Metabolite Biomarkers for Diseases Associated with Contact Activation Systems

Abstract

To provide methods and kits for analyzing a biological sample obtained from a subject having, suspected of having, or being at risk for a disease associated with the contact activation system.SOLUTION: A method for analyzing a sample includes: preparing a biological sample (for example, serum sample or plasma sample) obtained from a subject (human subject and the like) having, suspected of having, or being at risk for a disease associated with the contact activation system; and measuring the level of a metabolite biomarker set including at least one metabolite biomarker selected from Table 1.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 518,367, filed Jun. 12, 2017, and U.S. Provisional Application No. 62 / 395,770, filed Sep. 16, 2016, under 35 U.S.C. § 119(e). The entire contents of each of the aforementioned applications are hereby incorporated by reference into this specification.

Background Art

[0002] Background The plasma contact activation system is a pro - inflammatory and pro - coagulant system involving a group of plasma proteases. The plasma contact activation system is activated by factor XIIa upon exposure to foreign surfaces or negatively charged surfaces, or by prolyl carboxypeptidase on the endothelial cell surface (Sainz I.M. et al., Thromb. Haemost. (2007) 98, 77 - 83). Inappropriate or disordered activation of the contact system is involved in various diseases including hereditary angioedema (HAE).

[0003] HAE is a disease that causes sudden attacks of swelling that can affect multiple parts of the body (such as the face, limbs, genitals, gastrointestinal tract, and upper airway). Since the symptoms of HAE often resemble those of allergies or intestinal colic, patients with HAE are often difficult to identify until they exhibit severe or life - threatening symptoms. Early diagnosis would allow for better management of emergencies associated with acute HAE attacks and would also help manage HAE patients to prevent or attenuate acute HAE episodes (e.g., by having HAE patients avoid exposure to stimuli that may trigger HAE episodes).

[0004] Accordingly, it is highly interesting to develop reliable diagnostic and prognostic methods for identifying biomarkers of HAE and for identifying subjects having a particular type of HAE or at risk of suffering an acute HAE attack. Such biomarkers would also be useful in studies regarding the disease mechanism, which could facilitate the development of effective new therapies for this disease. SUMMARY OF THE INVENTION

[0005] SUMMARY OF THE DISCLOSURE The present disclosure is based on the identification of metabolite biomarkers that are differentially present in biological samples obtained from subjects having a disease associated with the contact activation system compared to healthy individuals, or that are differentially present in biological samples obtained from subjects at different stages of such a disease (e.g., attack vs. quiescent state).

[0006] Accordingly, one aspect of the present disclosure provides a method of analyzing a sample comprising: (i) providing a biological sample (e.g., a serum sample or a plasma sample) obtained from a subject (such as a human subject) having, suspected of having, or at risk of having a disease associated with the contact activation system; and (ii) measuring the levels of a set of metabolite biomarkers comprising at least one metabolite biomarker selected from Table 1. In some embodiments, the disease associated with the contact activation system is hereditary angioedema (HAE), such as type I HAE or type II HAE. In some examples, the set of biomarkers consists of 2 to 10 metabolite biomarkers selected from Table 1.

[0007] In any of the methods described herein, at least one metabolite is serotonin or a metabolite related to serotonin metabolism. In some embodiments, at least one metabolite biomarker is a metabolite related to fatty acid amide hydrolase activity (e.g., a fatty acid amide such as palmitic acid amide, oleic acid amide, or linoleic acid amide). In some embodiments, at least one metabolite biomarker is a lipid peroxide (e.g., 9-hydroxyoctadecadienoic acid (9-HODE), 13-hydroxyoctadecadienoic acid (13-HODE), 9,10-dihydroxy-12Z-octadecenoic acid (9,10-DiHOME), 12,13-dihydroxy-9Z-octadecenoic acid (12,13-DiHOME), or 19,20-dihydroxy-4Z,7Z,10Z,13Z,16Z-docosapentaenoic acid (19,20-DiHDPA)). In some embodiments, at least one metabolite biomarker is a metabolite involved in sulfur metabolism (e.g., N-acetylmethionine, methionine sulfone, S-adenosylhomocysteine, cystine, or cysteine sulfinic acid).In some embodiments, at least one metabolite biomarker is a metabolite involved in steroid metabolism (e.g., pregnenolone sulfate, 5α-pregnan-3β,20β-diol monosulfate, pregnenediol disulfate, pregn steroid monosulfate, prenanediol-3-glucuronide, cortisol, corticosterone, cortisone, dehydroepiandrosterone sulfate (DHEA-S), 16a-hydroxy DHEA 3-sulfate, epiandrosterone sulfate, androsterone sulfate, 4-androsten-3β,17β-diol monosulfate, 4-androsten-3α,17α-diol monosulfate, 4-androsten-3β,17β-diol disulfate, 5α-androstan-3α,17α-diol monosulfate, 5α-androstan-3β,17β-diol disulfate, androsteroid monosulfate, etiocholanolone glucuronide or pregnanolone / alloprenanolone sulfate). In some embodiments, at least one metabolite biomarker is a fatty acid (e.g., eicosapentaenoic acid (EPA), mead acid, or stearidonate). In some embodiments, at least one metabolite biomarker is a cofactor precursor (e.g., a cofactor precursor of coenzyme A (such as nicotinamide or pantothenic acid)).

[0008] In some embodiments, measuring the levels of a set of biomarkers involves mass spectrometry, chromatography or immunoassay. In some embodiments, measuring the levels of a set of biomarkers involves mass spectrometry, where the biological sample is subjected to a separation step (e.g., gas chromatography, liquid chromatography or capillary electrophoresis) prior to mass spectrometry.

[0009] In some embodiments, the method further comprises identifying that the subject has a disease associated with the contact system when the level of a set of target biomarkers deviates from the level of the same set of biomarkers in a control subject. In some embodiments, the method further comprises administering to the subject an effective amount of a therapeutic agent (such as a plasma kallikrein (pKal) inhibitor, a bradykinin 2 receptor inhibitor, and / or a C1 esterase inhibitor, etc.) for treating the disease when the subject is identified as having the disease. In some embodiments, the pKal inhibitor is an anti-pKal antibody. In some embodiments, the therapeutic agent is lanadelumab, ecallantide, icatibant or a human plasma-derived C1 esterase inhibitor.

[0010] In some embodiments, the subject is a human patient undergoing treatment for a disease, where the method further comprises evaluating the effectiveness of the treatment based on the level of a set of metabolite biomarkers, and a deviation of the level of the set of metabolite biomarkers in the subject from that of a control subject indicates the effectiveness of the treatment. In some embodiments, the method further comprises identifying a treatment suitable for the subject based on the level of a set of metabolite biomarkers. In some embodiments, the method further comprises identifying the subject as a candidate for treatment of the disease based on the level of a set of metabolite biomarkers.

[0011] In some embodiments, the human patient has a medical history of a disease (such as HAE). In some embodiments, the set of metabolite biomarkers comprises one or more metabolite biomarkers selected from the list consisting of corticosterone, eicosapentaenoic acid, mead acid, stearidonic acid, nicotinamide and pantothenic acid. In some embodiments, the method further comprises evaluating the risk of an attack of the disease (such as an HAE attack) based on the level of a set of metabolite biomarkers, and a deviation of the level of the set of metabolite biomarkers from that of a control subject indicates the risk of an attack of the disease. In some embodiments, the method further comprises administering a therapeutic agent to the subject when the subject is identified as having a risk of an attack of the disease.

[0012] The present disclosure provides biomarkers capable of identifying patients having a disease associated with the contact activation system (e.g., HAE). Measuring the levels of a set of biomarkers may also be useful in the evaluation and treatment of such diseases.

[0013] Another aspect of the present disclosure is a method of identifying whether a subject has or is at risk of having a disease associated with the contact activation system, comprising: (i) providing a biological sample obtained from the subject; (ii) measuring the levels of a set of metabolite biomarkers; and (iii) identifying that the subject has or is at risk of having the disease when the levels of the set of metabolite biomarkers deviate from the levels of the set of metabolite biomarkers in a control sample.

[0014] Another aspect is a method of assessing the risk of an attack of a disease associated with activation of the contact system (e.g., an HAE attack), comprising: (i) providing a biological sample obtained from the subject; (ii) measuring the levels of a set of metabolite biomarkers; and (iii) identifying that the subject is at risk of having an attack of the disease when the levels of the set of metabolite biomarkers deviate from the levels of the set of metabolite biomarkers in a control sample.

[0015] In another aspect, a kit for analyzing a sample of a subject having, suspected of having, or at risk of having a disease associated with a contact system, the kit comprising a first binding substance specific for a first metabolite marker selected from Table 1 and a second binding substance specific for a second metabolite marker selected from Table 1, wherein the first metabolite marker and the second metabolite marker are different. In some embodiments, the kit further comprises at least one additional binding substance for another metabolite marker selected from Table 1. In some examples, the first binding substance is an antibody specific for a first metabolite biomarker and / or the second binding substance is an antibody specific for a second metabolite biomarker. In some embodiments, the kit may further comprise a first detection substance that binds to the first binding substance and a second detection substance that binds to the second binding substance. In some embodiments, the first binding substance and the second binding substance may be immobilized on a support member.

[0016] Details of one or more embodiments of the present disclosure are set forth in the following description. Other features or advantages of the present disclosure will become apparent from the following drawings and detailed description of several embodiments, and even from the appended claims.

[0017] The following drawings form a part of this specification and are included to further illustrate certain aspects of the present disclosure that can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

Brief Description of the Drawings

[0018]

Figure 1-1

Figure 1-2

Figure 2

Mode for Carrying Out the Invention

[0019] Detailed Description The contact activation system initiates the intrinsic pathway of blood coagulation and promotes inflammation through the release of bradykinin, a pro-inflammatory peptide. Factor XII (FXII), also known as Hageman factor, is a serine protease that plays a role in the activation of the intrinsic pathway of blood coagulation as well as the kallikrein-kinin system. FXII is activated by negatively charged surfaces (e.g., polyanionic surfaces, glass, polyphosphate, ellagic acid) to yield the active form, FXIIa. Activated FXIIa has the ability to cleave prekallikrein to yield active pKal. Subsequently, activated pKal can cleave FXII to FXIIa, resulting in a positive feedback loop where FXIIa generates more pKal, which further activates additional FXII to FXIIa. Activated pKal can also cleave high-molecular weight kininogen (HMWK) to release bradykinin. In diseases associated with activation of the contact system, such as HAE, an increase in bradykinin levels can induce vasodilation and inflammation leading to edematous HAE attacks. For example, it is desirable to identify novel biomarkers that can be used to identify subjects having or at risk of having such diseases in order to identify diseases mediated by the contact activation system.

