who experience onset of symptom relief within four hours – was >90% for all attacks; and (iii) median time to minimal symptoms was eight hours. These findings suggested that rhC1INH treatment shortened acute attack symptoms in the patients.
Two randomised, placebo-controlled, double-blind studies, one in Europe and one in North America, in 70 HAE patients suffering from an acute angioedema attack confirmed the efficacy data suggested by the explorative studies. The pooled data of both studies revealed that 29 patients treated with 100U/kg rhC1INH and 12 patients with 50U/kg had significantly shorter times to onset of relief of symptoms and to minimal symptoms than 29 patients who had received saline.(17) Response rates were 93%, 100% and 41% in the group treated with 100U/kg, 50U/kg or saline, respectively. No relapses of attacks were observed. Thus, rhC1INH at 100U/kg was highly effective as a treatment of acute attacks in HAE patients. Moreover, 50U/kg was as effective as 100U/kg. In the open-label extension phase of the American study, patients were treated with either 50U/kg or 100U/kg (decision made by the clinician) whereas, in the European extension phase, a vial-based dosing was used to meet clinical practice preference. Most patients were treated with a single vial of 2100U rhC1INH but one or two additional doses could be given within four hours, at the discretion of the clinical investigator. Efficacy was maintained across these various dosing regimens, although response rate – the proportion of patients who achieve onset of relieve within four hours – was somewhat lower in the patients who received 2100U, in line with the pharmacokinetic simulation that, in approximately 25% of the patients receiving 2100U, C1INH activity level is restored in the normal range.
Moreover, rhC1INH was efficacious in all types of angioedema attack, including upper airway and abdominal attacks. Efficacy was also maintained in patients with repeat attacks. A detailed analysis of patients with peripheral attacks revealed that rhC1INH shortened times to onset of relief as well as to minimal symptoms, not only by overall severity VAS for the attack, but also when individual symptoms such as swelling, dysfunction or pain were considered.(6)
Immunogenicity is a safety concern of any therapeutic protein. HAE results from a heterozygous deficiency of C1INH and therefore HAE patients have a small amount of normal endogenous C1INH in the circulation. This amount is sufficient to induce immunological tolerance to rhC1INH. Antibody responses to exogenous pdC1INH products are expected to be rare in HAE patients. To evaluate the immunogenicity of rhC1INH, blood samples were collected from all subjects before and after exposure to rhC1INH and tested for IgM, IgG and IgA antibodies against C1INH or against host-related impurities (HRI) (rabbit proteins). Analysis of data from 155 HAE patients who received 424 treatments with rhC1INH revealed that the proportion of samples with positive anti-C1INH antibody results were similar before (1.6%) and after (1.3%) exposure to rhC1INH.(18) In six patients, the presence of anti-rhC1INH antibodies were confirmed; in two patients, these antibodies were pre-existing with no increase post-exposure; in three patients, antibodies were detected on a single occasion post exposure; and in only one patient, a treatment-emergent anti-C1INH response was observed with antibodies against C1INH occurred on subsequent occasions post-exposure. Neutralising anti-C1INH antibodies were not found in any of the patients. The proportion of plasma samples with positive anti-HRI antibodies was slightly higher after exposure than before (0.7% of the samples before exposure to rhC1INH, versus 1.9% of the samples after first exposure, and 3.1% after repeat treatment with rhC1INH).(18) Anti-HRI antibodies were confirmed in five patients, of whom one had pre-existing anti-HRI. In three of the 155 rhC1INH-treated patients, confirmed anti-HRI antibodies were present at additional time points. Anti-HRI findings did not correlate with clinical adverse events.
In the clinical programme of rhC1INH, one serious adverse event related to the administration of rhC1INH occurred. This event was an anaphylactic reaction upon first exposure to rhC1INH in a healthy control. No other anaphylactic reactions occurred in any of the subjects exposed to rhC1INH. Moreover, induction of IgE was not observed following administration of rhC1INH. All antibody data considered, rhC1INH has a reassuring immunogenicity profile. Patients should be asked of any pre-existing rabbit allergy and, in case of doubt, measurement of IgE levels against rabbit dander might be considered.
The viral safety of rhC1INH is ensured by control measures, including housing the rabbits under specific pathogen-free conditions, testing the milk and the drug substance for adventitious agents, and the inactivation and removal of known and unknown viruses through solvent/detergent treatment and nanofiltration.(13) No transmission of viruses or other pathogens by rhC1INH product has been reported to date.
