Methods for determining amidolytic activity

Abstract

The present invention provides methods relating to plasma kallikrein (PKa) biomarkers. In particular, the present invention relates to methods for determining a level of amidolytic activity of a plasma sample, which can be used in a method for determining specific PKa (sPKa) activity. The invention further provides methods for use in diagnosing (or identifying) subjects at risk of a disease or disorder relating to elevated PKa activity, and determining if a disease or disorder is susceptible to treatment with a PKa inhibitor. The invention also provides treatments of subjects identified by the methods described herein, as well as methods of evaluating the effectiveness of such treatments. Kits that can be used in methods described herein are also provided.

Description

[0001] M ETHODS FOR DETERMINING AM I DO LYTIC ACTIVITY

[0002] The present invention provides methods relating to plasma kallikrein (PKa) biomarkers. In particular, the present invention relates to methods for determining a level of amidolytic activity of a plasma sample, which can be used in a method for determining specific PKa (sPKa) activity. The invention further provides methods for use in diagnosing (or identifying) subjects at risk of a disease or disorder relating to elevated PKa activity, and determining if a disease or disorder is susceptible to treatment with a PKa inhibitor. The invention also provides treatments of subjects identified by the methods described herein, as well as methods of evaluating the effectiveness of such treatments. Kits that can be used in methods described herein are also provided.

[0003] BACKGROUND

[0004] PKa is a trypsin-like serine protease that can liberate kinins from kininogens (see K. D. Bhoola et al., "Kallikrein-Kinin Cascade", Encyclopedia of Respiratory Medicine, p483-493; J. W. Bryant et al., "Human plasma kallikrein-kinin system: physiological and biochemical parameters" Cardiovascular and haematological agents in medicinal chemistry, 7, p234-250, 2009; K. D. Bhoola et al., Pharmacological Rev., 1992, 44, 1; and D. J. Campbell, "Towards understanding the kallikrein-kinin system: insights from the measurement of kinin peptides", Brazilian Journal of Medical and Biological Research 2000, 33, 665-677). Plasma prekallikrein is encoded by a single gene and is mainly synthesized in the liver, as well as other tissues. It is secreted by hepatocytes as a zymogen that circulates in plasma as a heterodimer complex bound to high molecular weight kininogen (HK) which is activated to give the active PKa. Factor Xlla (FXIIa) mediates conversion of plasma prekallikrein to PKa, which is a serine protease that subsequent cleavage of high molecular weight kininogen (HK) to generate bradykinin, a potent inflammatory hormone. Kinins are potent mediators of inflammation that act through G protein-coupled receptors and antagonists of kinins (such as bradykinin receptor antagonists) have previously been investigated as potential therapeutic agents for the treatment of a number of disorders (F. Marceau and D. Regoli, Nature Rev., Drug Discovery, 2004, 3, 845-852).

[0005] Activation of the kallikrein-kinin system (KKS) is initiated by a variety of physiological (e.g. negatively charged surfaces) and pathological factors, which induce a conformational change in FXII leading to its cleavage and activation by certain proteases e.g. plasmin (Hofman et al Clin Rev Allergy Immunol 2016), which may not require negative surfaces, or by misfolded proteins (Maas et al J Clinical Invest 2008). Activation of plasma prekallikrein to PKa by FXIIa is followed by reciprocal activation of FXII to FXIIa by PKa and thereby provides positive feedback amplification of the KKS. The biologically active peptide bradykinin activates bradykinin 2 receptors (B2R) that are expressed on a variety of vascular, neuronal, and immune cell types (Kaplan AP, Joseph K. Pathogenic mechanisms of bradykinin mediated diseases: dysregulation of an innate inflammatory pathway. Adv Immunol. 2014;121:41-89). The interaction between bradykinin and B2R activates proinflammatory signalling pathways that mediate, for example, angioedema, pain, inflammation, chemotaxis of neutrophils, vascular hyperpermeability, and vasodilatation (see Kaplan et aL, Adv Immunol. 2014;121:41-89; and Hopp et aL, J Neuroinflammation. 2017 Feb 20;14(l):39).

[0006] PKa is thought to play a role in a number of inflammatory disorders. The major inhibitor of PKa is the serpin Cl esterase inhibitor (C1INH). People with a genetic deficiency in C1INH levels or function due to mutation in SERPING1 can develop hereditary angioedema (HAE-C1INH) which can result in intermittent swelling of face, extremities, larynx, gastro-intestinal tract and genitals. Plasma from HAE patients have increased levels of cleaved HK, which indicates the generation of bradykinin (PLoS ONE 8(8): e74043. 2013). Treatment with a PKa inhibitors, (such as the approved medicinal products ecallantide, lanadelumab, and berotralstat) has been shown to effectively treat HAE-C1INH.

[0007] Hereditary angioedema (HAE) is a rare inherited disorder characterised by recurrent acute attacks where fluids accumulate outside of the blood vessels, blocking the normal flow of blood or lymphatic fluid and causing rapid swelling of tissues such as in the hands, feet, limbs, face, intestinal tract, or airway. "Hereditary angioedema" can thus be defined as any disorder characterised by recurrent episodes of bradykinin-mediated angioedema (e.g. severe swelling) caused by an inherited dysfunction / fault / mutation. There are currently three known categories of HAE: (i) HAE type 1, (ii) HAE type 2, and (iii) HAE with normal C1INH levels and function (HAE-nClINH). However, the HAE field is developing quickly so it is expected that further types of HAE might be defined in the future.

[0008] Without wishing to be bound by theory, it is thought that HAE type 1 is caused by mutations in the SERPING1 gene that lead to reduced levels of ClINHin the blood. Without wishing to be bound by theory, it is thought that HAE type 2 is caused by mutations in the SERPING1 gene that lead to dysfunction of the C1INH in the blood.

[0009] Unlike HAE types 1 and 2 (also called HAE-C1INH), the pathophysiology of HAE-nClINH is heterogeneous and not fully understood. Without wishing to be bound by theory, the cause of HAE-nClINH is less well-defined and the underlying genetic dysfunction / fault / mutation can sometimes remain unknown. What is known is that the cause of HAE-nClINH is not related to reduced levels or dysfunction of the C1INH (in contrast to HAE types 1 and 2).

[0010] Currently, there are difficulties in diagnosing patients with HAE-nClINH. Indeed, the absence of universally accepted criteria for the diagnosis of HAE-nClINH limits estimates of its prevalence. At present, HAE- nCHNH can be diagnosed by reviewing the family history and noting that angioedema has been inherited from a previous generation (and thus it is hereditary angioedema). HAE-nClINH can also be diagnosed by determining that there is a dysfunction / fault / mutation in a gene other than those related to C1INH. For example, it has been reported that dysfunction / fault / mutation with plasminogen can cause HAE-nClINH (see e.g. Veronez et al.. Front Med (Lausanne). 2019 Feb 21;6:28; or Recke et al., Clin Transl Allergy. 2019 Feb 14;9:9). It has also been reported that dysfunction / fault / mutation with F12 gene and Factor XII can cause HAE-nClINH (see e.g. Mansi et al. 2014 The Association for the Publication of the Journal of Internal Medicine Journal of Internal Medicine, 2015, 277; 585-593; or Maat et al. J Thromb Haemost. 2019 Jan;17(l):183-194). However, the genetic cause in most patients with HAE-nClINH is unknown, and this subtype is referred as HAE-UNK or AE-UNK (Bork K et al. Hereditary angioedema with normal Cl-INH with versus without specific F12 gene mutations. Allergy. 2015 Aug;70(8):1004-12, Bork K et al. Gene Mutations Linked to Hereditary Angioedema in Solitary Angioedema Patients With Normal Cl Inhibitor. J Allergy Clin Immunol Pract. 2023 Aug;ll(8):2441-2449). About 87% of patients diagnosed with HAE-nClINH have an unknown mutation, limiting the utility of genetic tests to establish the diagnosis.

[0011] Angioedemas are not necessarily inherited. Indeed, another class of angioedema is bradykinin-mediated angioedema non-hereditary (BK-AEnH), which are not caused by an inherited genetic dysfunction / fault / mutation. Often the underlying cause of BK-AEnH is unknown and / or undefined. There is therefore a need to better diagnose BK-AEnH.

[0012] Specific types of BK-AEnH include: nonhereditary angioedema with normal C1INH (AE-nCl Inh), which can be environmental, hormonal, or drug-induced; acquired angioedema; anaphylaxis associated angioedema; angiotensin converting enzyme (ACE) inhibitor-induced angioedema; dipeptidyl peptidase-4 inhibitor-induced angioedema; and tPA-induced angioedema (tissue plasminogen activator-induced angioedema). However, reasons why these factors and conditions cause angioedema in only a relatively small proportion of individuals are unknown.

[0013] Environmental factors that can induce AE-nClINH include air pollution (Kedarisetty et al, Otolaryngol Head Neck Surg. 2019 Apr 30:194599819846446. doi: 10.1177 / 0194599819846446) and silver nanoparticles such as those used as antibacterial components in healthcare, biomedical and consumer products (Long et al., Nanotoxicology. 2016;10(4):501-ll. doi: 10.3109 / 17435390.2015.1088589).