[0020] The present disclosure is based at least in part on the identification of metabolites that are differentially present in biological samples obtained from subjects having a disease associated with the contact activation system (e.g., in a basal state or an attack) compared to healthy individuals by metabolomic analysis. It was unexpectedly observed that metabolites belonging to specific cellular pathways or processes (e.g., metabolites involved in sulfur metabolism, steroid metabolism, serotonin metabolism, metabolites associated with fatty acid amide hydrolase activity) and metabolites belonging to metabolite families (e.g., fatty acids) tend to have a similar trend (e.g., elevated or decreased levels) in samples from subjects having the disease compared to healthy individuals. Furthermore, several metabolites were identified as being differentially present in biological samples obtained from subjects during an attack of a disease of the contact activation system compared to subjects having the disease in a quiescent (basal) state.

[0021] Accordingly, provided herein is a method for analyzing a biological sample from a subject having, suspected of having, or at risk for a disease associated with the contact activation system (e.g., HAE) by detecting the presence or measuring the level of a set of metabolite biomarkers. Such methods can be used, for example, to identify patients at risk for a disease associated with the contact activation system (e.g., HAE), to select candidates for treatment, to monitor disease progression or condition, to evaluate the effectiveness of treatment for a disease, to determine the course of treatment, to evaluate whether a subject is at risk of disease onset, to identify whether a disease or disorder is associated with the contact activation system, and / or for research purposes (including, for example, research into the mechanisms of a disease and / or the biological pathways / processes involved in that disease, which may be utilized for the development of new therapies).

[0022] Metabolite Biomarkers of the Contact Activation System The methods and kits described herein are based at least in part on the identification of metabolites that are found to be present differently in samples from subjects with HAE compared to samples from healthy subjects and / or that are present differently in samples at different stages of such a disease (e.g., basal state vs. attack). As used herein, the term "metabolite biomarker" or "set of metabolite biomarkers" refers to a metabolite or set of metabolites that are present at different levels in samples from different groups of subjects (e.g., subjects with a disease associated with the contact system vs. healthy subjects (e.g., subjects without a disease), or subjects with a disease in a quiescent stage vs. subjects during an attack of the disease). Such biomarkers / sets of biomarkers may be used for both diagnostic / prognostic applications and non-clinical applications (e.g., for research purposes).

[0023] In some embodiments, a metabolite biomarker may be present at elevated levels in a sample from a subject having a disease associated with the contact activation system (e.g., HAE) compared to the levels of the same metabolite biomarker in a sample from a healthy subject. In some embodiments, a metabolite biomarker may be present at reduced levels in a sample from a subject having a disease associated with the contact activation system (e.g., HAE) compared to the levels of the biomarker in a sample from a healthy subject. In still other examples, a metabolite biomarker may be present at elevated levels in a sample obtained from a subject during an episode of a disease as described herein compared to the subject during a quiescent state of the disease. Alternatively, a metabolite biomarker may be present at reduced levels in a sample obtained from a subject during an episode of a disease as described herein compared to the subject during a quiescent state of the disease.

[0024] In some embodiments, a set of metabolite biomarkers comprising one or more biomarkers may be analyzed by the methods described herein. If the set of metabolite biomarkers comprises two or more biomarkers, all of those biomarkers may be present at elevated or reduced levels in a subject having a disease compared to a healthy subject. Alternatively, the set of metabolite biomarkers may comprise at least one biomarker that is elevated in a subject having a disease compared to a healthy subject and at least one biomarker that is reduced in a subject having a disease compared to a healthy subject.

[0025] Similarly, for a set of metabolic biomarkers, a set of biomarkers for distinguishing a subject during an episode of a disease from a subject in a quiescent state of the disease may comprise a plurality of biomarkers all of which are elevated or reduced in a first disease stage (e.g., episode) compared to a second disease stage (e.g., quiescent state). Alternatively, the set of biomarkers may comprise at least one biomarker that is elevated in a first disease stage compared to a second disease stage and at least one biomarker that is reduced in a first disease stage compared to a second disease stage.

[0026] Table 1 below presents metabolite biomarkers that can be evaluated by the methods described herein for evaluating a subject or a biological sample from a subject for diseases associated with the contact activation system.

[0027] [Table 1] TIFF0007692508000002.tif55162

[0028] In some embodiments, a set of biomarkers measured and analyzed by any of the methods described herein comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 or more) metabolite selected from Table 1.

[0029] As described in Example 1, it was unexpectedly found that several metabolites involved in different cellular processes and pathways are present differently in samples from subjects with HAE compared to healthy subjects. This data indicates that the metabolites or metabolite pathways shown in Table 1 may play a role in or be affected by diseases associated with the contact system.

[0030] The metabolite biomarkers described herein may be characterized as “involved in” or “associated with” a particular pathway or activity. As used herein, the terms “involved in” or “associated with” are applied to metabolites that contribute to a pathway. For example, a metabolite involved in or associated with a pathway may potentially function within that pathway, may be a precursor of another molecule within the pathway, may be a substrate for an enzyme within the pathway, or may be a product (including a by-product or product of degradation or molecule) of the pathway. In some embodiments, the pathway is a metabolic pathway such as a biosynthetic pathway or a catabolic pathway. In some embodiments, a metabolite may be described as being associated with the activity of a particular enzyme. For example, a metabolite associated with the activity of an enzyme may be a substrate of that enzyme, a product of the activity of that enzyme, or a cofactor whose presence regulates or enhances the activity of that enzyme. In some embodiments, the level of an enzyme substrate or enzyme product is an indicator of the level of activity of that enzyme (e.g., between diseases or between particular states of a disease).

[0031] As used herein, the term “precursor” such as a precursor of a cofactor refers to a metabolite that precedes another molecule (e.g., a product) in a synthetic pathway. For example, a precursor of a cofactor is a metabolite within the synthetic pathway of the cofactor and can be modified or used to generate the cofactor. Also as used herein, the term “cofactor” refers to a molecule that enhances the activity of an enzyme by interacting with it.

[0032] In some embodiments, a set of biomarkers includes serotonin, or one or more metabolites involved in serotonin production (e.g., one or more metabolites involved in serotonin production selected from Table 1).

[0033] In some embodiments, the set of biomarkers includes one or more metabolites related to fatty acid amide hydrolase activity (e.g., one or more metabolites involved in serotonin production selected from Table 1). Metabolites related to fatty acid amide hydrolase activity can be any metabolite involved in the reaction catalyzed by fatty acid amide hydrolase. Such metabolites can be the substrate of the enzyme, the product of the enzyme, or any intermediate thereof.

[0034] In some embodiments, the set of biomarkers includes one or more metabolites that are lipid peroxides, which refer to any product of the lipid peroxidation reaction. Examples include lipid peroxide compounds listed in Table 1. In some embodiments, the set of biomarkers includes one or more metabolites related to sulfur metabolism (e.g., one or more metabolites involved in sulfur metabolism selected from Table 1).

[0035] In some embodiments, the set of biomarkers includes one or more metabolites related to steroid metabolism (e.g., one or more metabolites involved in steroid metabolism selected from Table 1).

[0036] As also described in Example 1, it was also found that some metabolites are present differently in samples from subjects with HAE during an HAE attack compared to subjects with HAE during a resting (baseline) state. In some embodiments, the set of biomarkers includes one or more metabolites that are fatty acids (e.g., one or more metabolites that are fatty acids selected from Table 1). In some embodiments, the set of biomarkers includes one or more metabolites that are precursors of cofactors (e.g., one or more metabolites that are precursors of cofactors selected from Table 1). In some embodiments, the set of biomarkers includes corticosterone.

[0037] Usefulness of metabolite biomarkers One aspect of the present disclosure relates to a method for analyzing a sample obtained from a subject (e.g., a human patient) having, suspected of having, or at risk of a disease associated with a contact activation system by measuring the levels of a set of biomarkers as described herein in the sample. Results obtained from such an assay would be useful for diagnostic and / or prognostic uses as well as other non-clinical uses (such as research uses).

[0038] (i) Analysis of biological samples The methods described herein involve preparing a biological sample obtained from a subject. As used herein, "biological sample" refers to a composition comprising tissue from a subject (e.g., blood, plasma, or protein). Samples include both the initially untreated sample taken from the subject as well as subsequently processed (e.g., partially purified or preserved) forms thereof. Exemplary samples include blood, plasma, tears, or mucus. In some embodiments, the sample is a body fluid sample such as a serum sample or a plasma sample. In some embodiments, multiple (e.g., at least 2, 3, 4, 5, or 6 or more) biological samples may be taken from the subject over time or at specific time intervals, for example, to evaluate disease progression or the effectiveness of treatment.

[0039] A biological sample can be obtained from a subject using any means known in the art. In some embodiments, the sample is obtained from a subject by collecting the sample (e.g., a blood sample) into a vacuum collection tube (e.g., a vacuum blood collection tube). In some embodiments, the vacuum collection tube contains one or more protease inhibitors, e.g., to reduce or prevent ex vivo activation of the contact system during sample collection. Such protease inhibitors may be included in a liquid formulation. In some embodiments, the protease inhibitor includes at least one serine protease inhibitor and at least one cysteine protease inhibitor. Such vacuum collection tubes are known in the art. See, e.g., PCT Application No. US2016 / 046681. Optionally, the vacuum blood collection tube may further contain one or more anticoagulants.

[0040] The terms “patient,” “subject,” or “individual” may be used interchangeably and refer to a subject in need of the analysis as described herein. In some embodiments, the subject is a human or a non-human mammal. In some embodiments, the subject is suspected of having or at risk of a disease or disorder associated with the contact activation system (e.g., HAE). Such a subject may exhibit one or more symptoms associated with the disease. Alternatively, or in addition, such a subject may possess one or more risk factors for the disease (e.g., a genetic factor associated with the disease (e.g., a genetic defect in CI-INH)).

[0041] Alternatively, the subject in need of the analysis described herein may be a patient with the disease. Such a subject may currently be suffering from an attack of the disease or may have suffered from the disease in the past (e.g., currently in a quiescent state of the disease). In some examples, the subject is a human patient who may be undergoing treatment for the disease (e.g., treatment with a C1 esterase inhibitor (C1-INH), a plasma kallikrein inhibitor, or a bradykinin inhibitor). In other examples, such a human patient may not be undergoing such treatment.

