C1-inhibitor deficiency and angioedema: molecular mechanisms and clinical progress

Review

C1-inhibitor deficiency and angioedema: molecular mechanisms and clinical progress Massimo Cugno1, Andrea Zanichelli2, Fabrizio Foieni2, Sonia Caccia3 and Marco Cicardi2 1

Department of Internal Medicine, University of Milan, IRCCS Fondazione Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Milan 20122, Italy 2 Department of Clinical Sciences, University of Milan, Ospedale Luigi Sacco, Milan 20157, Italy 3 Department of Biomedical and Technological Science, University of Milan, Milan 20090, Italy

C1 inhibitor (C1-INH, also known as SERPING1) can be deficient in plasma as a result of genetic or acquired conditions, and this causes an episodic, local increase in vascular permeability in the subcutaneous and submucosal layers, identified as angioedema (hereditary or acquired). Bradykinin, the mediator of the increase in vascular permeability, is released on inappropriate activation of the contact system, which is controlled by C1 inhibitor. Therapy aims to reverse or prevent angioedema. Advances in understanding the complex effects of C1-INH deficiency at the molecular level have led to new molecular-targeted approaches. Three new treatments, an inhibitor of kallikrein to prevent bradykinin release, an antagonist of the bradykinin receptor to prevent its action and a recombinant human C1-INH produced in transgenic animals, are under clinical evaluation currently. Here, we review the molecular mechanisms underlying angioedema due to C1-inhibitor deficiency and clinical progress using molecular-targeted interventions. C1 inhibitor: a central role in vascular homeostasis C1 inhibitor (C1-INH) is a serine protease inhibitor (serpin), also known as SERPING1 according to the revised nomenclature [1], that blocks the activity of C1r, C1s and mannosebinding lectin-associated serine protease (MASP)-1 and MASP-2 in the complement system, factor XII and kallikrein in the contact system, factor XI and thrombin in the coagulation system and tissue plasminogen activator (tPA) and plasmin in the fibrinolytic system [1]. Thus, C1-INH is important in controlling a range of processes involved in vascular homeostasis, including inflammation, blood pressure and coagulation. As a consequence, C1-INH might have a role in different pathologic processes, such as ischemia reperfusion injury and septic shock [2,3]. A strong association between age-related macular degeneration and genetic variations in C1-INH has been reported recently [4]. Nevertheless, the most straightforward involvement of C1-INH in disease is owing to its deficiency. Subjects with functional C1-INH levels in plasma that are lower than 50% of normal develop a local, self-limiting, reversible increase in vascular permeability in the deeper layers of the skin and/or Corresponding author: Cicardi, M. ([email protected]).

the gastrointestinal and laryngeal mucosa that represents angioedema. Because of the angioedema, these patients suffer from episodic disfiguration of the face and extremities, severe abdominal pain and asphyxia from glottis obstruction. Even if the cause of C1-INH deficiency is a genetic defect that remains stable throughout life (hereditary angioedema), the frequency and severity of angioedema recurrences is extremely variable from patient to patient and even in the same patient from time to time; the reason for this variability remains unknown. When untreated, the disease is highly disabling and might also be fatal. Here, we review the conditions leading to C1-INH deficiency, the mechanisms that cause angioedema when C1-INH is deficient and the new therapies that are being evaluated for this condition. C1-inhibitor deficiency in hereditary or acquired angioedema C1-INH deficiency can be genetic or acquired [5]. The genetic form is due to mutations in one of the two alleles of the C1-INH gene that result in reduced protein levels in plasma [hereditary angioedema (HAE), type I] or in normal protein levels but always in reduced function (HAE type II). C1-INH belongs, with a-1 antitrypsin (SERPINF1) and several other regulatory proteins, to the serpin family [1]. Being a serpin, C1-INH uses a unique inhibitory mechanism, which is illustrated in Figure 1 [6,7]. Interaction with the target protease results in cleavage of the C1-INH molecule itself followed by a profound conformational change. As a consequence, the covalently bound protease is translocated to the other side of the inhibitor molecule, resulting in enzyme inhibition by active site distortion [8]. The crystal structure of the serpin domain of human C1INH in latent form has been reported recently [9]. Hereditary angioedema Understanding C1-INH structure–function correlates explains how mutations causing single amino acid substitutions almost invariably result in loss of inhibitory activity and HAE. The mutated allele and C1-INH deficiency segregate into HAE families according to Mendelian law and nearly 200 different mutations causing HAE have been described

1471-4914/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.molmed.2008.12.001 Available online 21 January 2009

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Figure 1. Serpin inhibitory mechanism. Active serpin (pink) is characterized by a 5-stranded b-sheet (b-sheet A, cyan) and an exposed reactive centre loop (RCL, green) presenting the P1 residue to the active site of the target protease (gold) [6–8] (a). The initial step is the binding of free protease and active serpin RCL (b). Subsequently, the protease cleaves the RCL. RCL cleavage triggers a profound conformational change within the serpin molecule: the RCL downstream of the scissile bond inserts between strands 3 and 5 of b-sheet A as strand 4, while the protease is translocated to the opposite pole of the inhibitor molecule and deformed, resulting in enzyme inhibition by active-site distortion (c). The complex displays new molecular epitopes that promote cellular internalization and degradation. Therefore, protease-serpin acyl-enzyme complex formation can be viewed generally as biologically irreversible. (The coordinates used are from Protein Data Bank (http://www.rcsb.org/pdb/home/home.do) entry 1K1L [92] for free protease, 1OO8 [93] for active serpin, 1OPH [93] for Michaelis complex and 1EZY [94] for covalent complex.).

so far. Most of them are listed in a web-based database (HAEdb, http://hae.enzim.hu) [10] and, recently, we reported 67 new mutations as a result of C1-INH genotyping in 102 families of HAE patients [11]. No genotype or phenotype correlation has been found. Mutations that cause type I HAE occur throughout the gene and result in truncated or misfolded proteins that are not secreted efficiently, with decreases in both antigenic and functional levels of C1 inhibitor. Mutations that cause type II HAE usually involve exon 8 at, or near, the active site, resulting in a mutant protein that is secreted but is dysfunctional. Among the molecular events that cause retention or impaired secretion of the mutated protein, misfolding and polymerization are common and highly relevant to pathologic processes. Misfolding of serpins resulting in serpin polymerization is also called serpinopathy [12]. The diseases that depend on changes in serpin conformation are grouped under the term ‘conformational diseases’ [13]. Intracellular accumulation of serpin polymers can lead to cell death and has been implicated as the cause of liver cirrhosis associated with a1-antitrypsin mutations and a form of dementia identified as ‘conformational’ [14,15]. The underlying molecular mechanism of these diseases is the direct effect of a vulnerability to misfolding. The molecular basis for this process has been elucidated recently [16]. By solving the crystallographic structure of a stable serpin dimer, explanations have been provided for the extreme stability of serpin polymers, the molecular basis of their rapid propagation and why serpin polymers do not invoke a protective signalling pathway called the misfolded-protein response in the cell. How serpin polymers cause cell death, 70