C1INH can inhibit, to some extent, serine proteases such as plasmin and tissue-type plasminogen activator of the fibrinolytic system. Hence, an interaction of rhC1INH with tPA can be expected. It is therefore recommended that rhC1INH and tPA are not used together.
Administration of high doses of plasma-derived C1INH has resulted in thromboembolic side effects.(19) In the clinical programme of rhC1INH, no thromboembolic events have been observed, except for a patient who experienced an acute myocardial infarction more than 70 days following treatment, but which was not estimated to be related to rhC1INH treatment. Furthermore, an evaluation of coagulation and fibrinolytic parameters in patients receiving rhC1INH at doses of 100U/kg and 50U/kg revealed mild inhibition of coagulation and no effect on fibrinolysis.(20) rhC1INH at doses of up to 100U/kg seems to be devoid of thrombogenic potential.
The majority of treatment-emergent adverse events (TEAEs) encountered in the clinical programme with rhC1INH were mild-to-moderate in severity, and no TEAEs led to withdrawal from the study. The overall incidence of TEAEs was lower in rhC1INH treatment groups as compared with the placebo group. The most frequent TEAE was headache. Only one serious adverse event directly related to treatment – the anaphylactic reaction described earlier – occurred in the clinical programme.(13)
It is generally accepted that HAE patients with acute angioedema attacks located in the abdomen or laryngeal–pharyngeal region need treatment with C1INH products, because the former can cause extreme pain and the latter can be lethal owing to asphyxiation. However, attacks in the extremities also can be painful and cause severe dysfunction, such as the inability to walk when the attack is located in the foot or to write when located in the hand. In addition, an attack located in the face can have a psychological impact on patients. Consequently, patients can be absent from work even in the case of an attack that is not considered to be clinically serious. The Hereditary Angioedema International Working Group recommends that all angioedema attacks irrespective of location are eligible for treatment. Appropriate treatment would give onset of relief of symptoms within four hours for up to 95% of the patients and at least 50% of the patients will have minimal symptoms, if any, within four-to-six hours, whereas, without treatment, this may be 24–72 hours or even longer. Therefore, treatment will shorten (if not abrogate) absence from work.
Several C1INH products, plasma‑derived and recombinant, and the bradykinin-2 receptor antagonist, icatibant, are available for the treatment of acute angioedema attacks in HAE. These products have not been compared with each other regarding efficacy. Therefore, a direct cost-effectiveness comparison of these treatment options is not possible. Comparison of randomised, controlled study results of the C1INH products suggests that efficacy is optimal when C1INH activity is restored into the normal range, independent of half-life of the C1INH administered.(21) In other words, it is the Units that matter and not the half-life. Therefore, C1INH products that are the cheapest by Units seem to be the best choice from a pharmacoeconomic point of view, moreso because none of the marketed products have significant safety issues. Sales prices of the various C1INH products as well as of icatibant may vary from country to country, and information on this is difficult to retrieve. However, per Unit, on average, recombinant C1INH would appear to cost approximately half that of the plasma-derived C1INH products. For example, in France one vial of Ruconest – containing 2100U – costs €840, whereas one vial of Berinert P – containing 500U pdC1INH – is €570. By comparison, Firazyr (30mg icatibant) costs €1715, and patients may need up to three injections per attack.
Other potential uses
RhC1INH has been licensed for the treatment of acute angioedema attacks in HAE. However, C1INH products have been evaluated for indications other than HAE in a number of preclinical and clinical studies.(22) The overall picture that emerges is that, in diseases where complement or contact system-mediated inflammatory reactions play a role, C1INH constitutes a therapeutic option. Doses to be administered for these alternative indications are 100U/kg or more. One group of indications is ischaemia-or reperfusion-related conditions such as acute myocardial infarction. In addition to a number of preclinical studies, three clinical trials have been performed with pd-C1INH in patients with acute myocardial infarction. The results suggest a cardioprotective effect of pdC1INH, although it not known whether such an effect can also be achieved with rhC1INH. RhC1INH has been evaluated in a stroke model in mice.(22) RhC1INH given dose-dependently reduced infarct size in this model, even when given as late as 18 hours after ischaemia. Moreover, rhC1INH was more potent in reducing infarct size than pdC1INH. RhC1INH has also been evaluated in a pig model of kidney ischaemia-reperfusion.(23) Treatment with 500U/kg largely prevented apoptosis induction of tubular cells. RhC1INH has also been studied in a baboon model of antibody-mediated kidney rejection.(24) Administration of 100 or 200U/kg resulted in a significant graft survival benefit. These promising results illustrate the potential of rhC1INH, but double-blind, controlled trials are needed to provide clinical proof of concept for these indications.