[0014] Various publications suggest a link between the bradykinin and contact system pathways and BK-AEnHs, and also the potential efficacy of treatments, see e.g.: Bas et al. (N Engl J Med 2015; Leibfried and Kovary. J Pharm Pract 2017); van den Elzen et al. (Clinic Rev Allerg Immunol 2018); Han et al (JCI 2002).

[0015] TPA-induced angioedema is discussed in various publications as being a potentially life-threatening complication following thrombolytic therapy in acute stroke victims (see e.g. Simao et al., Blood. 2017 Apr 20;129(16):2280-2290. doi: 10.1182 / blood-2016-09-740670; Frohlich et al.. Stroke. 2019 Jun 11:STROKEAHA119025260. doi: 10.1161 / STROKEAHA.119.025260; Rathbun, Oxf Med Case Reports. 2019 Jan 24;2019(l):omyll2. doi: 10.1093 / omcr / omyll2; Lekoubou et aL, Neurol Res. 2014 Jul;36(7):687-94. doi: 10.1179 / 1743132813Y.0000000302; Hill et aL, Neurology. 2003 May 13;60(9):1525-7).

[0016] Stone et al. (Immunol Allergy Clin North Am. 2017 Aug;37(3):483-495.) reports that certain drugs can cause angioedema.

[0017] Scott et al. (Curr Diabetes Rev. 2018;14(4):327-333. doi: 10.2174 / 1573399813666170214113856) reports cases of dipeptidyl Peptidase-4 Inhibitor induced angioedema.

[0018] Hermanrud et aL, (BMJ Case Rep. 2017 Jan 10;2017. pii: bcr2016217802) reports recurrent angioedema associated with pharmacological inhibition of dipeptidyl peptidase IV and also discusses acquired angioedema related to angiotensin-converting enzyme inhibitors (ACEI-AAE). Kim et al. (Basic Clin Pharmacol Toxicol. 2019 Jan;124(l):115-122. doi: 10.1111 / bcpt.l3097) reports angiotensin II receptor blocker (ARB)-related angioedema. Reichman et al., (Pharmacoepidemiol Drug Saf. 2017 Oct;26(10):1190- 1196. doi: 10.1002 / pds.4260) also reports angioedema risk for patients taking ACE inhibitors, ARB inhibitors and beta blockers. Diestro et al. (J Stroke Cerebrovasc Dis. 2019 May;28(5):e44-e45. doi: 10.1016 / j.jstrokecerebrovasdis.2019.01.030) also reports a possible association between certain angioedemas and ARBs.

[0019] Giard et al. (Dermatology. 2012;225(l):62-9. doi: 10.1159 / 000340029) reports that bradykinin-mediated angioedema can be precipitated by oestrogen contraception. There is a general need to provide effective methods for use in diagnosing diseases and disorders relating to elevated PKa activity. In particular, there is a need to provide effective methods for use in diagnosing patients with HAE-nClINH, so that treatment can be effectively administered.

[0020] THE INVENTION

[0021] Definitions

[0022] As used herein, "PKa" is an abbreviation of "plasma kallikrein". In addition, "sPKa" is an abbreviation of "specific plasma kallikrein" or "specific PKa".

[0023] The term "specific plasma kallikrein (sPKa) activity" of a plasma sample is the level of amidolytic activity of the plasma sample that is attributable to PKa i.e. excluding any contribution of amidolytic activity from other amidolytic enzymes. sPKa activity of a plasma sample can be determined by taking the level of amidolytic activity that is not inhibited by a PKa inhibitor (i.e. the level of amidolytic activity that is not attributable to PKa) and subtracting it from the total level of amidolytic activity of the sample as follows: sPKa activity = [total amidolytic activity] - [the level of amidolytic activity determined in the presence of a PKa inhibitor added ex vivo]

[0024] In this context, "a PKa inhibitor added ex vivo” refers to the addition of an exogenous PKa inhibitor during the methods as discussed herein under section I.

[0025] The term "hereditary angioedema" (HAE) means any disorder characterised by recurrent episodes of bradykinin-mediated angioedema (e.g. severe swelling) caused by an inherited dysfunction, fault, or mutation. As a result, the term "HAE" includes at least HAE type 1, HAE type 2, and HAE with normal C1INH levels and function (HAE-nClINH). Preferred aspects of the invention relate to bradykinin-mediated HAE-nClINH, especially HAE-nClINH of unknown genetic cause. However, the HAE field is developing quickly so it is expected that further types of HAE might be defined in the future. Hereditary angioedema is characterised by recurrent acute attacks where fluids accumulate outside of the blood vessels, blocking the normal flow of blood or lymphatic fluid and causing rapid swelling of tissues such as in the hands, feet, limbs, face, intestinal tract, or airway.

[0026] The term "HAE-nClINH" means HAE with normal C1INH levels and function. As used herein, "FXIIa" is an abbreviation of "Factor Xlla".

[0027] As used herein, "an elevated level" or "a level above a reference value" in the context of sPKa activity means that the level of sPKa activity is greater than a reference value of sPKa activity determined from at least one healthy subject (or a population of healthy subjects). An elevated level of sPKa activity in a subject can be indicative of a dysfunction in PKa activity, such that the subject may be at risk of a disease or disorder relating to elevated PKa activity. An elevated level of a sPKa activity includes a level of sPKa activity that is, for example, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% or more above a reference value.

[0028] The terms "subject", "patient" or "individual" may be used interchangeably and refer to a subject undergoing the methods described herein. The subject is typically a human or non-human animal e.g. mammal. Preferably, the subject is a human. The subject can be female. The subject can be male. The subject can be a female that is taking estrogen contraception.

[0029] The term "healthy subject" means a subject does not have a dysfunctional kail ikrein-kinin system (KKS). In particular, a subject that has not been diagnosed with, and is not suspected of having, any disease or disorder relating to a dysfunctional KKS e.g. a disease or disorder relating to elevated PKa activity such as HAE.

[0030] The term "PKa inhibitor" means a substance, molecule or compound that can bind to, block and / or inhibit PKa. A PKa inhibitor can be identified using PKa binding and / or inhibition assays. PKa binding and inhibition assays are known in the art. For example, the assay of Example 1 can be used to identify a PKa inhibitor. The PKa inhibitor can be an inhibitory polypeptide or peptide such as ecallantide. The PKa inhibitor can be an antibody, or antigen-binding fragment, such as lanadelumab or STAR-0215. The PKa inhibitor can be a small molecule that binds and inhibits PKa. Suitable PKa inhibitors can be berotralstat and sebetralstat. Exemplary PKa inhibitors include sebetralstat, berotralstat, avoralstat, KVD001, feniralstat, lanadelumab, STAR-0215, RZ-402 (ASP-440), ATN-249, and KV999272, or a salt and / or stereoisomer thereof.

[0031] The PKa inhibitor can be a PKa inhibitor as described in WO03 / 076458, W02013 / 005045, WO2014 / 108679, WO2015 / 022546, WO2015 / 022547, WO2014 / 188211, W02016 / 083820,

[0032] WO2016 / 083818, WO2016 / 083816, WO2017 / 207983, WO2019 / 106359, or WO2021 / 028649, the contents of which are incorporated by reference in their entirety. RZ-402 (ASP-440) is disclosed in W02008016883A2 (see e.g. Table 1 of W02008016883A2), the contents of which is incorporated herein by reference.

[0033] KVD001 is Example 3 in W02013 / 005045, the contents of which is incorporated herein by reference.

[0034] Sebetralstat (also known as KVD900) is Example 41 in W02016 / 083820, the contents of which are incorporated herein by reference.

[0035] Feniralstat is Example 30 in WO2017 / 207983, the contents of which is incorporated herein by reference.

[0036] The term "determining" in the context of "determining amidolytic activity" means measuring, detecting or evaluating the presence, absence, quantity, level or amount of the enzymatic activity in the sample.

[0037] The term "about" means plus or minus 10% of the indicated value.

[0038] Summary of the invention

[0039] The inventors have identified a new PKa biomarker assay, called specific plasma kallikrein activity (sPKa), that enables effective detection of subjects having elevated PKa activity (and therefore who are susceptible to suffering from a disease or disorder relating to elevated PKa activity). As shown in Examples 4, 4a and 5, when sPKa activity is used as the biomarker, there is a demonstrated improvement in the method's performance in detecting diseased plasma compared to using total amidolytic activity as the biomarker, where "diseased plasma" is plasma from a subject having a disease or disorder relating to elevated PKa activity. In particular. Examples 4, 4a and 5 show that the sensitivity of the assay to identify patients having elevated PKa activity was found to be improved when sPKa activity was used. Receiver operating characteristics (ROC) analysis also confirms that using sPKa activity as a biomarker is improved compared to using total amidolytic activity as the biomarker. An improved sensitivity reduces the number of false negative results, providing a clinically superior method, that may enable more reliable diagnosis and therefore more effective treatment.