however, remains an intriguing puzzle [17]. Examples of C1-INH oligomerization have been reported: point mutations affecting the C-terminal region relative to the reactive centre cause partial secretion owing to intracellular multimerization [18]. Another example of polymerization occurs when the C1-INH molecule carries a mutation in the reactive-centre proximal hinge [19]. Like most serpins with proximal-hinge mutations, this C1-INH loses its inhibitory activity. However, unlike other examples, this mutant is not converted to a substrate but exists in both monomeric and multimeric forms. No conformation-dependent consequences, other than plasma deficiency, have been related so far to C1-INH mutations. Nevertheless, HAE is characterized by large variability in its phenotypic expression and we expect that further understanding of the intracellular fate of C1-INH mutants will eventually reveal more about the molecular mechanisms of such variability. Despite the presence of a normal allele, HAE patients have levels of functional C1-INH that are markedly below the expected 50% of normal. As an explanation of this finding, it has been demonstrated that in vivo catabolism of normal C1-INH is faster in HAE patients than in normal subjects [20] owing to the formation of complexes with the target proteases [21]. However, detection of reduced levels of C1-INH mRNA by real-time reverse transcriptase polymerase chain reaction (RT-PCR) has opened the possibility of a coexistent downregulation of normal C1-INH [22]. Confirmation of this finding could represent a new target for therapy because C1-INH levels of 50% protect from angioedema.

Review Acquired angioedema The other cause of C1-INH deficiency is acquired and is usually identified as acquired angioedema (AAE). First described in 1972 by Caldwell [23], the mechanism(s) underlying acquired C1-INH deficiency has been a matter of controversy. This condition is characterized by massive activation of the classical complement pathway and accelerated catabolism of C1-INH, demonstrated clearly by in vivo turnover studies [24]. Owing to an association with Bcell malignancies, it has been suggested that the associated disease could lead to C1-INH deficiency, either by triggering massive activation of classical complement pathway causing secondary C1-INH consumption or by acting directly on C1-INH. Geha et al. [25] identified M components – immunoglobulins against the idiotypic determinants of monoclonal immunoglobulin expressed on the surface of B cells or in the cytoplasm of bone-marrow cells – in patients with both AAE and B-cell lymphoproliferative disease. These idiotype–anti-idiotype immune complexes fixed C1q and consumed C1-INH. However, this finding has not been further confirmed in subsequent patients. In 1986, Jackson et al. [26] described, for the first time, the presence of an autoreactive immunoglobulin G against C1INH in a patient with AAE. Because the first patients with autoantibodies to C1-INH looked otherwise healthy, it was proposed that two different forms of AAE existed: type I, paraneoplastic, associated mainly with lymphatic malignancies or other diseases; and type II, autoimmune, caused by autoantibodies to C1-INH in otherwise healthy patients. However, this distinction seems artificial because lymphoproliferation and autoimmunity coexist in most patients and might also develop one from the other [24]. The epitope specificity and pathogenic role of autoantibodies to C1-INH has been investigated subsequently [24]. These data indicate that autoantibodies that bind C1-INH around its reactive centre prevent the inhibitory activity on target proteases and convert the inhibitor into a substrate that can be cleaved into an inactive form. The existence of a relationship between acquired C1-INH deficiency and B-cell proliferation has been clear since the first description of this syndrome with the presence of antiC1-INH autoantibodies but the key question is whether the same B-cell clones producing autoantibodies are those that become neoplastic. Several lines of evidence suggest that autoreactive and neoplastic lymphoproliferation share part of the molecular mechanism that facilitates expansion of deviating clones [27]. Monoclonal immunoglobulins present in different conditions, including acquired C1-INH deficiency, might bind autoantigens [28] and the associations between certain autoimmune disorders and the risk of non-Hodgkin lymphoma (NHL) might not be general but rather mediated through specific NHL subtypes. These NHL subtypes develop during post-antigen exposure stages of lymphocyte differentiation, consistent with a role for antigenic drive in autoimmunity-related lymphomagenesis [29]. Thus, we have suggested that all acquired C1-INH deficiencies might depend on the proliferation of clones that recognize C1-INH. These clones might expand subsequently with characteristics of autoimmune or neoplastic proliferation. However, experimental proof of this hypothesis needs to be provided [30].

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The kinin system and the pathophysiology of angioedema in C1-INH deficiency The mechanism that leads C1-INH-deficient patients to develop angioedema involves the inappropriate generation of kinins. Kinins are peptides that stimulate vascular smooth-muscle relaxation and induce increased permeability. The term ‘kinin’ derives from the Greek word kinein, which means to move. The most important kinins are bradykinin – a nonapeptide derived from the action of the enzyme plasma kallikrein on high molecular weight kininogen (HK) – and lys-bradykinin or kallidin – a decapeptide derived from the action of tissue kallikrein on low molecular weight kininogen (LK). A detailed history of the kinin system has been provided by Bhoola et al. [31]. Mechanisms of bradykinin release and breakdown A simplified representation of the kinin system and its relationship with the contact system and the renin–angiotensin system in plasma is given in Figure 2 [31–34]. Bradykinin is generated through the cleavage of HK by plasma kallikrein during contact-system activation. The contact system consists of the substrate HK and the two zymogens prekallikrein and factor XII, which activate each other to form the enzymes kallikrein and activated factor XII (FXIIa), respectively [35]. In vitro, the system is activated after contact with negatively charged surfaces (hence the name) and the activation of prekallikrein to kallikrein depends on FXIIa. In vivo, other pathways independent of FXIIa can also activate prekallikrein on endothelial cells. For example, membrane-expressed enzyme prolylcarboxypeptidase (PRCP) [36] and/or the protein HSP90 [37] can directly activate prekallikrein bound to endothelial cell surfaces. The activation of the contact system results in blood coagulation and inflammation, however, its relevance in the coagulation cascade has been questioned because FXII-, prekallikrein- and HK-deficient patients do not suffer from bleeding disorders [35]. The cleavage of HK occurs at several points, enabling the release of the bradykinin nonapeptide – located inside the HK molecule - and other breakdown products (cleaved HK fragments) [38]. Bradykinin release is facilitated by the presence of plasmin (Figure 2). The most important inhibitor of the contact system is C1-INH, which inactivates both kallikrein and FXIIa. Tissue kallikrein cleaves LK to generate kallidin, which is converted to bradykinin by aminopeptidases. The physiological inhibitor of tissue kallikrein is kallistatin (SERPINA4). Tissue kallikrein activates prorenin to renin, which, in turn, activates angiotensinogen to angiotensin I. Bradykinin is degraded quickly by peptidases, such as human kininase I and kininase II, which are also called carboxypeptidase N and angiotensin-converting enzyme (ACE), respectively. Other important kininases are aminopeptidase P and neutral endopeptidase. It is interesting to note that kininase II (i.e. ACE) not only degrades bradykinin but also activates angiotensin I to angiotensin II and thus represents an important link between the kinin system and the renin–angiotensin system. The degradation of bradykinin occurs in several ways. Kininase I (carboxypeptidase N) degrades the intact nonapeptide bradykinin-(1–9) to bradykinin-(1–8), whereas 71