[0042] Examples of diseases associated with the contact activation system include, but are not limited to, kallikrein-mediated disorders such as bradykinin-mediated disorders (such as hereditary angioedema (HAE)), non-histamine-dependent idiopathic angioedema, rheumatoid arthritis, Crohn's disease, lupus, Alzheimer's disease, septic shock, burns, cerebral ischemia / reperfusion injury, cerebral edema, diabetic retinopathy, diabetic nephropathy, macular edema, vasculitis, arterial or venous thrombosis, thrombosis associated with ventricular assist devices or stents, heparin-induced thrombocytopenia with thrombosis, thromboembolic diseases, and coronary heart disease with unstable angina, edema, eye diseases, gout, intestinal bowel disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis - degenerative spinal disorders, postoperative ileus, aortic aneurysm, osteoarthritis, hereditary angioedema, pulmonary embolism, stroke, head trauma or peritumoral cerebral edema, sepsis, acute middle cerebral artery (MCA) ischemic events (stroke), restenosis (e.g., after angioplasty), systemic lupus erythematosus nephritis, autoimmune diseases, inflammatory diseases, cardiovascular diseases, neurological diseases, diseases associated with protein misfolding, diseases associated with angiogenesis, hypertensive nephropathy and diabetic nephropathy, allergic diseases and respiratory diseases (such as anaphylaxis, asthma, chronic obstructive pulmonary disease, acute respiratory distress syndrome, cystic fibrosis, persistent rhinitis), and tissue injury (such as burns or chemical injury).

[0043] In some embodiments, the disease or condition associated with the contact activation system is hereditary angioedema (HAE). Hereditary angioedema (HAE) is also known as "Quincke's edema", C1-esterase inhibitor deficiency, C1 inhibitor deficiency, and hereditary angioneurotic edema (HANE). HAE is characterized by recurrent episodes of severe swelling (angioedema) that can affect, for example, the limbs, face, genitals, gastrointestinal tract, and airway. Symptoms of HAE include, for example, swelling in the arms, legs, lips, eyes, tongue, and / or throat; airway obstruction that may be accompanied by swelling of the larynx and sudden hoarseness; repeated episodes of abdominal cramps without an obvious cause; and / or swelling of the intestine that can be severe and lead to abdominal cramps, vomiting, dehydration, diarrhea, pain, and / or shock. Approximately one-third of individuals with HAE develop a non-itchy rash called erythema marginatum during an attack.

[0044] Swelling of the airway can be life-threatening and, in some patients, can result in death. The mortality rate is estimated to be 15 - 33%. HAE results in approximately 15,000 - 30,000 emergency hospital visits per year. Trauma or stress (e.g., dental procedures, illnesses (e.g., viral diseases such as colds and influenza), menstruation, and surgery) can trigger an attack of angioedema. To prevent acute attacks of HAE, patients can try to avoid specific stimuli that have previously triggered an attack. However, in many cases, attacks occur without a known trigger. Typically, the symptoms of HAE first appear in childhood and worsen during puberty. On average, untreated individuals have an attack every 1 - 2 weeks, and most episodes last about 3 - 4 days (ghr.nlm.nih.gov / condition / hereditary-angioedema). The frequency and duration of attacks vary widely among people with hereditary angioedema, even among people in the same family.

[0045] There are three types of HAE known as type I, type II, and type III. One in 50,000 people has HAE. Type I accounts for approximately 85% of cases, type II accounts for approximately 15% of cases, and type III is presumed to be very rare. Type III is the most recently described form and was initially thought to occur only in women, but families with affected males have been identified.

[0046] HAE is inherited in an autosomal dominant pattern, so an affected person may inherit the mutation from one affected parent. Also, new mutations in the gene can occur, and thus HAE can occur in people with no family history of this disorder. 20 - 25% of cases are estimated to arise from new spontaneous mutations.

[0047] Mutations in the SERPING1 gene cause hereditary angioedema types I and II. The SERPING1 gene gives instructions to produce C1 inhibitor protein, which is important for the suppression of inflammation. C1 inhibitor blocks the activity of certain proteins that promote inflammation. Mutations that cause type I hereditary angioedema reduce the level of C1 inhibitor in the blood. In contrast, mutations that cause type II result in the production of abnormally functioning C1 inhibitor. When the level of functional C1 inhibitor is not appropriate, an excessive amount of bradykinin is produced. Bradykinin promotes inflammation by increasing the leakage of body fluids into body tissues through the blood vessel walls. Excessive accumulation of body fluids in tissues causes the swelling episodes seen in individuals with types I and II hereditary angioedema.

[0048] Mutations in the F12 gene are associated with some cases of type III hereditary angioedema. The F12 gene provides instructions for producing coagulation factor XII. In addition to playing a crucial role in blood clotting (coagulation), factor XII is also an important stimulator of inflammation and is involved in the production of bradykinin. Certain mutations in the F12 gene result in the production of factor XII with increased activity. As a result, more bradykinin is generated, increasing the leakiness of blood vessel walls, which leads to episodes of swelling. The cause of other cases of type III hereditary angioedema remains unknown. In these cases, mutations in one or more yet-to-be-identified genes may be responsible for this disorder.

[0049] HAE can present similarly to other forms of angioedema caused by allergies or other medical conditions, but HAE differs significantly in terms of cause and treatment. If HAE is misdiagnosed as an allergy, it is most commonly treated with antihistamines, steroids, and / or epinephrine, which are usually ineffective for HAE (although epinephrine can be used for life-threatening reactions). Misdiagnosis has also led to unnecessary exploratory laparotomies in patients with abdominal swelling, and in some HAE patients, abdominal pain has been wrongly diagnosed as being psychosomatic.

[0050] C1 inhibitor therapy and other therapies for HAE are described in Kaplan, A.P., J Allergy Clin Immunol, 2010, 126(5):918-925.

[0051] The acute treatment of HAE attacks is carried out to stop the progression of swelling as quickly as possible. Intravenous administration of C1 inhibitor concentrate derived from donor blood is one of the acute treatments. However, this treatment is not available in many countries. In emergencies where C1 inhibitor concentrate is not available, fresh frozen plasma (FFP) can be used as an alternative because fresh frozen plasma (FFP) also contains C1 inhibitor.

[0052] Purified C1 inhibitor derived from human blood has been used in Europe since 1979. Several C1 inhibitor therapies are currently available in the United States, and two C1 inhibitor products are currently available in Canada. Sterile Berinert (CSL Behring) was approved by the FDA in 2009 for acute attacks. Nanofiltrated Cinryze® was approved by the FDA in 2008 for prophylaxis. Ruconest (Pharming) is a recombinant C1 inhibitor under development that does not carry the risk of transmission of infections caused by human blood-derived pathogens.

[0053] Treatment of acute HAE attacks may also include medication and / or intravenous fluids for pain relief.

[0054] Other therapies may stimulate the synthesis of C1 inhibitor or reduce the consumption of C1 inhibitor. Androgenic drugs such as danazol can reduce the frequency and severity of attacks by stimulating the production of C1 inhibitor.

[0055] Helicobacter pylori can cause abdominal attacks. Antibiotics for treating H. pylori will reduce abdominal attacks.

[0056] Newer therapies attack the contact cascade. Ecallantide (KALBITOR®) inhibits plasma kallikrein and is approved in the United States. Icatibant (Firazyr®, Shire) inhibits the bradykinin B2 receptor and is approved in Europe and the United States.

[0057] The diagnosis of HAE can rely on, for example, family history and / or blood tests. The test findings associated with types I, II, and III HAE are described, for example, in Kaplan, A.P., J Allergy Clin Immunol, 2010, 126(5):918-925. In type I HAE, the levels of C1 inhibitor, as well as C4, are decreased, but the level of C1q is normal. In type II HAE, the level of C1 inhibitor is normal or increased. However, the function of C1 inhibitor is abnormal. The level of C4 is decreased and the level of C1q is normal. In type III, the levels of C1 inhibitor, C4, and C1q may all be normal. The present disclosure is at least partially based on the identification of metabolites (Table 1) that have different levels in samples from HAE patients compared to healthy individuals. Measuring the levels of a set of biomarkers of these metabolites can be utilized to identify whether a subject has a disease such as HAE. In some embodiments, the method may be utilized to determine whether a patient has had an HAE attack or is having an HAE attack.

[0058] The symptoms of HAE can be evaluated, for example, using a questionnaire (e.g., a questionnaire answered by the patient, clinician, or family). Such questionnaires are known in the art and include, for example, visual analog scales. See, for example, McMillan, C.V. et al. Patient. 2012;5(2):113-26.

[0059] The biological samples described herein may be subjected to analysis by measuring the levels of a set of biomarkers as described herein in the biological samples. The levels (e.g., amounts) of the biomarkers disclosed herein or changes in the levels of the biomarkers may be evaluated using the assays described herein and / or assays known in the art. One or more of the biomarkers described herein may be analyzed using conventional methods. In some embodiments, the level of a biomarker is evaluated or measured by directly detecting metabolites in the biological sample. Alternatively, or in addition, the level of a metabolite may be indirectly evaluated or measured in the biological sample, for example, by detecting the level of a binding substance (e.g., an antibody in an immunoassay) that binds to the metabolite or by detecting the activity of the metabolite.

[0060] The type of detection assay used for the detection and / or quantification of biomarkers of the contact activation system (such as those presented herein) will depend on several parameters, including the particular situation (e.g., clinical or research use) in which the assay will be used, the type and number of biomarkers to be detected, and the type and number of patient samples to be run in parallel.

[0061] In some embodiments, biomarkers are measured using mass spectrometry. Generally, mass spectrometry relies on separating ions of molecules based on their mass-to-charge ratio. In some embodiments, the mass spectrometry is tandem mass spectrometry involving two or more mass spectrometries. The metabolite levels may be measured by comparing the mass spectrum obtained from the mass spectrometry of a sample with the mass spectrum of another sample or a reference sample or a control sample. The identity of the metabolite in the spectrum may be confirmed by comparing the spectrum with a library of spectra or the spectra of known metabolites. In some embodiments, before detecting metabolite biomarkers, the components of the biological sample may be separated using, for example, chromatography methods as described herein. Examples of separation methods include, for example, gas chromatography, liquid chromatography, capillary electrophoresis, and ion mobility. In some embodiments, biomarkers are measured using liquid chromatography followed by mass spectrometry.

[0062] In some embodiments, biomarkers are measured using chromatography methods. Chromatography enables the separation of molecules (in the mobile phase) based on their movement through a medium (stationary phase). Examples of chromatography methods that may be compatible with the methods described herein include, but are not limited to, gas chromatography, liquid chromatography, reverse-phase chromatography, ion-exchange chromatography, size-exclusion chromatography, and affinity chromatography.

[0063] In some embodiments, the biomarker is measured using an immunoassay. Examples of immunoassays include, but are not limited to, immunoblotting assays (western blot), enzyme-linked immunosorbent assay (ELISA) (e.g., sandwich ELISA), radioimmunoassay, detection assays utilizing electrochemiluminescence, magnetic immunoassay, lateral flow assay, and related techniques. Additional suitable immunoassays for detecting the biomarkers presented herein will be apparent to those skilled in the art. However, it will be apparent to those skilled in the art that the present disclosure is not limited to immunoassays, and that detection assays that rely on chromogenic substrates may also be useful for detecting and / or quantifying contact-based biomarkers as presented herein.