[0040] The results demonstrated in Examples 4 and 4a show that the improvement in sensitivity when sPKa activity was used as the biomarker did not detrimentally affect the method's specificity. It is known that there is generally a trade-off between a method's sensitivity and specificity such that improving a method's sensitivity (to improve detection of subjects having a disease) generally reduces a method's specificity (ability to discount subjects that do not have the disease) due to an overlap in a biomarker's prevalence between healthy and diseased groups. Examples 4 and 4a show that methods of the invention do not suffer from this general tradeoff and underscore that sPKa activity is a superior biomarker for detecting diseases or disorders relating to elevated PKa activity.

[0041] The methods of the present invention are particularly useful in identifying subjects suffering from bradykinin-mediated angioedema, and in particular, HAE. As discussed above, there is a particular need to provide methods that can detect subjects suffering from HAE-nClINH and, as shown in Examples 4, 4a, 5 and 8, the methods of the present invention can detect subjects having HAE-C1INH, as well as HAE- nCHNH.

[0042] As shown in Examples 4 and 4a, the sPKa activity of a plasma sample can be determined by subtracting the level of amidolytic activity when measured in the presence of a PKa inhibitor, from the total amidolytic activity of the plasma sample. When measuring the level of amidolytic activity in the presence of an added (ex vivo) PKa inhibitor, the inventors have identified that it is important to inhibit PKa activity after incubation of the PKa sample, where incubation induces KKS activation. Without wishing to be bound by theory, inducing KKS activation and subsequently inhibiting PKa activity enhances the method's ability to differentiate between healthy and diseased subjects. This is supported by the data in Examples 4, 4a, 8, and 11.

[0043] The invention therefore provides a method for determining a level of amidolytic activity of a plasma sample (in the presence of a PKa inhibitor added ex vivo) comprising: a. incubating the plasma sample to induce kal likrein-kinin system activation in the sample; b. after step a, adding a PKa inhibitor to at least a portion of the incubated plasma sample to form an inhibitor / sample mixture; c. after step b, adding a substrate for determining the amidolytic activity to at least a portion of the inhibitor / sample mixture to form an inhibitor / sample / substrate mixture; and d. after step c, determining the amidolytic activity of the inhibitor / sample / substrate mixture, which provides the level of amidolytic activity of the plasma sample.

[0044] As described above, the level of amidolytic activity when measured in the presence of an added (ex vivo) PKa inhibitor enables the sPKa activity to be determined by subtracting the level of amidolytic activity when PKa activity is inhibited from the total amidolytic activity of the plasma sample.

[0045] The invention therefore provides a method for determining the sPKa activity of a plasma sample comprising: i. determining the total amidolytic activity of a plasma sample without adding any inhibitor of the kallikrein-kinin system by: a. incubating the plasma sample to induce kallikrein-kinin system activation in the sample; b. after step a, adding a substrate for determining amidolytic activity to at least a portion of the sample to form a sample / substrate mixture; and c. after step b, determining the amidolytic activity of the sample / substrate mixture, which is the total amidolytic activity of the plasma sample; and, ii. subtracting: (1) the level of amidolytic activity as determined by the method for determining a level of amidolytic activity of a plasma sample (when PKa activity is inhibited) as described herein, from (2) the total amidolytic activity of the plasma sample; to provide the sPKa activity of the sample.

[0046] As described above, sPKa activity can be used as an excellent biomarker for determining whether a subject has elevated PKa activity. For instance, using sPKa activity as the biomarker instead of total amidolytic activity has been demonstrated as providing a more sensitive method. To do this, the sPKa activity of a subject can be compared to the sPKa activity of a healthy subject(s), which can be considered as a reference value of sPKa activity above which indicates elevated PKa activity.

[0047] The invention therefore provides a method for determining a reference value of sPKa activity above which indicates elevated PKa activity comprising: conducting the method for determining sPKa activity as described herein (e.g. in section II) on multiple different plasma samples to identify multiple sPKa activity values; wherein the reference value of sPKa activity corresponds to the 95th percentile of the multiple sPKa activity values; and wherein the multiple different plasma samples are collected from at least one healthy subject.

[0048] Determining that a subject has elevated PKa activity can be used in the diagnosis of a subject with a disease or disorder relating to elevated PKa activity. As described above, there is a need to provide improved methods for use in diagnosing subjects with such diseases / disorders, particularly HAE, and more particularly HAE-nClINH. The new biomarker described herein can provide improved sensitivity to detecting such subjects, which makes it suitable for improving such diagnostic methods.

[0049] More specifically, the subject can be diagnosed with a disease or disorder relating to elevated PKa activity if their sPKa activity is greater than the sPKa activity of a healthy subject(s), i.e. a subject (or subjects) that has(have) not been diagnosed with, and is(are) not suspected of having, any disease or disorder related to a dysfunctional KKS, which can be considered as a reference value of sPKa activity above which indicates elevated PKa activity.

[0050] The invention therefore provides a method for use in diagnosing a subject with a disease or disorder relating to elevated PKa activity comprising: a. determining the sPKa activity of a plasma sample collected from the subject, which is the subject's sPKa activity; b. comparing the subject's sPKa activity with a reference value of sPKa activity above which indicates elevated PKa activity; and c. diagnosing the subject with a disease or disorder relating to elevated PKa activity if the subject's sPKa activity is greater than the reference value.

[0051] The invention also provides a method for identifying a subject at risk of having a disease or disorder relating to elevated PKa activity comprising: a. determining the sPKa activity of a plasma sample collected from the subject, which is the subject's sPKa activity; b. comparing the subject's sPKa activity with a reference value of sPKa activity above which indicates elevated PKa activity; and c. identifying a subject at risk of having a disease or disorder relating to elevated PKa activity if the subject's sPKa activity is greater than the reference value.

[0052] The invention also provides a method for determining if a disease or disorder is susceptible to treatment with a PKa inhibitor comprising: a. determining the sPKa activity of a plasma sample collected from a subject, which is the subject's sPKa activity; b. comparing the subject's sPKa activity with a reference value of sPKa activity above which indicates elevated PKa activity; and c. identifying the disease or disorder as being susceptible to treatment with a PKa inhibitor if the subject's sPKa activity is greater than the reference value.

[0053] A subject diagnosed with a disease or disorder relating to elevated PKa activity as described herein can be treated with a therapeutic agent for treating the disease or disorder. sPKa activity is an excellent biomarker for use in diagnosing subjects that may benefit from such treatment. As described above, there is a general need to improve the treatment regimens of subjects having a disease or disorder relating to elevated PKa activity, particularly HAE, more particularly HAE-nClINH. Also as described herein, methods utilising sPKa activity to detect elevated PKa activity can have high sensitivity and specificity, thereby minimising the rate of false negative and false positive results. sPKa activity can be used to provide more effective treatments of such diseases / disorders.

[0054] The invention therefore provides a method for treating a disease or disorder relating to elevated PKa activity in a subject, comprising administering to the subject a therapeutic agent for treating the disease or disorder; wherein the subject has a sPKa activity that is greater than a reference value, the reference value being a sPKa activity above which indicates elevated PKa activity; and wherein the sPKa activity of the subject is as described herein.

[0055] The invention also provides a therapeutic agent for use in the treatment of a disease or disorder relating to elevated PKa activity in a subject, comprising administering to the subject a therapeutic agent for treating the disease or disorder; wherein the subject has a sPKa activity that is greater than a reference value, the reference value being a sPKa activity above which indicates elevated PKa activity; and wherein the sPKa activity of the subject is as described herein.

[0056] As described above, identifying an effective treatment for subjects suffering from a disease or disorder relating to elevated PKa can be difficult. For instance, there is no universally accepted method to diagnose subjects having HAE-nClINH. Additionally, it is desirable to have effective methods to monitor and evaluate the effectiveness of any treatment administered to ensure that the subject is being treated adequately and is not at risk of symptomatic disease. sPKa activity is an advantageous biomarker to detect subjects having elevated PKa activity, as described above, and can be used in methods to monitor and evaluate the effectiveness of any treatment.

[0057] The invention therefore provides a method for evaluating treatment of a disease or disorder relating to elevated PKa in a subject comprising: a. determining the sPKa activity of a plasma sample collected from the subject, which is the subject's sPKa activity; b. comparing the subject's sPKa activity with a reference value of sPKa activity above which indicates elevated PKa activity; and c. evaluating effectiveness of the treatment based on whether the subject's sPKa activity is greater than the reference value. Detailed of the invention

[0058] I. Methods for determining a level of amidolvtic activity in the presence of a PKa inhibitor

[0059] One aspect of the present invention relates to methods for determining a level of amidolytic activity of a plasma sample comprising: (a) incubating the plasma sample to induce KKS activation in the sample; (b) after step a, adding a PKa inhibitor to at least a portion of the incubated plasma sample to form an inhibitor / sample mixture; (c) after step b, adding a substrate for determining the amidolytic activity to at least a portion of the inhibitor / sample mixture to form an inhibitor / sample / substrate mixture; and (d) after step c, determining the amidolytic activity of the inhibitor / sample / substrate mixture, which provides the level of amidolytic activity of the plasma sample. The level of amidolytic activity can be determined by measuring cleavage of a small peptide substrate.