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Figure 2. Simplified representation of the kinin system. Bradykinin is generated through the cleavage of high molecular weight kininogen (HK) by plasma kallikrein during contact-system activation [31–34]. The contact system (green box) consists of the substrate HK and the two zymogens prekallikrein and factor XII (FXII), which activate each other to form the enzymes kallikrein and activated factor XII (FXIIa), respectively. The cleavage of HK enables the release of bradykinin, which is located inside the HK molecule, and other breakdown products (cleaved HK). Bradykinin release is facilitated by the presence of plasmin. The most important inhibitor of the contact system is C1INH, which inactivates kallikrein and FXIIa. Tissue kallikrein cleaves low molecular weight kininogen (LK), generating kallidin, which is converted to bradykinin by aminopeptidases. The physiological inhibitor of tissue kallikrein is kallistatin. Tissue kallikrein activates prorenin to rennin, which, in turn, activates angiotensinogen to angiotensin I (purple box). Bradykinin is degraded by peptidases, such as human kininase I, also called carboxypeptidase N, and kininase II, also called angiotensinconverting enzyme (ACE). Other important kininases are aminopeptidase P and neutral endopeptidase. kininase II (i.e. ACE) not only degrades bradykinin but also activates angiotensin I to angiotensin II.

kininase II (ACE) degrades bradykinin-(1–9) to bradykinin-(1–7), similar to the neutral endopeptidase. Both bradykinin-(1–8) and bradykinin-(1–7) are further degraded by kininase II to the bradykinin-(1–5) pentapeptide, which has a longer half-life. Aminopeptidase P inactivates bradykinin by removing the N-terminal Arg residue. Pathophysiology of bradykinin Bradykinin participates in various processes, including tissue permeability, vascular dilation and smooth muscle contraction. The biological effects of bradykinin are exerted through activation of the bradykinin B2 receptor, which is G-protein-coupled and generally expressed constitutively by vascular endothelial and smooth muscle cells [39]. On binding, the receptor is activated and transduces a signal cascade. Following activation, the receptor is desensitized, endocytosed and resensitized. In humans, there is another bradykinin receptor, named B1, which is closely related to the B2 receptor. It is activated by desArg(10)-kallidin or desArg(9)-bradykinin, which are metabolites of kallidin and bradykinin, respectively. The B1 receptor is induced following tissue injury or after treatment with bacterial endotoxins, such as lipopolysacharide or cytokines. The search for kinin receptors culminated in the 1990 s with the cloning of the rat B2 receptor cDNA by Jarnagin and coworkers. The B1 receptor was also cloned, 3 years later. The targeted ablation of the genes encoding the B2 and B1 receptor in mice has helped to define more precisely the physiological and pathophysiological roles of kinin receptors [39]. 72

Bradykinin in the pathogenesis of angioedema due to C1-INH deficiency The key experiments that led to the identification of bradykinin as the mediator in the pathogenesis of angioedema have been reviewed by Davis [40] and by Cugno et al. [41]. In 2002, B2 receptor-knockout mice were used to demonstrate that the increase in vascular permeability in C1INH deficiency was due to bradykinin. In fact, using Evans blue dye, Han et al. demonstrated an increase in vascular permeability in mice deficient in C1-INH but not in mice deficient in both C1-INH and bradykinin B2 receptors [42]. In 2007, the first report appeared demonstrating that angioedema could be reverted in HAE patients by antagonizing bradykinin binding to B2 [43]. Recently, Joseph et al. compared spontaneous and kaolin-induced activation in normal plasma and the plasma of patients with HAE. They found activation of the bradykinin-forming cascade in HAE plasma and demonstrated, for the first time in whole plasma, that production of factor XIIf might be responsible for C1 activation [44]. Our approach has been to study the mediator involved in the pathogenesis of acute attacks of angioedema by measuring the levels of bradykinin and markers of contact-system activation in plasma from patients with C1-INH deficiency. All these studies have been reviewed previously [41]. Plasma levels of the bradykinin-(1–9) nonapeptide can be measured by radioimmunoassay after liquid-phase extraction and high-performance liquid chromatography

Review [45]. This approach solves several pre-analytical and analytical problems that have made the measurement of plasma kinins extremely difficult in the past. Pre-analytical problems include the low concentration of bradykinin (pM), its short half-life (seconds) and easy enzymatic generation and degradation during sampling and handling procedures. Analytical problems concern the interference of proteins, unrelated plasma components and extraction chemicals, as well as the cross-reactivity of anti-bradykinin antibodies with precursor molecules and bradykinin catabolic products [45]. In addition, the cleavage of HK can be assessed by sodium dodecyl sulphate- polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis [46]. In patients with hereditary or AAE due to C1-INH deficiency, plasma bradykinin concentrations were higher during six different acute attacks of angioedema than during remission and they were also elevated in patients with angioedema related to ACE inhibitors, whereas they were normal in patients during acute attacks of angioedema that responded to antihistamines [47]. The increase in bradykinin levels during acute attacks of angioedema due to C1-INH deficiency was as high as 2 to 12 times the upper limit of normal [45]. Acute attacks of angioedema in patients with C1-INH deficiency are also associated with high levels of cleaved HK, indicating that the increase of bradykinin is owing to an increase in its generation [48]. In a study of HAE patients with acute forearm angioedema, bradykinin levels were measured in blood samples taken simultaneously from the edematous area and from the contralateral unaffected forearm. Increased venous plasma bradykinin concentrations were found in the edematous forearm compared with the contralateral control arm [49]. The results indicated that increased bradykinin levels in the systemic circulation originated from a three- to