[0064] ELISA is known in the art (see, e.g., Crowther, John R (2009), "The ELISA Guidebook", 2nd ed., Humana Press and Lequin R (2005), "Enzyme immunoassay (EIA) / enzyme-linked immunosorbent assay (ELISA)", Clin.Chem. 51(12):2415-8), and an exemplary ELISA is described herein. Kits for performing ELISA are also known in the art and are commercially available (see, e.g., ELISA kits from Life Technologies and BD Biosciences).

[0065] In some embodiments, immunoassays are used to measure the levels of metabolite biomarker(s). The immunoassays described herein may be in the format of a sandwich ELISA where a first binding substance that specifically binds to metabolites of a set of biomarkers is immobilized on a support member. The support member may then be incubated with a biological sample as described herein for a suitable time under conditions that allow for the formation of a complex between the binding substance and the metabolite in the sample. The complex may then be detected using a detection substance that binds to the metabolite, the binding substance-metabolite complex, or the binding substance. The detection substance may be conjugated to a label that can emit a signal directly or indirectly. The intensity of the signal represents the level of the metabolite in the sample. In some embodiments, the detection substance is detected and its level represents the level of the metabolite in the sample.

[0066] Any binding substance that specifically binds to the desired metabolite may be used in the methods and kits described herein to measure the level of the metabolite in a biological sample. In some embodiments, the binding substance is an antibody that specifically binds to the desired metabolite. In some embodiments, the binding substance is an aptamer antibody that specifically binds to the desired metabolite. In some embodiments, the sample may be contacted with two or more binding substances that bind to different metabolites, simultaneously or sequentially (e.g., multiplex analysis; e.g., SOMAScan™ assay (SOMALogic)). The biological sample is contacted with the binding substance under appropriate conditions. Generally, the term "contact" refers to bringing the binding substance into contact with the biological sample or agent for a suitable time sufficient for the formation of a complex between the substance and the metabolite (if present) in the sample. In some embodiments, the contact is effected by capillary action across the surface of a support membrane by the biological sample or agent.

[0067] In some embodiments, the immunoassay may be performed on a low-throughput platform that includes a single immunoassay format. For example, using a low-throughput platform, the presence and amount of metabolites in a biological sample (e.g., a biological tissue, a tissue extract) may be measured for diagnostic methods, for monitoring the progression of a disease and / or treatment, and / or for predicting whether a disease or disorder may benefit from a particular treatment.

[0068] In some embodiments, it may be necessary to immobilize a binding substance on a support member. The method for immobilizing the binding substance will depend on factors such as the nature of the binding substance and the material of the support member, and may require a specific buffer. Such methods will be apparent to those skilled in the art. For example, a set of biomarkers in a biological sample as described herein may also be measured using any of the kits and / or detection devices described herein.

[0069] In some embodiments, the biomarker is measured by an enzyme-coupled assay. For example, a biomarker present in a biological sample may be used as a substrate for an enzymatic reaction that can be coupled to a second reaction. Such a reaction results in the production of a product (e.g., a cofactor) that can be detected, for example, by a change in absorbance or fluorescence.

[0070] As used herein, the terms "measuring" or "measurement" or "detecting" or "detection" mean evaluating the presence, absence, quantity or amount (which may be an effective amount) of a substance in a sample (including deriving a qualitative or quantitative concentration level of such a substance), or evaluating a value or classification of a subject.

[0071] An assay (e.g., Western blot assay) may further involve the use of a commercially available quantitative imaging system (e.g., LI-COR imaging technology) (see, e.g., the Odyssey® CLx infrared imaging system from LI-COR Biosciences). In some embodiments, an electrochemiluminescence detection assay, or an assay that relies on a combination of electrochemiluminescence and patterned array technology, is used (e.g., the ECL or MULTI-ARRAY technology assay from Meso Scale Discovery (MSD)).

[0072] In any of the methods described herein, the levels of metabolites of a set of biomarkers may be compared to the levels of metabolites in a control sample or a reference sample.

[0073] The methods and kits described herein (also with any of the sets of metabolite biomarkers described herein) may be applied to the assessment of diseases associated with the contact activation system (such as those described herein).

[0074] (ii) Diagnostic and / or prognostic uses The levels of the metabolites presented in Table 1 detected in a sample from a subject can be used as reliable biomarkers for diagnosing a disease associated with the contact activation system (e.g., HAE), for monitoring the progression of such a disease, for evaluating the effectiveness of treatment for the disease, for identifying patients suitable for a particular treatment, and / or for predicting the onset of the disease in a subject.

[0075] Accordingly, methods for diagnosing and prognosticating diseases related to contact activation systems based on the levels of sets of biomarkers in biological samples obtained from a subject are described herein. In some embodiments, the levels of biomarkers measured using any of the methods described herein can be utilized to assess whether a subject (e.g., a human patient) from whom a biological sample was obtained has or is at risk of having a disease related to a contact activation system, such as a disease related to plasma kallikrein (e.g., HAE) or an autoimmune disease (such as RA, UC, and Crohn's disease).

[0076] In some embodiments, the level of the biomarker may then be compared to a reference sample or a control sample to determine a value indicative of the amount of metabolite in the sample. In some embodiments, the value for the biomarker is obtained by comparing the level of the metabolite in the sample to the level of another metabolite in the sample (e.g., an internal control or an internal standard). Such a value for the biomarker may be a value normalized against an internal control or an internal standard. The value of the biomarker may be compared to a reference value to determine whether the subject has or is at risk of having a disease related to the contact system. The reference value may represent the level of the corresponding biomarker in a subject (e.g., a human subject) without the target disease. In some embodiments, if the level or value of the biomarker is higher than the reference level or reference value, the subject may be identified as having or being at risk of having a disease related to the contact activation system. In some embodiments, if the level or value of the biomarker is lower than the reference level or reference value, the subject may be identified as having or being at risk of having a disease related to the contact activation system.

[0077] In some embodiments, the level of the biomarker may be compared to a predetermined threshold for the metabolite such that deviation therefrom may indicate that the subject has a disease associated with the contact system. The predetermined threshold may represent a biomarker value that distinguishes the level of the biomarker in patients with the target disease from the level of the biomarker in patients without the target disease.

[0078] In some embodiments, the set of biomarkers includes two or more metabolites that are present differently (increased and / or decreased) in a subject having or at risk of having a disease compared to a healthy individual. In some examples, the set of biomarkers includes at least one metabolite biomarker that is increased in level in a subject having or at risk of having a disease and at least one metabolite biomarker that is decreased in level in a subject having or at risk of having a disease. In some embodiments, the set of biomarkers includes two or more metabolite biomarkers that have an increased level in a subject having or at risk of having a disease compared to that of a healthy control. In other embodiments, the set of biomarkers includes two or more metabolite biomarkers that have a decreased level in a subject having or at risk of having a disease compared to that of a healthy control.

[0079] In some embodiments, the control sample or reference sample is a biological sample obtained from a healthy individual. In some embodiments, the control sample or reference sample includes a known amount of the metabolite being evaluated.

[0080] As used herein, a control sample may be a healthy subject or healthy individual who is apparently free of the target disease (e.g., a disease associated with the contact system) at the time the level of the metabolite(s) is measured or a healthy subject or healthy individual without a medical history of the disease.

[0081] The term "control subject" encompasses an individual subject, or a group of subjects having similar characteristics (e.g., a group of healthy individuals having certain characteristics (e.g., age, gender, ethnicity, etc.) that match those of the candidate subject). In some embodiments, the level of a biomarker in a control subject is used to establish a reference value (e.g., the average level of the biomarker in control subjects encompassing the group of subjects) against which the level of the biomarker in a candidate subject or test subject can be compared.

[0082] The control level refers to the level of a set of metabolite biomarkers as described herein in a control subject. The control level may be a predetermined level or threshold. Such a predetermined level may represent the level of that metabolite in a population of subjects without or at low risk of having the target disease (e.g., the average level in a population of healthy subjects). It may also represent the level of that metabolite in a population of subjects having the target disease.

[0083] The predetermined level can take various forms. For example, it may be a single cut-off value such as a median or an average value. In some embodiments, such a predetermined level may be established based on a comparison group (e.g., a comparison group where one defined group is known to have the target disease and another defined group is known not to have the target disease). Alternatively, the predetermined level may be a range (e.g., a range representing the level of that metabolite in the control population).

[0084] The control level as described herein can be determined by conventional techniques. In some examples, the control level can be obtained by performing a conventional method (e.g., the same assay as used to obtain the level of that metabolite in a test sample as described herein) on a control sample also as described herein. In other examples, the level of that metabolite can be obtained from members of a control population and the results analyzed, for example, by a computational program, to obtain a control level (predetermined level) representing the level of that metabolite in the control population.

[0085] By comparing the level of a biomarker in a sample obtained from a candidate subject with a reference value as described herein, it can be determined whether the candidate subject has a disease associated with the contact system (e.g., HAE) or is at risk thereof. For example, if the level of a biomarker(s) in a sample of a candidate subject deviates from the reference value (e.g., is increased compared to the reference value), the candidate subject may be identified as having the disease or being at risk thereof. When the reference value represents a range of values of the level of a biomarker in a population of subjects having the target disease, the fact that the value of the biomarker in the candidate's sample is within that range indicates that the candidate subject has the target disease or is at risk thereof.

[0086] As used herein, "elevated level" or "level above the reference value" means that the level of a biomarker is higher than the reference value (such as a predetermined threshold of the level of the biomarker in a control sample). The control level is described in detail herein. Elevated levels of a biomarker include levels of the biomarker that exceed the reference value by, for example, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400% or 500% or more. In some embodiments, the level of a biomarker in a test sample is at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, 100-fold, 150-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold or 10000-fold or more higher than the level of the biomarker in a reference sample.

[0087] As used herein, "reduced level" or "level below a reference value" means that the level of a biomarker is lower than a reference value (such as a predetermined threshold of the biomarker in a control sample). Control levels are described in detail herein. Reduced levels of a biomarker include levels of the biomarker that are lower than the reference value by, for example, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, or 500% or more. In some embodiments, the level of the biomarker in the test sample is at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, 100-fold, 150-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold, or 10000-fold or more lower than the level of the biomarker in the reference sample.

[0088] In some embodiments, the subject of interest is a human patient having symptoms of a disease associated with a contact activation system, such as a pKal-mediated disorder (e.g., HAE) or an autoimmune disease (such as RA, UC, and Crohn's disease). For example, the subject has edema; swelling that is complete or mainly peripheral; urticaria; erythema, pain, and swelling in the absence of evidence of infection; non-histamine-mediated edema; recurrent episodes of swelling; or combinations thereof. In other embodiments, the subject does not have symptoms of a pKal-mediated disorder, does not have a history of symptoms of a pKal-mediated disorder, or does not have a history of a pKal-mediated disorder such as HAE at the time the sample is taken. In still other embodiments, the subject is resistant to antihistamine therapy, corticosteroid therapy, or both.