- rhC1INH constitutes an alternative to plasma-derived C1INH products without a risk of transmission of blood-borne pathogens.
- rhC1INH is efficacious for the treatment of the full spectrum of acute angioedema attacks in patients with HAE.
- For optimal efficacy in symptomatic HAE patients, rhC1INH should be dosed at 50U/kg
- rhC1INH should not be used in patients with known rabbit allergy or with elevated IgE against rabbit dander.
- rhC1INH does not induce clinically significant antibody responses.
- Gettins PG. Serpin structure, mechanism, and function. Chem Rev 2002;102(12):4751–804.
- Donaldson VH, Evans RR. A biochemical abnormality in hereditary angioneurotic edema. Am J Med 1963;35:37–44.
- Bygum A. Hereditary angioedema in Denmark: a nationwide survey. Br J Dermatol 2009;161:1153–8.
- *Zuraw BJ. Hereditary angioedema. N Engl J Med 2008;359:1027–36.
- Longhurst H, Cicardi M. Hereditary angioedema. Lancet 2012;379:474–81.
- Kusuma A et al. Clinical impact of peripheral attacks in hereditary angioedema patients. Am J Med 2012;125(9):937.e17–24.
- Bork K et al Treatment with C1 inhibitor concentrate in abdominal pain attacks of patients with hereditary angioedema. Transfusion 2005;45:1774–84.
- Bork K et al. Asphyxiation by laryngeal edema in patients with hereditary angioedema. Mayo Clin Proc 2000;75:349–54.
- Craig TJ et al. Efficacy of human C1 esterase inhibitor concentrate compared with placebo in acute hereditary angioedema attacks. J Allergy Clin Immunol 2009;124:801–8.
- Zuraw BL et al. Nanofiltered C1 inhibitor concentrate for treatment of hereditary angioedema. N Engl J Med 2010;363:513–22.
- Cicardi M et al. Ecallantide for the treatment of acute attacks in hereditary angioedema. N Engl J Med 2010;363:523–31.
- Cicardi M et al. Icatibant: a new bradykinin-receptor antagonist, in hereditary angioedema. N Engl J Med 2010;363:532–41.
- *European Medicines Agency. CHMP assessment report. www.ema.europa.eu.
- Van Doorn MBA et al. A phase I study of recombinant human C1 inhibitor in asymptomatic patients with hereditary angioedema. J Allergy Clin Immunol 2005;116:876–83.
- Koles K et al. N- and O-glycans of recombinant human C1 inhibitor expressed in the milk of transgenic rabbits. Glycobiology 2004;14:51–64.
- Choi G et al. Recombinant human C1-inhibitor in the treatment of acute angioedema attacks. Transfusion 2007;47:1028–32.
- *Zuraw B et al. Recombinant human C1-inhibitor for the treatment of acute angioedema attacks in patients with hereditary angioedema. J Allergy Clin Immunol 2010;126:821–7.
- Hack CE et al. Immunogenicity assessment of recombinant human C1-inhibitor: an integrated analysis of clinical studies. BioDrugs 2012;26(5):303–13.
- Arzneimittelkommission der deutschen Arzteschaft. Schwerwiegende Thrombenbildung nach Berinert HS. Deutsches Ärzteblatt 2000 Apr; 97 (Heft 15):A-1016.
- Relan A et al. Recombinant C1-inhibitor: effects on coagulation and fibrinolysis in patients with hereditary angioedema. BioDrugs 2012;26:43–52.
- Hack CE et al. Target levels of functional C1-inhibitor in hereditary angioedema. Allergy 2012;67:123–30.
- Caliezi C et al. C1-Esterase inhibitor: an anti-inflammatory agent and its potential use in the treatment of diseases other than hereditary angioedema. Pharmacol Rev 2000;52:91–112.
- Castellano G et al.Therapeutic targeting of classical and lectin pathways of complement protects from ischemia-reperfusion-induced renal damage. Am J Pathol 2010;176:1648–59.
- Tillou X et al. Recombinant human C1-inhibitor prevents acute antibody-mediated rejection in alloimmunized baboons. Kidney Int 2010;78:152–9.