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eight-fold increase in bradykinin concentrations in the blood draining the affected arm. This finding strengthens our previous observations that bradykinin generation is enhanced during angioedema attacks and suggests that the event leading to bradykinin generation is local. In conclusion, our findings, together with those of others, argue in favour of bradykinin being the mediator of angioedema due to C1-INH deficiency and of local activation being the event that leads to bradykinin generation (Figure 3). Bradykinin might also be involved in angioedema related to ACE inhibitors owing to a reduction in its catabolism but does not appear to be involved in cases of angioedema that are responsive to antihistamines [41]. The treatment of hereditary C1-INH deficiency Angioedema attacks are disabling and life threatening, so the search for an adequate treatment has always represented a crucial priority. Anti-fibrinolytic agents, which inhibit fibrinolysis, have been used to prevent angioedema symptoms but with low efficacy [50]. The first drugs that really changed the life of HAE patients positively were androgens and their attenuated derivatives. Methyltestosterone was introduced for treatment of HAE based on its hypothetical capacity to protect from some of the effects of histamine [51]. Despite the fact that the antihistamine effect of testosterone has never been confirmed and that, a few years later, it was shown that HAE symptoms were not histamine mediated [52], androgen derivatives represent a milestone in HAE treatment and are still largely and successfully used long term to prevent symptoms [53]. The mechanism of action of androgens in HAE is not clear. High doses (e.g. of danazol) can cause a significant increase in C1-INH plasma levels but much lower doses,

Figure 3. Representation of the pathogenesis of angioedema due to C1-INH deficiency. In HAE, the deficiency of C1-INH is due to a mutation in the C1-INH gene, which impairs C1-INH synthesis or function. In AAE, C1-INH deficiency is due to the cleavage of C1-INH by autoantibodies or to its consumption by neoplastic, mainly lymphoproliferative, tissue. Reduced C1-INH plasma levels result in hyperactivation of the classical complement pathway with increased consumption of C1-INH and further reduction of its plasma level. Impaired inhibition of activated Factor XII (FXIIa) and kallikrein, as a result of a lack of their principal inhibitor C1-INH, enables cleavage of high molecular weight kininogen (HK) by kallikrein and release of bradykinin, which binds to its B2 receptors, causing edema.

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Review which do not change the plasma levels of C1-INH, are clinically effective [54]. By measuring the effect of androgen therapy on the levels of C1-INH mRNA extracted from peripheral blood mononuclear cells (PBMCs) of HAE patients on and off treatment, it was demonstrated that minimal effective doses upregulate C1-INH gene expression [55]. However, it remains to be determined whether androgens exert any of their therapeutic effects by C1INH-dependent mechanisms. By contrast, androgens increase plasma levels of aminopeptidase P, an enzyme that has an important role in the catabolism of kinins [56]. This increase, by reducing kinin levels, could contribute to the improvement of the symptoms of angioedema during androgen treatment in HAE patients. With the exception of androgens, the treatments that have been, or are in the process of being, introduced for the treatment of HAE were developed in tight conjunction with the expanding knowledge of the molecular events underlying the generation of angioedema as a consequence of C1INH deficiency. When it became clear that C1-INH deficiency was the genetic defect in HAE patients, replacement therapy with fresh frozen plasma (FFP) was the first rational therapeutic approach [57]. As expected, this treatment has some efficacy and it is still used when alternatives are not available [58]. In the early 1970 s, preparations of C1-INH partially purified from pooled human plasma became available. The main advantages of partially purified C1-INH over FFP are: 1) to provide the deficient protein devoid of the enzymatic substrates that sustain angioedema; 2) to infuse much lower volumes so that higher C1-INH plasma concentrations can be reached in a shorter period of time (this is of relevance in life threatening situations, such as laryngeal edema); and 3) to guarantee higher safety levels in terms of viral transmission [59]. Evidence for the efficacy of C1-INH concentrates comes from a few controlled studies [60,61], the retrospective analysis of large case lists of patients treated for a long time [50,62–64] and clinical experience of nearly 30 years following HAE patients at major centres. Consensus publications provide guidelines for appropriate treatment of

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angioedema with C1-INH concentrate [65–67]. Retrospective studies have confirmed that C1-INH is suitable for the treatment of emergency situations, such as laryngeal edema and severe abdominal attacks [63,64]. Efficacy in peripheral cutaneous attacks has also been shown, although the time to beginning of relief of symptoms is longer. Thus, the available data provide sufficient evidence to consider C1-INH concentrate the treatment of choice for acute attacks in HAE patients. Accordingly, the largest accepted policy among HAE-treating physicians is to provide patients with C1INH concentrate at home for rapid use in case of attacks. To further facilitate the use of replacement therapy, some centres have started self-infusion programs. Experience with self administration of C1-INH suggests that this is a feasible and safe option, which provides more rapid and more effective treatment or prevention of severe angioedema attacks in patients with C1-INH deficiency [68,69]. Four different C1-INH concentrates have been used in humans but only two of them are available currently [59,70–72] (Table 1). Controlled Phase III clinical trials of CinryzeTM (Table 1) were performed for use in prophylaxis [73] as well as in the treatment of angioedema attacks in patients with HAE. Consequently, in October 2008, the U.S. Food and Drug Administration (FDA) approved CinryzeTM for routine prophylaxis against angioedema attacks in adolescent and adult patients with HAE. In addition, recently, Berinert1 P was shown to provide faster relief of acute symptoms of abdominal and facial attacks compared with placebo in a large controlled clinical trial [74]. A recombinant human C1 inhibitor (Rhucin1) has also been developed using transgenic rabbits. Replacement therapy with a recombinant product provides correction of the genetic defect with advantages over plasma-derived products of no risk of transmitting human bloodborne infections and unlimited supply, providing a lack of dependence on human plasma collection. However, recombinant products may carry higher immunogenic potential, with risk of causing increased levels of neutralizing antibodies and/or allergic reactions owing to species-dependent differences in glycosylation patterns. The efficacy in treating angioedema in HAE was assessed initially in an open label