[0060] The presence of an added PKa inhibitor (i.e. a PKa inhibitor that is added ex vivo) means that the level of amidolytic activity of a plasma sample is reduced by an amount corresponding to the inhibitory effect of the PKa inhibitor. As described above, subtracting this level of activity from the total activity of the sample provides the sPKa activity of the sample.

[0061] The plasma samples used in the present invention are obtained / collected from a subject. Methods described herein that are conducted on a plasma sample are ex vivo methods. A sample can be obtained from a subject using any means known in the art. The plasma sample is obtained from a blood sample collected from the subject. The blood sample can be obtained from a subject by collecting the blood sample into a collection tube. The blood sample collection tube can contain an anticoagulant e.g. sodium citrate.

[0062] In some aspects of the methods described herein, the subject from whom the plasma sample is obtained is not receiving treatment e.g. the subject is not taking any medication. For example, the subject may not be administered medication that could interfere with the methods described herein. In particular, the subject may not be administered any such medications within the previous 30 days, 20 days, 10 days or 5 days prior to obtaining the blood sample from the subject. Exemplary medications that could interfere with the methods described herein include inhibitors of the KKS, PKa inhibitors (such ecallantide, lanadelumab, berotralstat or sebetralstat), FXIIa inhibitors, C1INH replacement therapy (such as Cinryze®, Ruconest®, Berinert®, or Haegarda®), or other medications that may elevate plasma kallikrein such as estrogen, immune checkpoint inhibitors, and ACE inhibitors. In other aspects of the methods described herein, the subject from whom the plasma sample is obtained is receiving treatment e.g. the subject is taking medication. For instance, the methods may be utilised to assess the effect of the treatment on sPKa activity. The subject may have been administered medication at any point prior to obtaining the blood sample. For example, the subject may have been administered medicaments such as inhibitors of the KKS, PKa inhibitors (such ecallantide, lanadelumab, berotralstat, sebetralstat), FXIIa inhibitors, C1INH replacement therapy (such as Cinryze®, Ruconest®, Berinert®, or Haegarda®), or other medications that may elevate plasma kallikrein activity such as estrogen, immune checkpoint inhibitors, and ACE inhibitors.

[0063] Females on estrogen contraception can demonstrate elevated sPKa activity, which is confirmed by Example 9. Specifically, as shown in Example 9, the methods described herein (e.g. a method for determining a level of amidolytic activity of a plasma sample, and a method for determining the sPKa activity of a plasma sample) can be carried out on plasma samples obtained from female subjects on estrogen contraception. For instance, the method for determining the sPKa activity of a plasma sample can detect an increase in sPKa activity in a plasma sample obtained from a female subject on estrogen contraception.

[0064] Preferably the blood sample is processed to obtain the plasma sample shortly after obtaining the blood sample from the subject. Doing this can minimise spontaneous activation of the KKS in the sample, which can be desirable. The plasma sample can be separated from whole blood within 72 hours of collecting the whole blood from a subject. For example, the plasma sample can be separated from whole blood within 60 hours, within 48 hours, within 36 hours, within 24 hours or within 12 hours of collecting the whole blood from a subject. Preferably, the plasma sample is separated from whole blood within 48 hours.

[0065] Once the plasma sample has been separated from the whole blood, the plasma sample can be used in the methods described herein. The plasma sample can be used within 12 hours of obtaining the plasma sample from the whole blood sample. For example, the plasma sample can be used in the methods described herein, within 6 hours, within 4 hours, within 2 hours or within 1 hour of obtaining the plasma sample from the whole blood sample, or immediately after obtaining the plasma sample.

[0066] The plasma sample can be frozen after collection (e.g. at -80°C). Freezing the plasma sample can minimise (or even prevent) spontaneous activation of the KKS. Freezing the plasma sample can also prevent deterioration or degradation of blood components (e.g. components of the KKS) and can therefore enable more accurate determination of the level of amidolytic activity in the sample. The plasma sample can be frozen soon after (e.g. immediately after) separating the plasma sample from whole blood. The plasma sample can be frozen within 2 hours of separating the plasma sample from whole blood. For example, the plasma sample can be frozen within 1 hour or within 0.5 hours of obtaining the plasma sample from the whole blood sample, or immediately after obtaining the plasma sample.

[0067] Prior to carrying out the methods described herein, the frozen plasma sample can be thawed. Preferably, the frozen plasma sample is thawed on ice i.e. frozen water. The frozen plasma sample can be thawed at a temperature greater than about 0°C. The frozen plasma sample can be thawed at a temperature less than about 15°C. The frozen plasma sample can be thawed at a temperature of about 4°C. The plasma sample used in the methods described herein can be a liquid. Incubating the plasma sample in step a can commence as soon as the plasma sample has been thawed. The plasma sample can be frozen after thawing i.e. the plasma sample can be frozen and thawed multiple times prior to carrying out the methods described herein.

[0068] In some aspects, the subject from whom the blood sample is collected is a healthy human subject. This can be the case to determine a reference value of a healthy population, as described in more detail below.

[0069] In other aspects, the subject from whom the blood sample is collected can be a human patient having, suspected of having, or at risk for developing a disease or disorder relating to elevated PKa activity. In some aspects, the subject can be a human patient having, suspected of having, or at risk for developing HAE e.g. HAE-nClINH. In some aspects, the sample can be obtained from a subject having, or at risk for developing HAE (e.g. HAE-nClINH) during the intercritical remission period, which is the period between HAE attacks.

[0070] In step a of the method for determining a level of amidolytic activity of a plasma sample, activation of the KKS in the plasma sample can be induced by incubating the plasma sample. In this context, "incubating" means subjecting the sample to conditions (e.g. time and temperature) to induce (or allow) activation of the KKS. That is, incubating the plasma sample in step a can allow for KKS activation to occur such that KKS activity after incubation is greater than KKS activity at baseline. In particular, incubation (e.g. cold incubation) provides conditions that are suitable for inducing (or allowing) KKS activation in a plasma sample obtained from a subject having a disease or disorder relating to elevated PKa activity e.g. plasma obtained from an HAE patient such as HAE type 1 or type 2. The level of activation detected following incubation can be dependent on whether the plasma sample has been collected from a healthy or diseased patient and the severity of any disease. For instance, incubation (e.g. cold incubation) can induce (or allow for) amplification of KKS activity in the sample e.g. when the sample is taken from a diseased subject such as an HAE patient, thus allowing for differentiation from KKS activity in healthy plasma, which may show little (or no) activation. In addition, in the context of HAE, the level of activation detected following incubation can be dependent on when the sample was obtained from the HAE patient e.g. during remission or during an HAE attack.

[0071] Activation of the KKS can occur at cold temperatures i.e. cold activation of the KKS. In step a, the plasma sample can be incubated at a temperature sufficiently low to induce KKS activation. For example, KKS activation can be induced in the plasma sample by incubating the sample at a temperature of less than about 15°C, such as less than about 10°C, less than about 9°C, less than about 8°C, less than about 7°C, less than about 6°C, or less than about 5°C. The plasma sample can be incubated at a temperature of between about 0°C and about 15°C. The plasma sample can be incubated at a temperature of between about 0°C and about 10°C. Preferably, the plasma sample is incubated at a temperature of between about 0°C and about 5°C. More preferably, the plasma sample is incubated at a temperature of about 4°C in step a.

[0072] The plasma sample can be incubated on wet ice. The plasma sample can be incubated on wet ice at a temperature of less than or equal to about 4°C. The plasma sample can be incubated on wet ice at a temperature of between about 0°C and about 4°C. Preferably, the plasma sample can be incubated on wet ice at a temperature of about 4°C. The plasma sample is in liquid form when it is incubated in step a.

[0073] The plasma sample can be incubated for a length of time sufficient to induce or allow for KKS activation (e.g. amplification of KKS activity). The plasma sample can be incubated in step a for at least about 2 hours, at least about 3 hours or at least about 4 hours. The plasma sample can be incubated in step a for between about 2 hours and about 20 hours. For example, the plasma sample can be incubated in step a for between about 4 hours and about 18 hours, between about 4 hours and about 12 hours, between about 4 hours and about 10 hours, or between about 4 hours and about 8 hours. Preferably, the plasma sample is incubated in step a for between about 5 hours and about 7 hours. More preferably, the plasma sample is incubated in step a for about 6 hours. As shown in Example 11, incubating the plasma sample for about 6 hours in step a allows for an assay with excellent sensitivity and specificity.

[0074] The plasma sample can be incubated in step a at a temperature of between about 0°C and about 15°C for between about 4 hours and about 18 hours. The plasma sample can be incubated in step a at a temperature of between about 0°C and about 10°C for between about 4 hours and about 18 hours. The plasma sample can be incubated in step a at a temperature of between about 0°C and about 5°C for between about 4 hours and about 18 hours. The plasma sample can be incubated in step a at a temperature of about 4°C for between about 4 hours and about 18 hours. The plasma sample can be incubated in step a at a temperature of between about 0°C and about 15°C for between about 4 hours and about 12 hours. The plasma sample can be incubated in step a at a temperature of between about 0°C and about 10°C for between about 4 hours and about 12 hours. The plasma sample can be incubated in step a at a temperature of between about 0°C and about 5°C for between about 4 hours and about 12 hours. The plasma sample can be incubated in step a at a temperature of about 4°C for between about 4 hours and about 12 hours.