[0089] Subjects identified by the methods described herein may receive a suitable treatment, such as treatment with a pKal inhibitor, as described herein.

[0090] Considering the correlation between the level of a biomarker and such a disease, the assay methods and kits described herein can also be applied to evaluate the effectiveness of treatment for diseases related to the contact system (such as those described herein). For example, a plurality of biological samples (e.g., blood samples or plasma samples) may be collected from a subject to be treated before and after treatment, or during the course of treatment. The level of the biomarker may be measured by any of the assay methods as described herein, and accordingly, the value (e.g., amount) of the biomarker may be determined. For example, if an elevated level of the biomarker indicates that the subject has the target disease and the level of the biomarker decreases after treatment or over the course of treatment (compared to the level of the biomarker in a previously collected sample, the level of the biomarker in a later collected sample), this indicates that the treatment is effective. As another example, if a decreased level of the biomarker indicates that the subject has the target disease and the level of the biomarker increases after treatment or over the course of treatment (compared to the level of the biomarker in a previously collected sample, the level of the biomarker in a later collected sample), this indicates that the treatment is effective. In some examples, the treatment involves an effective amount of a therapeutic agent (such as a plasma kallikrein inhibitor, a bradykinin B2 receptor antagonist, or a C1 esterase inhibitor (C1-INH)). Examples of therapeutic agents include, but are not limited to, lanadelumab, ecallantide, icatibant, and human plasma-derived C1-INH.

[0091] If a subject is identified as not responding to treatment, a higher dose and / or more frequent dosing of the therapeutic agent is administered to the identified subject. In some embodiments, the dose or frequency of dosing of the therapeutic agent is maintained, reduced, or discontinued in a subject identified as responding to treatment or not requiring further treatment. Alternatively, a different treatment may be applied to a subject found not to respond to the initial treatment.

[0092] In other embodiments, the value of a biomarker or set of biomarkers can also be used to identify that the disorder is related to the contact system or that the disorder may be treatable, for example, by a pKal inhibitor. To carry out this method, the level of a biomarker in a sample (e.g., a blood sample or plasma sample) taken from a subject having the target disease may be measured by a suitable method (e.g., those described herein such as mass spectrometry, chromatography, immunoassay, etc.). If the level of the biomarker deviates from a reference value (e.g., is elevated or decreased), this indicates that a pKal inhibitor may be effective in treating the disease. If the disease is identified as being sensitive to a pKal inhibitor (treatable by a pKal inhibitor), the method may further comprise administering to a subject having the disease an effective amount of a pKal inhibitor (such as an anti-pKal antibody or an inhibitory peptide (e.g., lanadelumab, ecallantide, etc.)), a bradykinin 2 receptor inhibitor (e.g., icatibant), and / or C1-INH (e.g., human plasma-derived C1-INH).

[0093] Also within the scope of the present disclosure is a method of assessing the severity or condition of a disorder related to the contact system. For example, as described herein, HAE may be in a quiescent state (baseline state) during which the subject may not experience symptoms of the disease. HAE attacks are typically recurrent episodes, lasting, for example, 2 - 5 days, during which the subject may experience pain and swelling, for example, in the hands, feet, face, gastrointestinal tract, genitals, and pharynx (throat). In some embodiments, the level of one or more biomarkers indicates whether a subject is going to experience an HAE attack, is experiencing an HAE attack, or is about to experience an HAE attack soon. In some embodiments, the method involves comparing the level of a biomarker in a sample obtained from a subject having HAE with the level of that biomarker in a sample obtained from the same subject (e.g., a sample obtained from the same subject in a baseline state or a sample obtained from the same subject during an HAE attack).

[0094] Other aspects of the present disclosure provide a method for assessing the risk of an episode of a disease (e.g., an HAE episode). As described herein, an HAE episode is typically a recurrent episode during which a subject may experience symptoms (such as pain and swelling). In some embodiments, the level of one or more biomarkers indicates whether a subject is at risk of having an HAE episode. In some embodiments, the method involves comparing the level of a biomarker in a sample obtained from a subject having HAE to the level of that biomarker in a sample obtained from the same subject (e.g., a sample obtained from the same subject in a basal state or a sample obtained from the same subject during an HAE episode). For example, an increase (or decrease) in the level of a biomarker associated with an HAE episode compared to the level of the biomarker in another sample from the subject may indicate that the subject is at risk of having an HAE episode.

[0095] In some embodiments, the method involves comparing the level of a biomarker in a sample obtained from a subject having HAE to the level of the biomarker in a control sample or a reference sample. In some embodiments, the level of the biomarker in the control sample or the reference sample represents the level of the biomarker indicative of the attack state of HAE. For example, a level of a biomarker in a sample obtained from a subject having HAE that is similar to the level of the biomarker for reference indicative of the attack state of HAE may indicate that the subject is at risk of having an HAE episode. In some embodiments, the level of the biomarker in the control sample or the reference sample represents the level of the biomarker indicative of HAE in a basal state. For example, a level of a biomarker in a sample obtained from a subject having HAE that deviates (increases or decreases) from the level of the biomarker for reference indicative of HAE in a basal state may indicate that the subject is at risk of having an HAE episode.

[0096] (iii) Non-clinical uses Furthermore, any level of the set of biomarkers described herein may be used for research purposes. A number of diseases associated with the contact activation system have been identified, but there is a possibility that other diseases are mediated by similar mechanisms or involve similar components. In some embodiments, the methods described herein may be used to identify a disease as being associated with the contact activation system or a component of the contact activation system. In some embodiments, the methods described herein may be used to study the mechanism (e.g., discovery of a novel biological pathway or process involved in the development of the disease) or progression of a disease.

[0097] In some embodiments, the levels of a set of biomarkers as described herein may be utilized in the development of new therapies for diseases associated with the contact activation system. For example, the levels of a set of biomarkers may be measured in samples obtained from subjects undergoing a new treatment (e.g., a clinical trial). In some embodiments, the levels of the set of biomarkers may indicate the effectiveness of a new therapy or the progression of a disease in a subject before, during, or after a new treatment.

[0098] Kits and detection devices for measuring a set of metabolite biomarkers The present disclosure also provides kits and detection devices for use in measuring the levels of a set of biomarkers as described herein. Such a kit or detection device may include a binding substance that specifically binds to a metabolite biomarker (such as those listed in Table 1). For example, such a kit or detection device may include at least two binding substances that are specific for two different metabolite biomarkers selected from Table 1. In some examples, the kit or detection device includes a binding substance that is specific for all members of the set of metabolite biomarkers described herein.

[0099] In some embodiments, one or more of the binding substances are antibodies that specifically bind to metabolites of a set of biomarkers. In some embodiments, one or more binding substances are aptamers (such as peptide aptamers or oligonucleotide aptamers) that specifically bind to metabolites of a set of biomarkers.

[0100] In some embodiments, the kit further comprises a detection substance (e.g., an antibody that binds to the binding substance) for detecting the binding of the binding substance to metabolites (s) of a set of biomarkers. The detection substance may be conjugated to a label. In some embodiments, the detection substance is an antibody that specifically binds to at least one of the binding substances. In some embodiments, the binding substance comprises a tag that can be recognized by the detection substance and to which the detection substance can bind directly or indirectly.

[0101] In some embodiments, the kit or device further comprises a support member. In some embodiments, the support member is a membrane such as a nitrocellulose membrane, a polyvinylidene fluoride (PVDF) membrane, or a cellulose acetate membrane. In some examples, the immunoassay may be in the format of a Western blot assay or a lateral flow assay format.

[0102] In some embodiments, the support member is a multi-well plate, such as an ELISA plate. In some embodiments, the immunoassays described herein may be performed on a high-throughput platform. In some embodiments, multi-well plates (e.g., 24-well, 48-well, 96-well or 384-well or greater plates) may be used for high-throughput immunoassays. Individual immunoassays may be performed simultaneously in each well. Thus, generally, it is desirable to use a plate reader to measure multiple wells simultaneously to increase the throughput of the assay. In some embodiments, a plate reader capable of simultaneously imaging multiple wells (e.g., 4 wells, 16 wells, 24 wells, 48 wells, 96 wells or 384 or more wells) may be used for this platform. For example, a commercially available plate reader (e.g., the plate::vision system available from Perkin Elmer (Waltham, MA)) may be used. This plate reader is capable of performing kinetic fluorescence analysis. The plate::vision system has an optical system with high light collection efficiency and a special optical system designed for simultaneous analysis of 96 wells. Further suitable parallel plate readers include, but are not limited to, SAFIRE (Tecan, San Jose, CA), FLIPRTETRA® (Molecular Devices, Union City, CA), FDSS7000 (Hamamatsu, Bridgewater, NJ) and CellLux (Perkin Elmer, Waltham, MA).

[0103] In a kit or detection device, one or more of the binding substances may be immobilized on a support member, which may be a membrane, beads, slides or multi-well plate. The selection of a suitable support member for an immunoassay will depend on various factors, such as the number of samples and the method of detecting the signal emitted from the label conjugated to the second agent.

[0104] The kit may also include one or more buffers as described herein (including but not limited to coating buffers, blocking buffers, washing buffers, and / or stopping buffers).

[0105] In some embodiments, the kit may include instructions for use according to any of the methods described herein. The instructions included may include instructions on the use of the components included in the kit for measuring the levels of metabolites of a set of biomarkers in a biological sample taken from a subject such as a human patient.

[0106] Instructions regarding the use of the kit generally include information about the amounts of each component and suitable conditions for performing the assay methods described herein. The components in the kit may be in unit doses, bulk packages (e.g., multi-dose packages), or sub-unit doses. The instructions provided in the kits of the present disclosure are typically instructions written on a label or package insert (e.g., a sheet of paper included in the kit), but machine-readable instructions (e.g., instructions stored on a magnetic or optical storage disk) are also acceptable.

[0107] The label or package insert indicates that the kit is used to evaluate the levels of metabolites of a set of biomarkers. Instructions may be provided for performing any of the methods described herein.

[0108] The kits of the present disclosure are contained within a suitable package. Suitable packages include, but are not limited to, vials, bottles, jars, flexible packages (e.g., sealed Mylar or plastic bags), etc. Also contemplated are packages for use in combination with certain devices such as inhalers, nasal administration devices (e.g., nebulizers) or infusion devices (such as mini pumps). The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper penetrable by a hypodermic needle). The container may also have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper penetrable by a hypodermic needle).

[0109] Optionally, the kit may provide additional components such as information for determination (such as control samples and / or standard or reference samples). Typically, the kit includes a container and a label or package insert(s) on or associated with the container. In some embodiments, the present disclosure provides a product comprising the contents of the above-described kit.

[0110] Treatment of diseases associated with contact activation systems Subjects at risk of or suffering from a disease associated with a contact activation system identified using the methods described herein may be treated with any suitable therapeutic agent. In some embodiments, the methods provided include selecting treatment for a subject based on the output of the methods described (e.g., measurement of the levels of a set of biomarkers).