Table 1. Current and potentially new treatments for angioedema due to hereditary or acquired C1-INH deficiency Drug Mechanism Indications Attenuated androgens (Danazol, Increase in plasma levels Long-term and short-term of C1-INH prophylaxis of acute attacks Stanozolol) in HAE and AAE Long-term and short-term Inhibition of fibrinolysis Anti-fibrinolytic agents prophylaxis of acute attacks (fibrinolysis facilitates (Tranexamic acid in HAE and AAE bradykinin generation) e-aminocaproic acid) Replacement of the HAE and AAE acute attacks Fresh frozen plasma deficient protein when C1-INH plasma concentrate is not available Replacement of the HAE and AAE acute attacks. Plasma derived human C1-INH deficient protein Short-term prophylaxis of (Berinert1 P [CSL Behring], attacks CinryzeTM [ViroPharma Inc.]) Replacement of the HAE acute attacks and Recombinant human C1-INH deficient protein potentially AAE acute from transgenic animals attacks (Rhucin1 [Pharming Group NV]) HAE acute attacks and Recombinant kallikrein inhibitor Inhibition of plasma kallikrein potentially AAE acute (Ecallantide [Dyax Corp.]) attacks Bradykinin antagonist (Icatibant Antagonist of bradykinin HAE acute attacks and B2 receptors potentially AAE acute [Firazyr1, Jerini AG]) attacks 74

Main advantages Main disadvantages Oral administration. Virilization, menstrual Low cost irregularities, liver damage

Refs [53]

Oral administration. Low efficacy in HAE, potential [89–91] Low cost thrombophilia [57]

Physiological approach

Risk of infections, potential transmission of edemainducing substrates Potential transmission of blood-borne infections

Physiological approach

Potential immunogenicity, infectious agents

[76]

Subcutaneous administration. No infectious risk Subcutaneous administration. No infectious risk

Rare anaphylactic reactions

[80]

Local discomfort at site of injection

[43]

Easy availability

[60,61,74]

Review study [75] and was then demonstrated by interim analysis of a controlled study in which the endpoint was achieved [76]. Two new compounds that are not based on C1-INH have been developed recently for the treatment of acute angioedema attacks in patients with HAE. For both of them, the plasma kallikrein inhibitor Dx-88 (also known as Ecallantide) and the bradykinin B2 receptor antagonist Icatibant (Firazyr1), development has been a direct consequence of the achievements of several years of research that has led to a better understanding of the pathogenesis of angioedema due to C1-INH deficiency. Dx-88, a recombinant peptide that has been produced for the treatment of HAE [77], is based on the reactive site (the first Kunitz domain) of aprotinine, a broad-spectrum protease inhibitor extracted from bovine lungs, which was used successfully in the past to treat angioedema in HAE patients [71,78]. Five clinical studies with Dx-88 in HAE (EDEMA 0, 1, 2, 3 and 4) have been completed and, although the results have not been published yet, the data that have so far been communicated to meetings suggest that Dx88 might be effective in reverting HAE attacks given either intravenously (i.v.) or subcutaneously (s.c.) [79–81]. The safety profile has also been investigated: a few acute dosing reactions (rhinitis, flushing and/or nausea) and one anaphylactoid reaction have so far been reported [82,83]. Icatibant [84] is a potent, specific and selective peptidomimetic bradykinin B2-receptor antagonist that is being tested currently for treatment of HAE. An open label pilot study showed that Icatibant, given either i.v. or s.c., considerably shortened the duration of attacks compared with similar untreated attacks in the same patients [43]. Two Phase III clinical studies (FAST 1 and FAST 2) of Icatibant for the treatment of HAE have been completed. Neither of these studies has yet been published as full paper, but results have been presented during international meetings (Symposium on C1 inhibitor and hereditary angioedema at the XXII International Complement Workshop: Basel, Switzerland; 2008 September 28–October 2). In the FAST 2 study, the primary endpoint was reached, showing a significant reduction in the time to onset of symptom relief. In the FAST 1 study, the primary endpoint was not reached, although all secondary endpoints were met and the drug showed a clinically relevant higher efficacy compared with placebo. A supportive analysis combining both studies showed a significant reduction in the time to onset of symptom relief. In life-threatening laryngeal attacks, which were allowed to be treated only open label with Icatibant, there was clinically relevant reduction in the time to symptom relief [85]. The treatment of acquired C1-INH deficiency This condition might be alleviated by treatment of the associated disease, by chemotherapy and other cytoreducing approaches (radiotherapy, surgery, antibodies to specific cell populations) or by acting directly on the angioedema symptoms by preventing their recurrence or reversing them on appearance [24]. The first option might be successful [86,87] but the resolution of remission might only be temporary or partial and this approach carries

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obvious intrinsic risks. C1-INH deficiency becomes the indication to treat the associated disease only if symptoms reduce the quality of life or result in life-threatening situations. Control of angioedema recurrences and treatment of acute attacks is performed using attenuated androgens, antifibrinolytic agents and C1-INH concentrate, as in HAE. However, patients with the acquired defect respond differently to these treatments: antifibrinolytic agents tend to be more effective than androgens in prophylaxis and some patients might become refractory to C1-INH concentrate. This difference probably relies on the rapid clearance of C1-INH from the circulation owing to the avidity of autoantibodies. It is possible, in the near future, that patients not responding to C1-INH concentrate will be treated successfully with the kallikrein inhibitor Ecallantide or the bradykinin receptor-antagonist Icatibant. These drugs are effective in HAE (as discussed earlier) and Ecallantide has also been used successfully to treat angioedema attacks in two AAE patients refractory to C1-INH concentrate [88]. Efficacy of these drugs should not be influenced by anti-C1-INH autoantibodies and therefore these might represent promising alternatives for the management of angioedema attacks in AAE patients. Concluding remarks The aetiology and pathogenesis of angioedema due to C1INH deficiency is largely understood. However, several important questions remain unanswered (Box 1). Mutations in one of the two alleles controlling C1-INH synthesis is the underlying cause of the genetic defect; Bcell proliferative disorders, through the production of neutralizing antibodies, can generate an acquired form of C1INH deficiency. Nevertheless, for the C1-INH genetic defect, we still do not understand the mechanisms that lead this heterozygous protein deficiency to result in plasma levels that are significantly below the predicted 50%. We have suggested that this could depend on an altered regulation of the normal allele, although other mechanisms may be involved. Understanding these mechanisms is an important task for the future because unBox 1. Outstanding questions  HAE is caused by a heterozygous defect in the gene encoding plasma protein C1-INH. What are the underlying mechanisms that lead HAE patients to present plasma levels of this protein that are approximately 10–20% of the normal values, instead of the predicted 50%?  HAE patients express a high interindividual variability in disease severity that does not segregate with the C1-INH mutation responsible for the disease. What factors are responsible for the variation in phenotypic expression of HAE?  Acquired C1-INH deficiency is associated with neutralizing autoantibodies against C1-INH and benign/malignant B-lymphocyte proliferation. Are these effects different steps of a unique process of altered B-cell proliferation driven by C1-INH acting as an autoantigen?  Bradykinin, released through activation of the contact system, mediates the episodic increase in vascular permeability that causes symptoms in C1-INH deficient patients. What are the specific events that can trigger bradykinin generation and what are the molecular mechanisms that initiate contact system activation in vivo?