[0075] The plasma sample can be incubated in step a at a temperature of between about 0°C and about 15°C for between about 4 hours and about 10 hours. The plasma sample can be incubated in step a at a temperature of between about 0°C and about 10°C for between about 4 hours and about 10 hours. The plasma sample can be incubated in step a at a temperature of between about 0°C and about 5°C for between about 4 hours and about 10 hours. The plasma sample can be incubated in step a at a temperature of about 4°C for between about 4 hours and about 10 hours.

[0076] The plasma sample can be incubated in step a at a temperature of between about 0°C and about 15°C for between about 4 hours and about 8 hours. The plasma sample can be incubated in step a at a temperature of between about 0°C and about 10°C for between about 4 hours and about 8 hours. The plasma sample can be incubated in step a at a temperature of between about 0°C and about 5°C for between about 4 hours and about 8 hours. The plasma sample can be incubated in step a at a temperature of about 4°C for between about 4 hours and about 8 hours.

[0077] The plasma sample can be incubated in step a at a temperature of between about 0°C and about 15°C for between about 5 hours and about 7 hours. The plasma sample can be incubated in step a at a temperature of between about 0°C and about 15°C for about 6 hours. The plasma sample can be incubated in step a at a temperature of between about 0°C and about 10°C for between about 5 hours and about 7 hours. The plasma sample can be incubated in step a at a temperature of between about 0°C and about 10°C for about 6 hours.

[0078] The plasma sample is incubated in step a at a temperature of between about 0°C and about 5°C for between about 5 hours and about 7 hours. Preferably, the plasma sample is incubated in step a at a temperature of between about 0°C and about 5°C for about 6 hours. Preferably, the plasma sample is incubated in step a at a temperature of about 4°C for between about 5 hours and about 7 hours. More preferably, the plasma sample is incubated in step a at a temperature of about 4°C for about 6 hours. Activation of the KKS in the plasma sample can be actively induced by any method known in the art. In the methods described herein, the KKS can be activated by external KKS-activating factors or reagents such as kaolin, silica, ellagic acid, dextran sulfate, polyanionic surfaces, glass or polyphosphate. Preferably, the methods described herein are carried out in the absence of any external KKS-activating agent (e.g. a KKS activating- factor or reagent such as dextran sulfate).

[0079] In step b of the method for determining a level of amidolytic activity of a plasma sample, a PKa inhibitor is added to at least a portion of the incubated plasma sample after step a. An inhibitor / sample mixture is formed in step b upon adding the PKa inhibitor to the incubated plasma sample from step a. Preferably, the PKa inhibitor added in step b is the only PKa inhibitor added in the methods. In addition, preferably no inhibitor of any of the enzymes of the KKS (e.g. a PKa inhibitor) is added ex vivo between the time that the blood sample is obtained / collected from the subject and the completion of step a. This means that incubation in step a can induce KKS activation in the plasma sample that is not inhibited by any added (exogenous) inhibitor of the KKS i.e. where no inhibitor of the KKS is added in step a.

[0080] The PKa inhibitor used in step b of the methods described herein is preferably a selective PKa inhibitor. The term "selective inhibitor" is a term well understood in the art. Preferably, the PKa inhibitor is selective for PKa over any of the other enzymes of the KKS e.g. FXIIa or protease enzymes that may be active in the plasma e.g thrombin, trypsin, plasmin, factor Xia. Therefore, the PKa inhibitor can specifically (preferentially) inhibit the activity of PKa. In the methods described herein, the PKa inhibitor may not inhibit the activity of FXIIa, thrombin or any other KKS proteases. In step b, the PKa inhibitor can be at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 1000-fold, at least 2000-fold, at least 3000-fold, at least 4000-fold, at least 5000-fold or at least 6000-fold selective for PKa over any of the other proteases of the KKS.

[0081] The PKa inhibitor can be added to the incubated plasma in step b in an amount sufficient to inhibit the activity of PKa in the sample. This amount can be determined by methods known in the art such as those described in Example 1 below. For example, the PKa inhibitor can be added to the incubated plasma in step b in an amount sufficient to inhibit the activity of PKa in the sample by at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80% or at least about 85%. Preferably, the PKa inhibitor is added to the incubated plasma in step b in an amount sufficient to inhibit the activity of PKa in the sample by at least about 90%. For example, the PKa inhibitor can be added to the incubated plasma in step b in an amount sufficient to inhibit the activity of PKa in the sample by at least about 90%, at least about 91%, at least about 92%, at least about 93% or at least about 94%. More preferably, the PKa inhibitor is added to the incubated plasma in step b in an amount sufficient to inhibit the activity of PKa in the sample by at least about 95%. The extent of PKa inhibition can be determined by a routine assay. For example, the assay described in Example 1 can be used to determine the extent of inhibition achieved by an amount of PKa inhibitor and thus the amount of PKa inhibitor required to inhibit the activity of PKa in the sample by a desired amount.

[0082] In step b, the PKa inhibitor can be selected from sebetralstat, berotralstat, avoralstat, KVD001, feniralstat, lanadelumab, STAR-0215, RZ-402 (ASP-440), ATN-249, and KV999272. In step b, the PKa inhibitor can be KV999272, which has the following structure:

[0083] KV999272.

[0084] KV999272 is also referred to in the art as FE999272 or VA999272, and is described in Clermont et al. (Investigative Ophthalmology & Visual Science May 2016, Vol.57, 2390-2399). KV999272 is (2'S,2"R)-4-(2'- (2"-amino-3"-(4’"-ethoxyphenyl)propanoylamino)-3'-phenylpropanoylamino)piperidine-l- carboxamidine. The PKa inhibitor can also be a salt and / or stereoisomer of KV999272, for example (2'S,2"R)-4-(2'-(2"-amino-3"-(4"'-ethoxyphenyl)propanoylamino)-3'-phenylpropanoylamino)piperidine- 1-carboxamidine trifluoroacetate. KV999272 is described in Example 1 of WO03 / 076458, the contents of which are incorporated herein by reference. The inhibition constant Ki of KV999272 for PKa can be determined according to Example 59 of WO03 / 076458 (see compound 1). KV999272, or a salt and / or stereoisomer thereof, can be added in step b of the method for determining a level of amidolytic activity of a plasma sample in an amount sufficient to inhibit the activity of PKa in the sample by at least about 90%. Preferably, KV999272, or a salt and / or stereoisomer thereof, is added in step b in an amount sufficient to inhibit the activity of PKa in the sample by at least about 95%. For example, KV999272, or a salt and / or stereoisomer thereof, can be added in step b at a final assay concentration of about 1 pM. Preferably, the PKa inhibitor used in step b is KV999272, or a salt and / or stereoisomer thereof.

[0085] The inhibitor / sample mixture (i.e. the incubated plasma sample from step a plus the PKa inhibitor from step b) can be incubated prior to commencing step c. Incubating the inhibitor / sample mixture in step b allows time for the PKa inhibitor to interact with the PKa in the sample.

[0086] In step b, the inhibitor / sample mixture can be incubated at room temperature e.g. ambient room temperature. The term "room temperature" is understood in the art to be a temperature at which laboratory experiments are usually performed. Therefore, the inhibitor / sample mixture can be incubated in step b at a temperature of between about 15°C and about 30°C. For example, the inhibitor / sample mixture can be incubated in step b at a temperature of between about 15°C and about 25°C. Preferably, the inhibitor / sample mixture is incubated in step b at a temperature of between about 18°C and about 23°C. More preferably, the inhibitor / sample mixture is incubated in step b at a temperature of between about 21°C and about 23°C.

[0087] In step b, the inhibitor / sample mixture can be incubated for a length of time sufficient to allow the PKa inhibitor to interact with the PKa present in the sample. Therefore, the inhibitor / sample mixture can be incubated in step b for between about 5 minutes and about 30 minutes. For example, the inhibitor / sample mixture can be incubated in step b for between about 5 minutes and about 25 minutes. Preferably, the inhibitor / sample mixture is incubated in step b for between about 10 minutes and about 20 minutes. More preferably, the inhibitor / sample mixture is incubated in step b for about 15 minutes.

[0088] The inhibitor / sample mixture can be incubated in step b at a temperature of between about 15°C and about 30°C for between about 5 minutes and about 30 minutes. The inhibitor / sample mixture can be incubated in step b at a temperature of between about 15°C and about 25°C for between about 5 minutes and about 30 minutes. The inhibitor / sample mixture can be incubated in step b at a temperature of between about 18°C and about 23°C for between about 5 minutes and about 30 minutes. The inhibitor / sample mixture can be incubated in step b at a temperature of between about 21°C and about 23°C for between about 5 minutes and about 30 minutes.

[0089] The inhibitor / sample mixture can be incubated in step b at a temperature of between about 15°C and about 30°C for between about 5 minutes and about 25 minutes. The inhibitor / sample mixture can be incubated in step b at a temperature of between about 15°C and about 25°C for between about 5 minutes and about 25 minutes. The inhibitor / sample mixture can be incubated in step b at a temperature of between about 18°C and about 23°C for between about 5 minutes and about 25 minutes. The inhibitor / sample mixture can be incubated in step b at a temperature of between about 21°C and about 23°C for between about 5 minutes and about 25 minutes.