[0111] In some embodiments, the methods described herein provide a method for identifying a subject as a candidate for prophylactic treatment. As used herein, "prophylactic" treatment encompasses any therapy or treatment regimen aimed at preventing or reducing the occurrence of a disease (e.g., an HAE attack). In some embodiments, the method further comprises administering prophylactic treatment to the subject. Any of the therapeutic agents described herein (e.g., a pKal inhibitor such as lanadelumab). In some embodiments, the levels of a set of metabolite biomarkers in a sample obtained from a subject indicate that the patient has or is at risk of having HAE (e.g., by comparing the levels of the metabolite biomarkers to the levels in a reference sample or control sample). Any subject who has or is at risk of having HAE may be administered prophylactic treatment. The selection of an appropriate therapeutic agent and dosing regimen for prophylactic treatment will be apparent to those of skill in the art.

[0112] In some embodiments, the method comprises either or both of the selection or administration of a therapeutic agent (e.g., a kallikrein inhibitor, a bradykinin B2 receptor inhibitor, and / or a C1 esterase inhibitor) for administration to the subject based on the output of an assay (e.g., detection of a biomarker).

[0113] In some embodiments, the therapeutic agent is administered to the subject one or more times. In some embodiments, a plasma kallikrein inhibitor is administered to the subject. In some embodiments, the kallikrein inhibitor is a peptide, a small molecule inhibitor, a kallikrein antibody, or a fragment thereof. In some embodiments, an antagonist of the bradykinin B2 receptor is administered to the subject. In some embodiments, C1-INH is administered to the subject.

[0114] Therapeutic agents (e.g., kallikrein inhibitors, bradykinin B2 receptor inhibitors and / or C1-INH) may be given together with another therapy as part of a combination therapy for the treatment of diseases or conditions involving the contact activation system. The combination therapy (e.g., combination therapy with one or more of a kallikrein inhibitor, a bradykinin B2 receptor antagonist or a C1-INH replacement agent, e.g., combination therapy with one or more of a kallikrein inhibitor, a bradykinin B2 receptor antagonist or a C1-INH replacement agent and another therapy) may be provided in a plurality of different configurations. The first agent may be administered before or after the administration of the other therapy. In some situations, the first agent and another therapy (e.g., a therapeutic agent) are given simultaneously or in close temporal proximity (e.g., during the same treatment session, at short injection intervals). The first agent and the other therapy may also be given at longer time intervals.

[0115] Therapeutic agent Plasma kallikrein-binding substances (e.g., binding proteins, e.g., polypeptides, e.g., inhibitory polypeptides, e.g., antibodies, e.g., inhibitory antibodies, or other binding substances (e.g., small molecules)) are useful therapeutic agents for various diseases and conditions (e.g., diseases and conditions involving plasma kallikrein activity). For example, in some embodiments, the diseases and conditions involving plasma kallikrein activity are hereditary angioedema (HAE). In some embodiments, plasma kallikrein-binding substances such as plasma kallikrein inhibitors are administered to subjects at risk of or suffering from diseases associated with the contact activation system.

[0116] Some useful proteinaceous inhibitors of kallikrein (either tissue kallikrein and / or plasma kallikrein) contain a Kunitz domain. As used herein, a "Kunitz domain" is a polypeptide domain having at least 51 amino acids and containing at least two (preferably three) disulfides. This domain is folded such that the first and sixth cysteines, the second and fourth, and the third and fifth cysteines form disulfide bonds (e.g., in a Kunitz domain having 58 amino acids, the cysteines may be at positions corresponding to amino acids 5, 14, 30, 38, 51 and 55 of the BPTI homology sequence presented below, and the disulfides may be formed between cysteines at positions 5 and 55, 14 and 38, and 30 and 51), or, if two disulfides are present, they may be formed between the corresponding subsets of those cysteines. The spacing between each pair of cysteines can be within 7, 5, 4, 3, 2, 1 or 0 amino acids of the spacing between the positions corresponding to 5-55, 14-38 and 30-51 according to the numbering of the BPTI sequence presented below. The BPTI sequence can be used as a reference for indicating specific positions in any common Kunitz domain. Comparison of a Kunitz domain of interest with BPTI can be performed by identifying the best aligned alignment that maximizes the number of matching cysteines.

[0117] The (high-resolution) 3D structure of the knotted domain of BPTI is known. One of the X-ray structures has been deposited in the Brookhaven Protein Data Bank as "6PTI". The 3D structures of several BPTI homologs (Eigenbrot et al., Protein Engineering (1990) 3(7):591-598; Hynes et al., Biochemistry (1990) 29:10018-10022) are known. The sequences of at least 81 knotted domains are known. Known human homologs include the three knotted domains of LACI, also known as tissue factor pathway inhibitor (TFPI) (Wun et al., J. Biol. Chem. (1988) 263(13):6001-6004; Girard et al., Nature (1989) 338:518-20; Novotny et al, J. Biol. Chem. (1989) 264(31):18832-18837), the two knotted domains of inter-α-trypsin inhibitor APP-I (Kido et al. J. Biol. Chem. (1988) 263(34):18104-18107), the knotted domain of collagen, the three knotted domains of TFPI-2 (Sprecher et al., PNAS USA (1994) 91:3353-3357), the knotted domain of hepatocyte growth factor activator inhibitor type 1, the knotted domain of hepatocyte growth factor activator inhibitor type 2, and the knotted domain described in US Patent Application Publication No. 2004-0152633. LACI is a human serum phosphoglycoprotein with a molecular weight of 39 kDa that contains three knotted domains (amino acid sequence in Table 2).

[0118]

Table 2

[0119] The above-mentioned knotted domains are designated as LACI-K1 (residues 50-107), LACI-K2 (residues 121-178), and LACI-K3 (213-270). The cDNA sequence of LACI was reported by Wun et al. (J. Biol. Chem. (1988) 263(13):6001-6004). Girard et al. (Nature (1989) 338:518-20) reported a mutagenesis study in which the P1 residue of each of the three knotted domains was altered. LACI-K1 inhibits factor VIIa (F.VIIa) when F.VIIa is complexed with tissue factor, and LACI-K2 inhibits factor Xa.

[0120] Proteins containing exemplary knotted domains include the following (the numbers in parentheses are SWISS-PROT accession numbers):

Chemical formula

[0121] Knitted domains can be identified from an array database using various methods. For example, a known amino acid sequence, consensus sequence, or motif of a knitted domain (e.g., ProSite Motif) can be searched against, for example, the Pfam database of HMMs (Hidden Markov Models) using BLAST (e.g., using default parameters for Pfam searches; against the SMART database; or against the ProDom database), against the GenBank sequence database (National Center for Biotechnology Information, National Institutes of Health, Bethesda MD). For example, the Pfam accession number PF00014 of Pfam Release 9 provides numerous knitted domains and HMMs for identifying knitted domains. The description of the Pfam database can be found in Sonhammer et al. Proteins (1997) 28(3):405 - 420, and a detailed description of HMMs can be found, for example, in Gribskov et al. Meth.Enzymol. (1990) 183:146 - 159; Gribskov et al. Proc.Natl.Acad.Sci.USA (1987) 84:4355 - 4358; Krogh et al. J.Mol.Biol. (1994) 235:1501 - 1531; and Stultz et al. Protein Sci. (1993) 2:305 - 314. The SMART database of HMMs (Simple Modular Architecture Research Tool, EMBL, Heidelberg, DE) is as described in Schultz et al. Proc.Natl.Acad.Sci.USA (1998) 95:5857 and Schultz et al. Nucl.Acids Res(2000) 28:231.The SMART database contains domains identified by profile analysis with the hidden Markov models of the HMMer2 search program (R. Durbin et al. (1998) "Biological sequence analysis: probabilistic models of proteins and nucleic acids", Cambridge University Press). This database is also annotated and monitored. The ProDom protein domain database consists of the automatic compilation of homologous domains (Corpet et al., Nucl. Acids Res. (1999) 27: 263-267). The current version of ProDom was constructed using recursive PSI-BLAST searches of the SWISS-PROT 38 and TREMBL protein databases (Altschul et al., Nucleic Acids Res. (1997) 25: 3389-3402; Gouzy et al., Computers and Chemistry (1999) 23: 333-340). This database automatically generates the consensus sequence of each domain. Prosite lists knitted domains as motifs and identifies proteins containing knitted domains. See, for example, Falquet et al., Nucleic Acids Res. (2002) 30: 235-238.

[0122] The knotted domain interacts with the target protease mainly using the amino acids within two loop regions (the "binding loops"). The first loop region is around the residues corresponding to amino acids 13 - 20 of BPTI. The second loop region is around the residues corresponding to amino acids 31 - 39 of BPTI. In an exemplary library of the knotted domain, one or more amino acid positions within the first and / or second loop regions are altered. When screening for a knotted domain that interacts with kallikrein or selecting a variant with improved affinity, particularly useful positions to be altered include positions 13, 15, 16, 17, 18, 19, 31, 32, 34 and 39 with respect to the sequence of BPTI. At least some of these positions are expected to be in close contact with the target protease. Also, it is useful to alter other positions (e.g., positions adjacent to the aforementioned positions in the three-dimensional structure).

[0123] The "framework region" of the knotted domain is defined as the residues that are part of the knotted domain, specifically excluding the residues within the first and second binding loop regions (i.e., around the residues corresponding to amino acids 13 - 20 of BPTI and amino acids 31 - 39 of BPTI). Conversely, residues outside the binding loops may allow a wider range of amino acid substitutions (e.g., conservative substitutions and / or non-conservative substitutions).

[0124] In one embodiment, these knotted domains are variant forms of a loop structure that includes knotted domain 1 of human lipoprotein-associated coagulation inhibitor (LACI). LACI contains three clearly defined internal peptide loop structures that are paradigmatic knotted domains (Girard, T. et al., Nature (1989) 338:518-520). Variants of LACI's knotted domain 1 described herein have been screened and isolated and these bind to kallikrein with improved affinity and specificity (see, e.g., U.S. Patent Nos. 5,795,865 and 6,057,287). These methods can also be applied to the frameworks of other knotted domains to obtain other knotted domains that interact with kallikrein (e.g., plasma kallikrein). A useful modulator of kallikrein function, as determined using kallikrein binding assays and kallikrein inhibition assays, typically binds to and / or inhibits kallikrein.