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Review derstanding how to restore C1-INH plasma levels to 50% could prevent angioedema symptoms. The relationship between B-cell proliferation, anti-C1-INH autoantibodies and the acquired C1-INH defect has been determined but the potential role of C1-INH as an autoantigen driving the proliferation of a B-cell clone, which might eventually become neoplastic, is not understood. In acquired C1INH deficiency autoimmunity, benign B-cell proliferation and malignant B-cell proliferation might all occur and, in some patients, there has been progression from one condition to the other. Unravelling the molecular events that underlie this progression could shed new light not only on the aetiology of acquired C1-INH deficiency but also on the mechanisms of lymphomagenesis. The role of bradykinin as the main mediator of symptoms deriving from C1-INH deficiency has been determined by in vitro experiments showing that bradykinin is required to induce increased vascular permeability together with in vivo evidence of its presence in patients’ plasma during symptoms. The demonstration that specific inhibition of bradykinin significantly modifies the course of angioedema in HAE patients is the final confirmation of its importance in edema formation. Furthermore, the sequence of events that, through the activation of coagulation factor XII and kallikrein, cleave high molecularweight kininogen to generate bradykinin has also been shown: specific inhibitors of enzymes involved in this pathway also modify the course of angioedema. The missing piece of the puzzle to fully understand the series of events leading to angioedema remains the initiating event. Factor XII is at the top of the cascade, so its activation is important in initiating angioedema. There are several efficient in vitro activators of factor XII and there exists an amplification loop that generates activated factor XII from active kallikrein and active kallikrein from activated factor XII. From clinical observations, microtrauma (e.g. small injuries or prolonged pressure on a specific body part) is known to be a contact-system activator and can trigger angioedema symptoms, however, angioedema attacks can occur without an identified trigger. Determining the initiator(s) of these events, that is, the molecular mechanisms that lead from trauma and/or other still unidentified angioedema triggers to contact-system activation, will be an important challenge for future research and might disclose new therapeutic targets. In conclusion, a better understanding of the molecular mechanisms leading from C1-INH deficiency to angioedema has enabled the development of therapies that are enabling us to counteract the disease effectively. Further understanding of the initiating events in angioedema is expected to definitively provide C1-INH-deficient patients with a quality of life that is indistinguishable from that of ‘normal’ subjects. Disclosure statement Marco Cicardi has a research grant from CSL Behring, and has consultancy agreements with Dyax, Jerini and Pharming. Marco Cicardi is also an advisory board member for CSL Behring, Dyax and Jerini and has been an invited speaker for CSL Behring, Dyax, Jerini and Pharming. 76

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References 1 Silverman, G.A. et al. (2001) The serpins are an expanding superfamily of structurally similar but functionally diverse proteins. Evolution, mechanism of inhibition, novel functions, and a revised nomenclature. J. Biol. Chem. 276, 33293–33296 2 Davis, A.E. and 3rd et al. (2007) C1 inhibitor: biologic activities that are independent of protease inhibition. Immunobiology 212, 313–323 3 Bergamaschini, L. and Cicardi, M. (2003) Recent advances in the use of C1 inhibitor as a therapeutic agent. Mol. Immunol. 40, 155–158 4 Ennis, S. et al. (2008) Association between the SERPING1 gene and age-related macular degeneration: a two-stage case-control study. Lancet 372, 1828–1834 5 Pappalardo, E. et al. (2002) Mechanisms of C1-inhibitor deficiency. Immunobiology 205, 542–551 6 Patston, P.A. et al. (1991) Mechanism of serpin action: evidence that C1 inhibitor functions as a suicide substrate. Biochemistry 30, 8876– 8882 7 Gettins, P.G. (2002) Serpin structure, mechanism, and function. Chem. Rev. 102, 4751–4804 8 Huntington, J.A. et al. (2000) Structure of a serpin-protease complex shows inhibition by deformation. Nature 407, 923–926 9 Beinrohr, L. et al. (2007) C1 inhibitor serpin domain structure reveals the likely mechanism of heparin potentiation and conformational disease. J. Biol. Chem. 282, 21100–21109 10 Kalmar, L. et al. (2005) HAEdb: a novel interactive, locus-specific mutation database for the C1 inhibitor gene. Hum. Mutat. 25, 1–5 11 Pappalardo, E. et al. (2008) Mutation screening of C1 inhibitor gene in 108 unrelated families with hereditary angioedema: functional and structural correlates. Mol. Immunol. 45, 3536–3544 12 Davies, M.J. and Lomas, D.A. (2008) The molecular aetiology of the serpinopathies. Int. J. Biochem. Cell Biol. 40, 1273–1286 13 Lomas, D.A. et al. (2005) Molecular mousetraps and the serpinopathies. Biochem. Soc. Trans. 33, 321–330 14 Lomas, D.A. and Carrell, R.W. (2002) Serpinopathies and the conformational dementias. Nat. Rev. Genet. 3, 759–768 15 Carrell, R.W. and Lomas, D.A. (2002) Alpha1-antitrypsin deficiency – a model for conformational diseases. N. Engl. J. Med. 346, 45–53 16 Yamasaki, M. et al. (2008) Crystal structure of a stable dimer reveals the molecular basis of serpin polymerization. Nature 455, 1255–1258 17 Whisstock, J.C. and Bottomley, S.P. (2008) Structural biology: Serpins’ mystery solved. Nature 455, 1189–1190 18 Eldering, E. et al. (1995) COOH-terminal substitutions in the serpin C1 inhibitor that cause loop overinsertion and subsequent multimerization. J. Biol. Chem. 270, 2579–2587 19 Aulak, K.S. et al. (1993) A hinge region mutation in C1-inhibitor (Ala436!Thr) results in nonsubstrate-like behavior and in polymerization of the molecule. J. Biol. Chem. 268, 18088–18094 20 Quastel, M. et al. (1983) Behavior in vivo of normal and dysfunctional C1 inhibitor in normal subjects and patients with hereditary angioneurotic edema. J. Clin. Invest. 71, 1041–1046 21 Cugno, M. et al. (1990) Plasma levels of C1- inhibitor complexes and cleaved C1- inhibitor in patients with hereditary angioneurotic edema. J. Clin. Invest. 85, 1215–1220 22 Pappalardo, E. et al. (2004) C1 inhibitor gene expression in patients with hereditary angioedema: quantitative evaluation by means of realtime RT-PCR. J. Allergy Clin. Immunol. 114, 638–644 23 Caldwell, J.R. et al. (1972) Acquired C1 inhibitor deficiency in lymphosarcoma. Clin. Immunol. Immunopathol. 1, 39–52 24 Zingale, L.C. et al. (2006) Acquired deficiency of the inhibitor of the first complement component: presentation, diagnosis, course, and conventional management. Immunol. Allergy Clin. North Am. 26, 669–690 25 Geha, R.S. et al. (1985) Acquired C1-inhibitor deficiency associated with antiidiotypic antibody to monoclonal immunoglobulins. N. Engl. J. Med. 312, 534–540 26 Jackson, J. et al. (1986) An IgG autoantibody which inactivates C1inhibitor. Nature 323, 722–724 27 Chatila, T.A. (2005) Role of regulatory T cells in human diseases. J. Allergy Clin. Immunol. 116, 949–959 28 Cicardi, M. et al. (1996) Relevance of lymphoproliferative disorders and of anti-C1 inhibitor autoantibodies in acquired angio-oedema. Clin. Exp. Immunol. 106, 475–480