[0090] The inhibitor / sample mixture can be incubated in step b at a temperature of between about 15°C and about 30°C for between about 10 minutes and about 20 minutes. The inhibitor / sample mixture can be incubated in step b at a temperature of between about 15°C and about 25°C for between about 10 minutes and about 20 minutes. The inhibitor / sample mixture can be incubated in step b at a temperature of between about 18°C and about 23°C for between about 10 minutes and about 20 minutes. The inhibitor / sample mixture can be incubated in step b at a temperature of between about 15°C and about 30°C for about 15 minutes. The inhibitor / sample mixture can be incubated in step b at a temperature of between about 15°C and about 25°C for about 15 minutes. The inhibitor / sample mixture can be incubated in step b at a temperature of between about 18°C and about 23°C for about 15 minutes.

[0091] Preferably, the inhibitor / sample mixture is incubated in step b at a temperature of between about 21°C and about 23°C for between about 10 minutes and about 20 minutes. Preferably, the inhibitor / sample mixture is incubated in step b at a temperature of between about 18°C and about 23°C for about 15 minutes. More preferably, the inhibitor / sample mixture is incubated in step b at a temperature of between about 21°C and about 23°C for about 15 minutes.

[0092] In the methods described herein, the PKa inhibitor may be the only inhibitor of the KKS added (ex vivo). The final mixture obtained at the end of the methods described herein may contain no other inhibitor of the KKS that has been added ex vivo. In other words, the methods described herein can be carried out in the absence of any inhibitor of the KKS added ex vivo, except for the PKa inhibitor of step b. For example, inhibitors of FXIIa, thrombin or trypsin activity may not be added to the methods described herein. Endogenous inhibitors of the KKS (e.g. C1INH which is an endogenous inhibitor of FXIIa) may be present in the plasma sample obtained from the subject. Exogenous inhibitors of the KKS other than the PKa inhibitor of step b may be present in the sample if e.g. the subject from whom the plasma sample has been obtained has been administered such inhibitor prior to obtaining the plasma sample.

[0093] In step c of the method for determining a level of amidolytic activity of a plasma sample, a substrate for determining the amidolytic activity is added to at least a portion of the inhibitor / sample mixture formed in step b. An inhibitor / sample / substrate mixture is formed in step c by adding a substrate for determining the amidolytic activity to the inhibitor / sample mixture. The substrate added in step c can be a substrate that is capable of detecting amidolytic activity. The substrate can be a substrate of amidolytic enzymes in the plasma sample that enables the activity of said enzymes to be determined. The substrate added in step c can be a substrate that is capable of detecting at least the activity of PKa. The substrate may not be specific to PKa. For example, other proteases of the KKS may be capable of acting on the substrate. Any substrate known in the art suitable for enabling the amidolytic activity of the inhibitor / sample mixture to be determined may be used (e.g. see Schapira, Scott and Colman. Biochemistry 1981, 20, 2738-2743; or Kluft C. Determination of prekallikrein in human plasma: optimal conditions for activating prekallikrein. J Lab Clin Med. 1978 Jan;91(l):83-95). In some aspects, the substrate is a peptide. The substrate can be a chromogenic substrate or a fluorogenic substrate. For example, the substrate can be a peptide (e.g. Pro-Phe-Arg) linked to a chromophore or fluorophore. Suitable chromogenic substrates are known in the art e.g. the chromogenic substrate can be H-D-Pro-Phe-Arg-pNA-2HCI. As used herein, the terms "H-D-Pro-Phe-Arg-pNA", "H-D-Pro-Phe-Arg- pNA-2HCI" and "H-D-Pro-Phe-Arg-pNA (hydrochloride)" may be used interchangeably. Suitable fluorogenic substrates are known in the art such as Pro-Phe-Arg-AFC, Pro-Phe-Arg-MCA and Z-Phe-Arg-AMC (e.g. Z-Phe-Arg-AMC.HCI). Preferably the substrate is a chromogenic substrate. More preferably, the substrate is H-D-Pro-Phe-Arg-pNA.

[0094] H-D-Pro-Phe-Arg-pNA is a chromogenic substrate for at least PKa. PKa binds to and cleaves H-D-Pro-Phe- Arg-pNA to release p-nitroanilide (pNA). Release of pNA can be quantified by colorimetric detection because pNA is a free chromophore. Therefore, release of pNA can provide a quantitative measure of at least PKa activity. Determining the release of pNA from H-D-Pro-Phe-Arg-pNA has previously been used as a proxy for the level of PKa activity. However, PKa is not the only amidolytic enzyme present in a subject's plasma sample (e.g. a human plasma sample) that is capable of cleaving H-D-Pro-Phe-Arg-pNA to release pNA. Multiple plasma proteases can cleave exogenous substrates used in PKa assays, which can reduce assay specificity and sensitivity, as shown in Example 2. An advantage of the present invention is that the activity specific to PKa can be determined thus providing a more specific and sensitive assay.

[0095] In step c, the substrate for determining the amidolytic activity can be incubated prior to adding the substrate to the inhibitor / sample mixture. Incubating the substrate in step c prior to adding the substrate to the inhibitor / sample mixture can improve absorbance readings, as shown in Example 10.

[0096] In step c, the substrate for determining the amidolytic activity can be incubated at a temperature of between about 15°C and about 40°C prior to adding the substrate to the inhibitor / sample mixture. Preferably, the substrate for determining the amidolytic activity is incubated at a temperature of between about 20°C and about 35°C prior to adding the substrate to the inhibitor / sample mixture. More preferably, the substrate for determining the amidolytic activity is incubated at a temperature of between about 25°C and about 35°C prior to adding the substrate to the inhibitor / sample mixture. Most preferably, the substrate for determining the amidolytic activity is incubated at a temperature of about 30°C prior to adding the substrate to the inhibitor / sample mixture.

[0097] The substrate for determining the amidolytic activity can be added in step c at substantially the same temperature as the temperature of the incubation of the substrate. Thus, the substrate for determining the amidolytic activity can be incubated immediately prior to adding the substrate to the inhibitor / sample mixture in step c. The substrate for determining the amidolytic activity can be added to the inhibitor / sample mixture in step c at a temperature of between about 15°C and about 40°C. Preferably, the substrate for determining the amidolytic activity is added to the inhibitor / sample mixture in step c at a temperature of between about 20°C and about 35°C. More preferably, the substrate for determining the amidolytic activity is added to the inhibitor / sample mixture in step c at a temperature of between about 25°C and about 35°C. Most preferably, the substrate for determining the amidolytic activity is added to the inhibitor / sample mixture in step c at a temperature of about 30°C.

[0098] Accordingly, the substrate for determining the amidolytic activity can be incubated at a temperature of between about 15°C and about 40°C, and then added to the inhibitor / sample mixture in step c at a temperature of between about 15°C and about 40°C. Preferably, the substrate for determining the amidolytic activity is incubated at a temperature of between about 20°C and about 35°C, and then added to the inhibitor / sample mixture in step c at a temperature of between about 20°C and about 35°C. More preferably, the substrate for determining the amidolytic activity is incubated at a temperature of between about 25°C and about 35°C, and then added to the inhibitor / sample mixture in step c at a temperature of between about 25°C and about 35°C. Most preferably, the substrate for determining the amidolytic activity is incubated at a temperature of about 30°C, and then added to the inhibitor / sample mixture in step c at a temperature of about 30°C.

[0099] In step c, the substrate for determining the amidolytic activity can be incubated for between about 5 minutes and about 40 minutes prior to adding the substrate to the inhibitor / sample mixture. For example, the substrate for determining the amidolytic activity can be incubated for between about 5 minutes and about 35 minutes, between about 5 minutes and about 30 minutes or between about 5 minutes and about 25 minutes prior to adding the substrate to the inhibitor / sample mixture. Preferably, the substrate for determining the amidolytic activity is incubated for between about 5 minutes and about 20 minutes prior to adding the substrate to the inhibitor / sample mixture. More preferably, the substrate for determining the amidolytic activity is incubated for between about 5 minutes and about 15 minutes, prior to adding the substrate to the inhibitor / sample mixture. Most preferably, the substrate for determining the amidolytic activity is incubated in step c for about 10 minutes prior to adding the substrate to the inhibitor / sample mixture.

[0100] The substrate for determining the amidolytic activity can be incubated at a temperature of between about 15°C and about 40°C for between about 5 minutes and about 40 minutes prior to adding the substrate to the inhibitor / sample mixture. The substrate for determining the amidolytic activity can be incubated at a temperature of between about 20°C and about 35°C for between about 5 minutes and about 40 minutes prior to adding the substrate to the inhibitor / sample mixture. The substrate for determining the amidolytic activity can be incubated at a temperature of between about 25°C and about 35°C for between about 5 minutes and about 40 minutes prior to adding the substrate to the inhibitor / sample mixture. The substrate for determining the amidolytic activity is incubated at a temperature of about 30°C for between about 5 minutes and about 40 minutes prior to adding the substrate to the inhibitor / sample mixture.