[0125] In some embodiments, the plasma kallikrein inhibitor binds to the active form of plasma kallikrein. In some embodiments, the plasma kallikrein inhibitor binds to and inhibits plasma kallikrein (e.g., human plasma kallikrein and / or mouse kallikrein). Exemplary polypeptide-based plasma kallikrein agents are disclosed in U.S. Patent No. 5,795,865, U.S. Patent No. 5,994,125, U.S. Patent No. 6,057,287, U.S. Patent No. 6,333,402, U.S. Patent No. 7,628,983, and U.S. Patent No. 8,283,321, U.S. Patent No. 7,064,107, U.S. Patent No. 7,276,480, U.S. Patent No. 7,851,442, U.S. Patent No. 8,124,586, U.S. Patent No. 7,811,991 and U.S. Patent Application Publication No. 20110086801, the entire contents of each of which are incorporated herein by reference. In some embodiments, the plasma kallikrein inhibitor is an inhibitory polypeptide or an inhibitory peptide. In some embodiments, the inhibitory peptide is ecallantide (also referred to as DX-88 or KALBITOR®; SEQ ID NO: 3). In some embodiments, the kallikrein inhibitor comprises, or consists of, a sequence of about 58 amino acids of amino acids 3 to 60 of SEQ ID NO: 3, or a DX-88 polypeptide having the 60 amino acid sequence of SEQ ID NO: 3.

[0126] Glu Ala Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg Ala Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp (SEQ ID NO: 3).

[0127] The plasma kallikrein inhibitor may be a full-length antibody (e.g., IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA (e.g., IgA1, IgA2), IgD, and IgE), or may only contain an antigen-binding fragment (e.g., Fab fragment, F(ab’)2 fragment, or scFv fragment). The binding protein may contain two heavy-chain immunoglobulins and two light-chain immunoglobulins, or may be a single-chain antibody. The plasma kallikrein inhibitor may be a recombinant protein such as a humanized antibody, CDR-grafted antibody, chimeric antibody, deimmunized antibody, or in vitro generated antibody, and optionally may contain a constant region derived from the sequence of a human germline immunoglobulin. In one embodiment, the plasma kallikrein inhibitor is a monoclonal antibody.

[0128] Exemplary plasma kallikrein-binding proteins are disclosed in U.S. Patent Application Publication No. 20120201756, the entire contents of which are incorporated herein by reference. In some embodiments, the kallikrein-binding protein is an antibody (e.g., a human antibody) having a light chain and / or a heavy chain of an antibody selected from the group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01 (also referred to as DX-2922), X81-B01, X67-D03, X67-G04, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X124-G01 (also referred to herein as DX-2930 or ranalizumab), X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04. In some embodiments, the plasma kallikrein-binding protein competes with or binds to the same epitope as M162-A04, M160-G12, M142-H08, X63-G06, X101-A01 (also referred to as DX-2922), X81-B01, X67-D03, X67-G04, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X124-G01, X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04. In some embodiments, the plasma kallikrein-binding protein is ranalizumab. See U.S. Patent Application Publication No. 20110200611 and U.S. Patent Application Publication No. 20120201756, which are incorporated herein by reference.

[0129] An example of a plasma kallikrein inhibitory antibody is ranalizumab. The amino acid sequences of the heavy chain variable region and the light chain variable region of ranalizumab are presented below, with the CDR regions identified in bold and underlined.

[0130] Sequence of the heavy chain variable region of ranalizumab (SEQ ID NO: 4) EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR IGVPRRDEFD IWGQGTMVTV SS

[0131] The sequence of the light chain variable region of ranalizumab (SEQ ID NO: 5) DIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPKLLIY KASTLESGVP SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEI

[0132] In some embodiments, the plasma kallikrein inhibitor may have at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the plasma kallikrein inhibitors described herein. In some embodiments, the plasma kallikrein inhibitor may have at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity in the framework regions of the HC and / or LC (e.g., FR1, 2, 3 and / or 4 of the HC and / or LC) to the plasma kallikrein inhibitors described herein. In some embodiments, the plasma kallikrein inhibitor may have at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity in the CDRs of the HC and / or LC (e.g., CDR1, 2 and / or 3 of the HC and / or LC) to the plasma kallikrein inhibitors described herein. In some embodiments, the plasma kallikrein inhibitor may have at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity in the constant regions (e.g., CH1, CH2, CH3 and / or CL1) to the plasma kallikrein inhibitors described herein.

[0133] In one aspect, a small molecule binds to and inhibits the active form of plasma kallikrein.

[0134] Bradykinin B2 receptor inhibitor In some embodiments, a bradykinin B2 receptor inhibitor (e.g., an antagonist) is administered to a subject. Exemplary bradykinin B2 receptor antagonists include icatibant (Firazyr®), a peptidomimetic that contains 10 amino acids that block the binding of native bradykinin to the bradykinin B2 receptor.

[0135] C1-INH replacement drug In some embodiments, a C1 esterase inhibitor (C1-INH), such as a C1-INH replacement drug, is administered to a subject. Exemplary C1-INH replacement drugs are publicly available and include, for example, human plasma-derived C1-INH (e.g., Berinert® and Cinryze®).

[0136] Without further elaboration, based on the above description, those skilled in the art should be able to make the fullest use of the present disclosure. Accordingly, the following specific embodiments should be construed as merely illustrative and not in any way limiting of other parts of the present disclosure. All publications cited herein are hereby incorporated by reference for the purposes or subject matter referred to herein.

Examples

[0137] Examples Example 1: Identification of metabolites that are differentially present in samples from HAE patients compared to healthy individuals To investigate novel metabolic biomarkers for hereditary angioedema, metabolite compositions of plasma samples obtained from patients with HAE were analyzed by comparing them with those of samples obtained from healthy individuals. Citrated plasma was collected from 20 healthy individuals and 20 patients with HAE during the disease's quiescent state ("baseline state") and during edematous attacks ("attack"). The samples were subjected to a validated liquid chromatography - mass spectrometry (LC - MS) method to detect metabolites present in the samples. This analysis detected 1,069 different plasma metabolites. The abundance of each metabolite was compared between samples and subjected to statistical analysis.

[0138] As shown in Table 3, 187 different metabolites were differentially present in samples from HAE patients in the baseline state compared to healthy individuals (73 were increased and 114 were decreased), 156 different metabolites were differentially present in samples from HAE patients during attacks compared to healthy individuals (51 were increased and 105 were decreased), and 59 different metabolites were differentially present in samples from HAE patients during attacks compared to HAE patients in the baseline state (25 were increased during attacks and 34 were decreased during attacks).

[0139]

Table 3

[0140] Random forest analysis showed separation of HAE patients from healthy individuals with 78% classification accuracy (Panel A of Figure 1) and separation of HAE patients in the baseline state from HAE patients during attacks with 20% classification accuracy (Panel B of Figure 1).

[0141] Metabolomic analysis comparing samples from HAE patients with healthy individuals Metabolome comparison of samples from HAE patients and healthy individuals identified several promising metabolites that showed disease-specific differences in levels detected in plasma samples. For example, the neurotransmitter serotonin was significantly elevated in plasma samples from HAE patients compared to those from healthy individuals (P < 0.05, Welch's two-sample t-test), and was found to be particularly elevated during HAE attacks (Table 4; Panel A of Figure 2). Tryptophan, a serotonin precursor, was found to be decreased in plasma samples from HAE patients compared to those from healthy individuals, further indicating that serotonin metabolism changes during HAE. Other metabolites involved in serotonin production (e.g., indolelactic acid and indolepropionic acid) were also found to be significantly different between HAE patients and healthy individuals (Table 4).

[0142]

Table 4

[0143] Serotonin is a neurotransmitter and a vasoactive amine that acts on the central nervous system, gastrointestinal tract, and platelets, and is involved in the regulation of vasoconstriction. When serotonin levels were classified based on the attack rate of HAE, it was observed that serotonin levels were highest in patients who had more frequent HAE attacks (more than 2 attacks per month) (Panel B of Figure 2). Therefore, detecting serotonin levels may be useful for identifying patients with more active disease or patients in whom the disease is less likely to be suppressed by treatment.

[0144] Several metabolites related to fatty acid amide hydrolase (FAAH) activity were significantly increased in samples from patients with HAE, suggesting a decrease in fatty acid amide hydrolase activity in HAE patients (Table 5). For example, palmitic acid amide, oleic acid amide, and linoleic acid amide (18:2n6) were significantly elevated in HAE plasma compared to healthy plasma (Table 5).

[0145]

Table 5

[0146] Several lipid peroxide markers, including 9 / 13-HODE, 9,10-DiHOME, 12,13-DiHOME and 19,20 DiHDPA, were found to be decreased in samples from HAE patients compared to healthy individuals (Table 6). This is consistent with the decreased membrane-associated oxidative stress predicted in patients with HAE.

[0147]

Table 6

[0148] The lipid-bound antioxidants γ / β-tocopherol was also found to have significantly elevated levels in samples from HAE patients (Table 7).

[0149]

Table 7

[0150] Metabolome data further suggested that sulfur metabolism and steroid metabolism may be involved in HAE. As shown in Table 8, many metabolites related to sulfur metabolism, including N-acetylmethionine, methionine sulfone, S-adenosylhomocysteine (SAH), cystine and cysteine sulfinic acid, were detected at elevated levels. Several steroid metabolites, including cortisol, were detected at decreased levels in HAE patients compared to healthy individuals (Table 9).

[0151]

Table 8

[0152] These results indicate that any of the metabolites identified in the metabolomics survey could be used as biomarkers to distinguish samples from patients with HAE from those from healthy individuals. Furthermore, this survey identified that the anti-inflammatory pathway, oxidative stress pathway, and steroid metabolism pathway are altered in HAE patients. These pathways may provide potential sources of novel biomarkers.

[0153] Metabolome analysis comparing samples from HAE patients in the basal state with samples from HAE patients during an attack Metabolomics analysis also identified several metabolites that were present at different levels between HAE patients in the basal state compared to patients with HAE during an attack. Corticosterone, an intermediate in the formation of aldosterone, was found to be significantly decreased during HAE attacks (Table 9). Furthermore, several polyunsaturated fatty acids, including eicosapentaenoic acid, mead acid, and stearidonic acid, were found to be significantly elevated during an attack compared to the basal state (Table 10). Finally, it was observed that two metabolic precursors of coenzyme A cofactors (nicotinamide and pantothenic acid) were significantly elevated during an attack compared to the basal state (Panels A - C of Figure 10).

[0154] [Table 9] TIFF0007692508000012.tif195162

[0155] [Table 10]

[0156] [Table 11]

[0157] These results indicate that these metabolites (e.g., those with significant fold change ratios) can be used, either alone or in combination, as reliable biomarkers for HAE and other diseases related to the contact system.

[0158] Metabolome data also provided new insights into the pathophysiology of HAE. For example, a subset of metabolites, including serotonin and lipid amides; metabolites related to oxidative stress such as oxidative limits, γ-tocopherol / β-tocopherol; and steroids, were associated with edema. This analysis also provided new insights into the pathophysiology of HAE in the basal state compared to during an attack. For example, some of the differences between the basal state and during an attack included anti-inflammatory lipid precursors, mineralocorticoids (corticosterone), and regulators of water and salt balance.