Review 29 Smedby, K.E. et al. (2006) Autoimmune and chronic inflammatory disorders and risk of non-Hodgkin lymphoma by subtype. J. Natl. Cancer Inst. 98, 51–60 30 Castelli, R. et al. (2007) Lymphoproliferative disease and acquired C1 inhibitor deficiency. Haematologica 92, 716–718 31 Bhoola, K.D. et al. (1992) Bioregulation of kinins: kallikreins, kininogens, and kininases. Pharmacol. Rev. 44, 1–80 32 Cugno, M. et al. (2001) Bradykinin in the ascitic fluid of patients with liver cirrhosis. Clin. Sci. 101, 651–657 33 Kaplan, A.P. et al. (2002) Pathways for bradykinin formation and inflammatory disease. J. Allergy Clin. Immunol. 109, 195–209 34 Schmaier, A.H. and McCrae, K.R. (2007) The plasma kallikrein–kinin system: its evolution from contact activation. J. Thromb. Haemost. 5, 2323–2329 35 Schmaier, A.H. (2008) The elusive physiologic role of Factor XII. J. Clin. Invest. 118, 3006–3009 36 Shariat-Madar, Z. (2002) Identification and characterization of prolylcarboxypeptidase as an endothelial cell prekallikrein activator. J. Biol. Chem. 277, 17962–17969 37 Joseph, K. et al. (2002) Heat shock protein 90 catalyzes activation of the prekallikrein–kininogen complex in the absence of factor XII. Proc. Natl. Acad. Sci. U. S. A. 99, 896–900 38 Schmaier, A.H. (2008) Assembly, activation, and physiologic influence of the plasma kallikrein/kinin system. Int. Immunopharmacol. 8, 161–165 39 Leeb-Lundberg, L.M. et al. (2005) International union of pharmacology. XLV. Classification of the kinin receptor family: from molecular mechanisms to pathophysiological consequences. Pharmacol. Rev. 57, 27–77 40 Davis, A.E., 3rd (2008) Hereditary angioedema: a current state-of-theart review. III: mechanisms of hereditary angioedema. Ann. Allergy Asthma Immunol. 100, S7–S12 41 Cugno, M. et al. (2003) Bradykinin and the pathophysiology of angioedema. Int. Immunopharmacol. 3, 311–317 42 Han, E.D. et al. (2002) Increased vascular permeability in C1 inhibitordeficient mice mediated by the bradykinin type 2 receptor. J. Clin. Invest. 109, 1057–1063 43 Bork, K. et al. (2007) Treatment of acute edema attacks in hereditary angioedema with a bradykinin receptor-2 antagonist (Icatibant). J. Allergy Clin. Immunol. 119, 1497–1503 44 Joseph, K. et al. (2008) Studies of the mechanisms of bradykinin generation in hereditary angioedema plasma. Ann. Allergy Asthma Immunol. 101, 279–286 45 Nussberger, J. et al. (1998) Plasma bradykinin in angio-oedema. Lancet 351, 1693–1697 46 Cugno, M. et al. (1994) Activation of the contact system and fibrinolysis in autoimmune acquired angioedema: a rationale for prophylactic use of tranexamic acid. J. Allergy Clin. Immunol. 93, 870–876 47 Nussberger, J. et al. (2002) Bradykinin-mediated angioedema. N. Engl. J. Med. 347, 621–622 48 Cugno, M. et al. (1997) Activation of the coagulation cascade in C1inhibitor deficiencies. Blood 89, 3213–3218 49 Nussberger, J. et al. (1999) Local bradykinin generation in hereditary angioedema. J. Allergy Clin. Immunol. 104, 1321–1322 50 Agostoni, A. et al. (2004) Hereditary and acquired angioedema: problems and progress: proceedings of the third C1 esterase inhibitor deficiency workshop and beyond. J. Allergy Clin. Immunol. 114, S51–S131 51 Spaulding, W.B. (1960) Methyltestosterone therapy for hereditary episodic edema (hereditary angioneurotic edema). Ann. Intern. Med. 53, 739–745 52 Donaldson, V.H. et al. (1969) Permeability-increasing activity in hereditary angioneurotic edema plasma. II. Mechanism of formation and partial characterization. J. Clin. Invest. 48, 642–653 53 Banerji, A. et al. (2008) Hereditary angioedema: a current state-of-theart review, V: attenuated androgens for the treatment of hereditary angioedema. Ann. Allergy Asthma Immunol. 100, S19–S22 54 Gelfand, J.A. et al. (1976) Treatment of hereditary angioedema with danazol. Reversal of clinical and biochemical abnormalities. N. Engl. J. Med. 295, 1444–1448 55 Pappalardo, E. et al. (2003) Increased expression of C1-inhibitor mRNA in patients with hereditary angioedema treated with Danazol. Immunol. Lett. 86, 271–276