[0101] The substrate for determining the amidolytic activity can be incubated at a temperature of between about 15°C and about 40°C for between about 5 minutes and about 30 minutes prior to adding the substrate to the inhibitor / sample mixture. The substrate for determining the amidolytic activity can be incubated at a temperature of between about 20°C and about 35°C for between about 5 minutes and about 30 minutes prior to adding the substrate to the inhibitor / sample mixture. The substrate for determining the amidolytic activity can be incubated at a temperature of between about 25°C and about 35°C for between about 5 minutes and about 30 minutes prior to adding the substrate to the inhibitor / sample mixture. The substrate for determining the amidolytic activity is incubated at a temperature of about 30°C for between about 5 minutes and about 30 minutes prior to adding the substrate to the inhibitor / sample mixture.

[0102] The substrate for determining the amidolytic activity can be incubated at a temperature of between about 15°C and about 40°C for between about 5 minutes and about 20 minutes prior to adding the substrate to the inhibitor / sample mixture. The substrate for determining the amidolytic activity can be incubated at a temperature of between about 20°C and about 35°C for between about 5 minutes and about 20 minutes prior to adding the substrate to the inhibitor / sample mixture. The substrate for determining the amidolytic activity can be incubated at a temperature of between about 25°C and about 35°C for between about 5 minutes and about 20 minutes prior to adding the substrate to the inhibitor / sample mixture. The substrate for determining the amidolytic activity is incubated at a temperature of about 30°C for between about 5 minutes and about 20 minutes prior to adding the substrate to the inhibitor / sample mixture. The substrate for determining the amidolytic activity can be incubated at a temperature of between about 15°C and about 40°C for between about 5 minutes and about 15 minutes prior to adding the substrate to the inhibitor / sample mixture. The substrate for determining the amidolytic activity can be incubated at a temperature of between about 20°C and about 35°C for between about 5 minutes and about 15 minutes prior to adding the substrate to the inhibitor / sample mixture. The substrate for determining the amidolytic activity can be incubated at a temperature of between about 25°C and about 35°C for between about 5 minutes and about 15 minutes prior to adding the substrate to the inhibitor / sample mixture. Preferably, the substrate for determining the amidolytic activity is incubated at a temperature of between about 25°C and about 35°C for about 10 minutes prior to adding the substrate to the inhibitor / sample mixture. Preferably, the substrate for determining the amidolytic activity is incubated at a temperature of about 30°C for between about 5 minutes and about 15 minutes prior to adding the substrate to the inhibitor / sample mixture. More preferably, the substrate for determining the amidolytic activity can be incubated at a temperature of about 30°C for between about 10 minutes prior to adding the substrate to the inhibitor / sample mixture.

[0103] The substrate for determining the amidolytic activity can be incubated at a temperature of between about 15°C and about 40°C for between about 5 minutes and about 40 minutes, and then added to the inhibitor / sample mixture in step c at a temperature of between about 15°C and about 40°C. The substrate for determining the amidolytic activity can be incubated at a temperature of between about 20°C and about 35°C for between about 5 minutes and about 40 minutes, and then added to the inhibitor / sample mixture in step c at a temperature of between about 20°C and about 35°C. The substrate for determining the amidolytic activity can be incubated at a temperature of between about 25°C and about 35°C for between about 5 minutes and about 40 minutes, and then added to the inhibitor / sample mixture in step c at a temperature of between about 25°C and about 35°C. The substrate for determining the amidolytic activity is incubated at a temperature of about 30°C for between about 5 minutes and about 40 minutes, and then added to the inhibitor / sample mixture in step c at a temperature of about 30°C.

[0104] The substrate for determining the amidolytic activity can be incubated at a temperature of between about 15°C and about 40°C for between about 5 minutes and about 30 minutes, and then added to the inhibitor / sample mixture in step c at a temperature of between about 15°C and about 40°C. The substrate for determining the amidolytic activity can be incubated at a temperature of between about 20°C and about 35°C for between about 5 minutes and about 30 minutes, and then added to the inhibitor / sample mixture in step c at a temperature of between about 20°C and about 35°C. The substrate for determining the amidolytic activity can be incubated at a temperature of between about 25°C and about 35°C for between about 5 minutes and about 30 minutes, and then added to the inhibitor / sample mixture in step c at a temperature of between about 25°C and about 35°C. The substrate for determining the amidolytic activity is incubated at a temperature of about 30°C for between about 5 minutes and about 30 minutes, and then added to the inhibitor / sample mixture in step c at a temperature of about 30°C.

[0105] The substrate for determining the amidolytic activity can be incubated at a temperature of between about 15°C and about 40°C for between about 5 minutes and about 20 minutes, and then added to the inhibitor / sample mixture in step c at a temperature of between about 15°C and about 40°C. The substrate for determining the amidolytic activity can be incubated at a temperature of between about 20°C and about 35°C for between about 5 minutes and about 20 minutes, and then added to the inhibitor / sample mixture in step c at a temperature of between about 20°C and about 35°C. The substrate for determining the amidolytic activity can be incubated at a temperature of between about 25°C and about 35°C for between about 5 minutes and about 20 minutes, and then added to the inhibitor / sample mixture in step c at a temperature of between about 25°C and about 35°C. The substrate for determining the amidolytic activity is incubated at a temperature of about 30°C for between about 5 minutes and about 20 minutes, and then added to the inhibitor / sample mixture in step c at a temperature of about 30°C.

[0106] The substrate for determining the amidolytic activity can be incubated at a temperature of between about 15°C and about 40°C for between about 5 minutes and about 15 minutes, and then added to the inhibitor / sample mixture in step c at a temperature of between about 15°C and about 40°C. The substrate for determining the amidolytic activity can be incubated at a temperature of between about 20°C and about 35°C for between about 5 minutes and about 15 minutes, and then added to the inhibitor / sample mixture in step c at a temperature of between about 20°C and about 35°C.

[0107] The substrate for determining the amidolytic activity can be incubated at a temperature of between about 25°C and about 35°C for between about 5 minutes and about 15 minutes, and then added to the inhibitor / sample mixture in step c at a temperature of between about 25°C and about 35°C. Preferably, the substrate for determining the amidolytic activity is incubated at a temperature of between about 25°C and about 35°C for about 10 minutes, and then added to the inhibitor / sample mixture in step c at a temperature of between about 25°C and about 35°C. Preferably, the substrate for determining the amidolytic activity is incubated at a temperature of about 30°C for between about 5 minutes and about 15 minutes, and then added to the inhibitor / sample mixture in step c at a temperature of about 30°C. More preferably, the substrate for determining the amidolytic activity can be incubated at a temperature of about 30°C for between about 10 minutes, and then added to the inhibitor / sample mixture in step c at a temperature of about 30°C. The substrate can be present in a buffer. Any suitable buffer known in the art can be used. An exemplary buffer solution is Tris-NaCI e.g. 50mM Tris, 150mM NaCI, pH 7.8.

[0108] In step d of the method for determining a level of amidolytic activity of a plasma sample, the am idolytic activity of the inhibitor / sample / substrate mixture is determined. The amidolytic activity of the inhibitor / sample / substrate mixture can be determined by measuring the whole inhibitor / sample / substrate mixture, or by measuring a portion of the inhibitor / sample / substrate mixture. The level of amidolytic activity of a portion of the inhibitor / sample / substrate mixture is representative of the level of amidolytic activity of the whole inhibitor / sample / substrate mixture. The level of amidolytic activity of the inhibitor / sample / substrate mixture is representative of the level of amidolytic activity of the whole plasma sample. Therefore, the level of amidolytic activity of the plasma sample is the level of amidolytic activity of the inhibitor / sample / substrate mixture.

[0109] Any method known in the art can be used to measure the amidolytic activity of the inhibitor / sample / substrate mixture. The exact method for determining the amidolytic activity in step d will depend in part on the identity of the substrate added in step c. Step d of the described methods can be performed using a spectrophotometer e.g. when a chromogenic substrate such as H-D-Pro-Phe-Arg-pNA is added in step c. In some aspects, step d can be carried out on high throughput platforms, for example multi-well plates e.g. 24-, 48-, 96- or greater well plates can be used. In some aspects, plate readers that are capable of imaging multi-well plates in parallel can be used.

[0110] In the methods described herein which use a chromogenic or fluorogenic substrate, amidolytic activity can be determined by using a spectrophotometer or fluorometric microplate reader. Suitable spectrophotometers and fluorometric microplate readers are known in the art e.g. Tecan Spark®. For example, release of pNA from H-D-Pro-Phe-Arg-pNA can be determined by measuring absorbance at 405 nm.