[0159] Metabolome analysis also identified many metabolites that are present at different levels between patients with HAE and healthy individuals and between patients with HAE in the basal state compared to during an attack. Any of the metabolites identified herein (e.g., metabolites with significant fold changes between HAE patients and healthy individuals) can be used, for example, as biomarkers for diseases related to the contact activation system (e.g., HAE) in methods for identifying patients at risk of diseases related to the contact activation system, selecting candidates for treatment, monitoring disease progression or condition, assessing the effectiveness of treatment for a disease, determining the course of treatment, identifying whether a disease or disorder is related to the contact activation system, and / or for research purposes (including studying the mechanisms of diseases that may be utilized for the development of new therapies), either individually or in combination (as a set of biomarkers).

[0160] Other embodiments All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature that serves the same, equivalent, or similar purpose. Thus, unless otherwise specified, each disclosed feature is merely an example of a general series of equivalent or similar features.

[0161] From the above description, those skilled in the art can easily identify the essential features of this disclosure and make various changes and modifications to this disclosure to adapt to various applications and conditions without departing from their spirit and scope. Accordingly, other embodiments also fall within the scope of the claims.

[0162] Equivalents and Scope Those skilled in the art will recognize many equivalents to the specific embodiments of this disclosure described herein or will be able to confirm them using nothing more than routine experimentation. The scope of this disclosure is not intended to be limited to the above description, but rather is as set forth in the appended claims.

[0163] In the claims, articles such as "a", "an", and "the" may mean one or more unless otherwise indicated or unless clear from the context. A claim or description that includes "or" between one or more members of a group is considered to be satisfied when, unless otherwise indicated or unless clear from the context, one, two or more, or all of the members of the group are present in, used in, or related to a given product or process. This disclosure includes embodiments where exactly one member of the group is present in, used in, or related to a given product or process. This disclosure includes embodiments where two or more or all of the members of the group are present in, used in, or related to a given product or process.

[0164] Furthermore, this disclosure encompasses all variations, combinations, and substitutions in which one or more limitations, elements, clauses, and descriptive terms from one or more of the recited claims are introduced into another claim. For example, any claim that depends on another claim may be modified to include one or more limitations found in any other claim that depends on the same base claim. When elements are presented as a list (e.g., in Markush group format), each subgroup of the elements is also disclosed, and any element(s) may be deleted from the group. In general, when the disclosure or an aspect of the disclosure is referred to as including a particular element and / or feature, it should be understood that a particular embodiment of the disclosure or an aspect of the disclosure consists of or consists essentially of such element and / or feature. For the sake of brevity, those embodiments are not specifically described herein by those words. It should also be noted that the terms "comprising" and "containing" are intended to be open-ended and allow the inclusion of additional elements or steps. When a range is given, the endpoints are included. Furthermore, unless otherwise indicated or not apparent from the context and the understanding of one of ordinary skill in the art, values expressed as a range may assume any specific value or sub-range within the recited range, to the tenth of the unit of the lower limit of the range, in various embodiments of the disclosure, unless the context clearly indicates otherwise.

[0165] This application references various issued patents, published patent applications, academic papers, and other publications, all of which are hereby incorporated by reference. In the event of any conflict between any of the incorporated references and this specification, this specification shall prevail. Further, any particular embodiment of the present disclosure that is included in the prior art may be explicitly excluded from one or more of the claims. Such embodiments are considered to be known to those of ordinary skill in the art and may be excluded even if such exclusion is not explicitly recited herein. Any particular embodiment of the present disclosure may be excluded from any claim for any reason, whether or not related to the existence of the prior art.

[0166] Those of ordinary skill in the art will recognize many equivalents to the particular embodiments described herein or will be able to ascertain them using only routine experimentation. The scope of the embodiments described herein is not intended to be limited to the above description but rather is as set forth in the appended claims. Those of ordinary skill in the art will understand that various changes and modifications may be made to this description without departing from the spirit or scope of the present disclosure as defined by the following claims.

Claims

1. (i) providing a biological sample obtained from a subject having, suspected of having, or at risk of having hereditary angioedema (HAE); (ii) measuring the level of a set of metabolic biomarkers comprising at least one lipid peroxide selected from the group consisting of 9-hydroxyoctadecadienoic acid (9-HODE), 13-hydroxyoctadecadienoic acid (13-HODE), 9,10-dihydroxy-12Z-octadecenoic acid (9,10-DiHOME), 12,13-dihydroxy-9Z-octadecenoic acid (12,13-DiHOME), and 19,20-dihydroxy-4Z,7Z,10Z,13Z,16Z-docosapentaenoic acid (19,20-DiHDPA); 16. A method for analyzing a sample, comprising:

2. 2. The method of claim 1, wherein the set of metabolic biomarkers consists of 2 to 10 metabolic biomarkers.

3. The method of claim 1 or 2, wherein the biological sample is a serum sample or a plasma sample.

4. The method according to any one of claims 1 to 3, wherein the HAE is type I HAE or type II HAE.

5. The set of metabolic biomarkers comprises: (i) metabolites involved in the production of serotonin; (ii) metabolites associated with fatty acid amide hydrolase activity; (iii) metabolites involved in sulfur metabolism; (iv) metabolites involved in steroid metabolism; (v) a fatty acid; or (vi) a precursor of a cofactor; The method of any one of claims 1 to 4, further comprising:

6. (i) the metabolite involved in the production of serotonin is serotonin, tryptophan, indole lactic acid, or indole propionic acid; (ii) the metabolite involved in the fatty acid amide hydrolase activity is palmitamide, oleamide, or linoleamide; (iii) the metabolite involved in sulfur metabolism is N-acetylmethionine, methionine sulfone, S-adenosylhomocysteine, cystine, or cysteine ​​sulfinic acid; (iv) The metabolites involved in the steroid metabolism include pregnenolone sulfate, 5α-pregnane-3β,20β-diol monosulfate, pregnene-diol disulfate, pregnane steroid monosulfate, pregnanediol-3-glucuronide, cortisol, corticosterone, cortisone, dehydroisoandrosterone sulfate (DHEA-S), 16a-hydroxyDHEA 3-sulfate, epiandrosterone sulfate, androsterone sulfate, 4-androstene-3β,17β-diol monosulfate, 4-androstene-3α,17α-diol monosulfate, 4-androstene-3β,17β-diol disulfate, 5α-androstane-3α,17α-diol monosulfate, 5α-androstane-3β,17β-diol disulfate, androsteroid monosulfates, etiocholanolong glucuronide, and pregnanolone / allopregnanolone sulfate; (v) the fatty acid is eicosapentaenoic acid (EPA), mead acid or stearidonic acid; or (vi) the precursor of the cofactor is nicotinamide or pantothenic acid; The method according to claim 5.

7. The method of any one of claims 1 to 6, wherein step (ii) involves mass spectrometry, chromatography or immunoassay.

8. 8. The method of claim 7, wherein step (ii) involves mass spectrometry and the biological sample is subjected to a separation step prior to said mass spectrometry.

9. 9. The method of claim 8, wherein the separating step comprises gas chromatography, liquid chromatography, or capillary electrophoresis.

10. The method of any one of claims 1 to 9, wherein the subject is a human patient.

11. 11. The method of any one of claims 1 to 10, wherein a deviation in the levels of the set of metabolic biomarkers in the subject from the levels of the same set of metabolic biomarkers in a control subject is indicative of the subject having HAE.

12. 11. The method of any one of claims 1 to 10, wherein the subject is a human patient undergoing treatment for HAE, the method further comprising assessing the efficacy of the treatment based on the levels of the set of metabolic biomarkers, wherein deviation of the levels of the set of metabolic biomarkers in the subject from those of a control subject indicates efficacy of the treatment.

13. 11. The method of any one of claims 1-10, further comprising identifying the subject as a candidate for treatment of HAE based on the levels of the set of metabolic biomarkers if the levels of the set of metabolic biomarkers in the subject deviate from the levels of the same set of metabolic biomarkers in a control subject.

14. The method of claim 12, wherein the human patient has a history of HAE.

15. 15. The method of any one of claims 1 to 14, wherein the set of metabolic biomarkers comprises one or more metabolic biomarkers selected from the group consisting of corticosterone, eicosapentaenoic acid, mead acid, stearidonic acid, nicotinamide, and pantothenic acid.

16. The method of claim 14 or 15, further comprising assessing the risk of an attack of HAE in the subject based on the level of the set of metabolic biomarkers, wherein deviation of the level of the set of metabolic biomarkers in the subject from that of a control subject indicates a risk of an attack of HAE.

17. A kit for analyzing a sample from a subject having, suspected of having, or at risk for hereditary angioedema (HAE), comprising: (i) a first binding agent specific for a first metabolic biomarker that is a lipid peroxide selected from the group consisting of 9-hydroxyoctadecadienoic acid (9-HODE), 13-hydroxyoctadecadienoic acid (13-HODE), 9,10-dihydroxy-12Z-octadecenoic acid (9,10-DiHOME), 12,13-dihydroxy-9Z-octadecenoic acid (12,13-DiHOME), and 19,20-dihydroxy-4Z,7Z,10Z,13Z,16Z-docosapentaenoic acid (19,20-DiHDPA); (ii) serotonin, tryptophan, indole lactic acid, indole propionic acid, palmitic acid amide, oleic acid amide, linoleic acid amide, N-acetyl methionine, methionine sulfone, S-adenosyl homocysteine, cystine, cysteine ​​sulfinic acid, pregnenolone sulfate, 5α-pregnane-3β, 20β-diol monosulfate, pregnene-diol disulfate, pregnane steroid monosulfate, pregnanediol-3-glucuronide, cortisol, corticosterone, cortisone, dehydroisoandrosterone sulfate (DHEA-S), 16a-hydroxy DHEA a second binding agent specific for a second metabolic biomarker selected from the group consisting of 5-androstene-3β,17β-diol monosulfate, epiandrosterone sulfate, androsterone sulfate, 4-androstene-3β,17β-diol monosulfate, 4-androstene-3α,17α-diol monosulfate, 4-androstene-3β,17β-diol disulfate, 5α-androstane-3α,17α-diol monosulfate, 5α-androstane-3β,17β-diol disulfate, androsteroid monosulfates, etiocholanolong glucuronide, pregnanolone / allopregnanolone sulfate, eicosapentaenoic acid (EPA), mead acid, stearidonic acid, nicotinamide, and pantothenic acid; Including the kit.

18. 20. The kit of claim 17, further comprising a first detection substance that binds to the first binding substance and a second detection substance that binds to the second binding substance.

19. 19. The kit of claim 17 or 18, wherein the first binding agent is an antibody specific for the first metabolic biomarker and / or the second binding agent is an antibody specific for the second metabolic biomarker.

20. The kit according to any one of claims 17 to 19, wherein the first binding substance and the second binding substance are immobilized on a support member.

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