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56 Drouet, C. et al. (2008) Metallopeptidase activities in hereditary angioedema: effect of androgen prophylaxis on plasma aminopeptidase P. J. Allergy Clin. Immunol. 121, 429–433 57 Pickering, R.J. et al. (1969) Replacement therapy in hereditary angioedema. Successful treatment of two patients with fresh frozen plasma. Lancet 1, 326–330 58 Hill, B.J. et al. (2004) Fresh frozen plasma for acute exacerbations of hereditary angioedema. Am. J. Emerg. Med. 22, 633 59 Cicardi, M. et al. (2007) The use of plasma-derived C1 inhibitor in the treatment of hereditary angioedema. Expert Opin. Pharmacother. 8, 3173–3181 60 Kunschak, M. et al. (1998) A randomized, controlled trial to study the efficacy and safety of C1 inhibitor concentrate in treating hereditary angioedema. Transfusion 38, 540–549 61 Waytes, A.T. et al. (1996) Treatment of hereditary angioedema with a vapor-heated C1 inhibitor concentrate. N. Engl. J. Med. 334, 1630– 1634 62 Agostoni, A. and Cicardi, M. (1992) Hereditary and acquired C1inhibitor deficiency: biological and clinical characteristics in 235 patients. Medicine (Baltimore) 71, 206–215 63 Bork, K. et al. (2005) Treatment with C1 inhibitor concentrate in abdominal pain attacks of patients with hereditary angioedema. Transfusion 45, 1774–1784 64 Bork, K. and Barnstedt, S.E. (2001) Treatment of 193 episodes of laryngeal edema with C1 inhibitor concentrate in patients with hereditary angioedema. Arch. Intern. Med. 161, 714–718 65 Bowen, T. et al. (2008) Hereditary angiodema: a current state-of-theart review, VII: Canadian Hungarian 2007 International Consensus Algorithm for the Diagnosis, Therapy, and Management of Hereditary Angioedema. Ann. Allergy Asthma Immunol. 100 (1 Suppl. 2), S30–S40 66 Gompels, M.M. et al. (2005) C1 inhibitor deficiency: consensus document. Clin. Exp. Immunol. 139, 379–394 67 Bowen, T. et al. (2003) Management of hereditary angioedema: a Canadian approach. Transfus. Apheresis Sci. 29, 205–214 68 Longhurst, H.J. et al. (2007) C1-inhibitor concentrate home therapy for hereditary angioedema: a viable, effective treatment option. Clin. Exp. Immunol. 147, 11–17 69 Levi, M. et al. (2006) Self-administration of C1-inhibitor concentrate in patients with hereditary or acquired angioedema caused by C1inhibitor deficiency. J. Allergy Clin. Immunol. 117, 904–908 70 Marasini, B. et al. (1978) Treatment of hereditary angioedema. Klin. Wochenschr. 56, 819–823 71 Vogelaar, E.F. et al. (1974) Contributions to the optimal use of human blood. 3. Large-scale preparation of human c1 esterase inhibitor concentrate for clinical use. Vox Sang. 26, 118–127 72 Terpstra, F.G. et al. (2007) Viral safety of C1-inhibitor NF. Biologicals 35, 173–181 73 Zuraw, B. et al. (2008) Efficacy and safety of long-term prophylaxis with C1 inhibitor (C1INH) concentrate in patients with hereditary angioedema (HAE). J. Allergy Clin. Immunol. 121, S272 74 Bernstein, J.A. et al. (2008) Treatment of acute abdominal and facial attacks of hereditary angioedema (HAE) with human C1 esterase inhibitor (C1-INH): results of a global, multicenter, randomized, placebo-controlled, Phase II/III Study (I.M.P.A.C.T.1). J. Allergy Clin. Immunol. 121, 795 75 Choi, G. et al. (2007) Recombinant human C1-inhibitor in the treatment of acute angioedema attacks. Transfusion 47, 1028–1032 76 Cicardi, M. et al. (2008) A randomised, placebo-controlled, double blind Phase III study of the efficacy and safety of recombinant human C1 inhibitor for the treatment of acute attacks in patients with hereditary angioedema. Mol. Immunol. 45, 4118–4119 77 Williams, A. and Baird, L.G. (2003) DX-88 and HAE: a developmental perspective. Transfus. Apheresis Sci. 29, 255–258 78 Cicardi, M. et al. (1982) Hereditary angioedema: an appraisal of 104 cases. Am. J. Med. Sci. 284, 2–9 79 Gonzales-Quevedo, T. et al. (2002) The synthetic Kunitz domain protein Dx88 to treat angioedema in patients with hereditary angioedema. Int. Immunopharmacol. 2, 1318 80 Schneider, L. et al. (2007) Critical role of kallikrein in hereditary angioedema pathogenesis: a clinical trial of ecallantide, a novel kallikrein inhibitor. J. Allergy Clin. Immunol. 120, 416–422 81 Lumry, W. et al. (2006) Interim results of EDEMA2, a multicenter, open-label, repeat-dosing study of intravenous and subcutaneous

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Review

82

83

84 85

86 87

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administration of ecallantide (DX-88) in hereditary angioedema. J. Allergy Clin. Immunol. 117, S179 Williams, T. (2005) DX-88, a novel recombinant protein inhibitor of plasma kallikrein: safety and immunogenicity in early studies. J. Allergy Clin. Immunol. 115, S181 Caballero, T. and Lopez-Serrano, C. (2006) Anaphylactic reaction and antibodies to DX-88 (kallikrein inhibitor) in a patient with hereditary angioedema. J. Allergy Clin. Immunol. 117, 476–477 [No authors listed] (2004) Icatibant: HOE 140, JE 049, JE049. Drugs R D. 5, 343–348 Bas, M. et al. (2006) Novel pharmacotherapy of acute hereditary angioedema with bradykinin B2-receptor antagonist icatibant. Allergy 61, 1490–1492 Levi, M. et al. (2006) Rituximab-induced elimination of acquired angioedema due to C1-inhibitor deficiency. Am. J. Med. 119, e3–e5 Sanchez-Cano, D. et al. (2008) Successful use of rituximab in acquired C1 inhibitor deficiency secondary to Sjogren’s syndrome. Lupus 17, 228–229

Trends in Molecular Medicine Vol.15 No.2 88 Lehmann, A. (2006) Ecallantide (Dyax/Genzyme). Curr. Opin. Investig. Drugs 7, 282–290 89 Sheffer, A.L. et al. (1972) Tranexamic acid therapy in hereditary angioneurotic edema. N. Engl. J. Med. 287, 452–454 90 Blohme, G. et al. (1972) Treatment of hereditary angioneurotic oedema with tranexamic acid. A random double-blind cross-over study. Acta Med. Scand. 192, 293–298 91 Frank, M.M. et al. (1972) Epsilon aminocaproic acid therapy of hereditary angioneurotic edema. A double-blind study. N. Engl. J. Med. 286, 808–812 92 Dullweber, F. et al. (2001) Factorising ligand affinity: a combined thermodynamic and crystallographic study of trypsin and thrombin inhibition. J. Mol. Biol. 313, 593–614 93 Dementiev, A. et al. (2003) Canonical inhibitor-like interactions explain reactivity of a1-proteinase inhibitor Pittsburgh and antithrombin with proteinases. J. Biol. Chem. 278, 37881–37887 94 Moy, F.J. et al. (2000) NMR structure of free RGS4 reveals an induced conformational change upon binding Ga. Biochemistry 39, 7063–7073