[0111] In step d, the level of amidolytic activity of the inhibitor / sample / substrate mixture (i.e. the level of amidolytic activity of the plasma sample) can be determined for at least about 5 minutes. For example, the level of amidolytic activity of the inhibitor / sample / substrate mixture can be determined for between about 5 minutes and about 30 minutes or between about 10 minutes and about 25 minutes. Preferably, the level of amidolytic activity of the inhibitor / sample / substrate mixture is determined for between about 10 minutes and about 20 minutes. More preferably, the level of amidolytic activity of the inhibitor / sample / substrate mixture can be determined in step d for about 15 minutes. In step d, the level of amidolytic activity of the inhibitor / sample / substrate mixture (i.e. the level of amidolytic activity of the plasma sample) can be determined at a temperature of between about 15°C and about 40°C. For example, the level of amidolytic activity of the inhibitor / sample / substrate mixture can be determined at a temperature of between about 20°C and about 35°C. Preferably, the level of amidolytic activity of the inhibitor / sample / substrate mixture is determined at a temperature of between about 25°C and about 35°C. More preferably, the level of amidolytic activity of the inhibitor / sample / substrate mixture is determined in step d at a temperature of about 30°C.

[0112] Accordingly, in step d, the level of amidolytic activity of the inhibitor / sample / substrate mixture can be determined at a temperature of between about 15°C and about 40°C for at least about 5 minutes. The level of amidolytic activity of the inhibitor / sample / substrate mixture can be determined at a temperature of between about 20°C and about 35°C for at least about 5 minutes. The level of amidolytic activity of the inhibitor / sample / substrate mixture can be determined at a temperature of between about 25°C and about 35°C for at least about 5 minutes. The level of amidolytic activity of the inhibitor / sample / substrate mixture can be determined in step d at a temperature of about 30°C for at least about 5 minutes.

[0113] In step d, the level of amidolytic activity of the inhibitor / sample / substrate mixture can be determined at a temperature of between about 15°C and about 40°Cfor between about 5 minutes and about 30 minutes. The level of amidolytic activity of the inhibitor / sample / substrate mixture can be determined at a temperature of between about 20°C and about 35°C for between about 5 minutes and about 30 minutes. The level of amidolytic activity of the inhibitor / sample / substrate mixture can be determined at a temperature of between about 25°C and about 35°C for between about 5 minutes and about 30 minutes. The level of amidolytic activity of the inhibitor / sample / substrate mixture can be determined in step d at a temperature of about 30°C for between about 5 minutes and about 30 minutes.

[0114] In step d, the level of amidolytic activity of the inhibitor / sample / substrate mixture can be determined at a temperature of between about 15°C and about 40°C for between about 10 minutes and about 25 minutes. The level of amidolytic activity of the inhibitor / sample / substrate mixture can be determined at a temperature of between about 20°C and about 35°C for between about 10 minutes and about 25 minutes. The level of amidolytic activity of the inhibitor / sample / substrate mixture can be determined at a temperature of between about 25°C and about 35°C for between about 10 minutes and about 25 minutes. The level of amidolytic activity of the inhibitor / sample / substrate mixture can be determined in step d at a temperature of about 30°C for between about 10 minutes and about 25 minutes. In step d, the level of amidolytic activity of the inhibitor / sample / substrate mixture can be determined at a temperature of between about 15°C and about 40°C for between about 10 minutes and about 20 minutes. The level of amidolytic activity of the inhibitor / sample / substrate mixture can be determined at a temperature of between about 20°C and about 35°C for between about 10 minutes and about 20 minutes. The level of amidolytic activity of the inhibitor / sample / substrate mixture can be determined at a temperature of between about 15°C and about 40°C for about 15 minutes. The level of amidolytic activity of the inhibitor / sample / substrate mixture can be determined at a temperature of between about 20°C and about 35°C for about 15 minutes.

[0115] The level of amidolytic activity of the inhibitor / sample / substrate mixture is determined at a temperature of between about 25°C and about 35°C for between about 10 minutes and about 20 minutes. Preferably, the level of amidolytic activity of the inhibitor / sample / substrate mixture is determined in step d at a temperature of about 30°C for between about 10 minutes and about 20 minutes. Preferably, the level of amidolytic activity of the inhibitor / sample / substrate mixture is determined in step d at a temperature of between about 25°C and about 35°C for about 15 minutes. More preferably, the level of amidolytic activity of the inhibitor / sample / substrate mixture is determined in step d at a temperature of about 30°C for about 15 minutes.

[0116] The inhibitor / sample / substrate mixture can be shaken every 30 seconds for a period of 5 seconds in step d.

[0117] The invention therefore provides a method for determining a level of amidolytic activity of a plasma sample (in the presence of a PKa inhibitor added ex vivo) comprising: a. incubating the plasma sample at a temperature of between about 0°C and about 5°C for between about 5 hours and about 7 hours to induce kal likrein-kinin system activation in the sample; b. after step a, adding a PKa inhibitor to at least a portion of the incubated plasma sample to form an inhibitor / sample mixture and incubating the inhibitor / sample mixture at room temperature (e.g. a temperature of between about 18°C and about 23°C) for between about 10 minutes and about 20 minutes; c. after step b, adding a chromogenic substrate for determining the amidolytic activity to at least a portion of the inhibitor / sample mixture to form an inhibitor / sample / substrate mixture, wherein the substrate is incubated at a temperature of between about 25°C and about 35°C for between about 5 minutes and about 15 minutes and then added to the inhibitor / sample mixture at a temperature of between about 25°C and about 35°C; and d. after step c, determining the amidolytic activity of the inhibitor / sample / substrate mixture at a temperature of between about 25°C and about 35°C for between about 10 minutes and about 20 minutes, which provides the level of amidolytic activity of the plasma sample.

[0118] The invention also provides a method for determining a level of amidolytic activity of a plasma sample (in the presence of a PKa inhibitor added ex vivo) comprising: a. incubating the plasma sample at a temperature of about 4°C for about 6 hours to induce kal I ikre inkinin system activation in the sample; b. after step a, adding a PKa inhibitor to at least a portion of the incubated plasma sample to form an inhibitor / sample mixture and incubating the inhibitor / sample mixture at room temperature (e.g. a temperature of between about 21°C and about 23°C) for about 15 minutes; c. after step b, adding a chromogenic substrate for determining the amidolytic activity to at least a portion of the inhibitor / sample mixture to form an inhibitor / sample / substrate mixture, wherein the substrate is incubated at a temperature of about 30°C for about 10 minutes and then added to the inhibitor / sample mixture at a temperature of about 30°C; and d. after step c, determining the amidolytic activity of the inhibitor / sample / substrate mixture at a temperature of about 30°C for about 15 minutes, which provides the level of amidolytic activity of the plasma sample.

[0119] The invention also provides a method for determining a level of amidolytic activity of a plasma sample (in the presence of a PKa inhibitor added ex vivo) comprising: a. incubating the plasma sample at a temperature of about 4°C for about 6 hours to induce kal I ikre inkinin system activation in the sample; b. after step a, adding a PKa inhibitor to at least a portion of the incubated plasma sample to form an inhibitor / sample mixture and incubating the inhibitor / sample mixture at room temperature (e.g. a temperature of between about 21°C and about 23°C) for about 15 minutes; c. after step b, adding a chromogenic substrate for determining the amidolytic activity to at least a portion of the inhibitor / sample mixture to form an inhibitor / sample / substrate mixture, wherein the substrate is incubated at a temperature of about 30°C for about 10 minutes and then added to the inhibitor / sample mixture at a temperature of about 30°C; and d. after step c, determining the amidolytic activity of the inhibitor / sample / substrate mixture at a temperature of about 30°C for about 15 minutes, which provides the level of amidolytic activity of the plasma sample; wherein the PKa inhibitor is KV999272, or a salt and / or stereoisomer thereof, and the chromogenic substrate is H-D-Pro-Phe-Arg-pNA. The invention also provides a method for determining a level of amidolytic activity of a plasma sample (in the presence of a PKa inhibitor added ex vivo) comprising: a. incubating the plasma sample at a temperature of about 4°C for about 6 hours to induce kal I ikre inkinin system activation in the sample; b. after step a, adding a PKa inhibitor to at least a portion of the incubated plasma sample to form an inhibitor / sample mixture and incubating the inhibitor / sample mixture at room temperature (e.g. a temperature of between about 21°C and about 23°C) for about 15 minutes; c. after step b, adding a chromogenic substrate for determining the amidolytic activity to at least a portion of the inhibitor / sample mixture to form an inhibitor / sample / substrate mixture, wherein the substrate is incubated at a temperature of about 30°C for about 10 minutes and then added to the inhibitor / sample mixture at a temperature of about 30°C; and d. after step c, determining the amidolytic activity of the inhibitor / sample / substrate mixture, which provides the level of amidolytic activity of the plasma sample; wherein the PKa inhibitor is KV999272, or a salt and / or stereoisomer thereof, and the chromogenic substrate is H-D-Pro-Phe-Arg-pNA.

[0120] II. Methods for determining sPKa activity

[0121] Another aspect of the present invention relates to methods for determining the sPKa activity of a plasma sample comprising: (i) determining the total amidolytic activity of a plasma sample without adding (ex vivo) any inhibitor of the KKS by (a) incubating the plasma sample to induce KKS activation in the sample; (b) after step a, adding a substrate for determining amidolytic activity to at least a portion of the sample to form a sample / substrate mixture; and (c) after step b, determining the amidolytic activity of the sample / substrate mixture, which is the total amidolytic activity of the plasma sample; and (ii) subtracting (1) the level of amidolytic activity as determined by the methods described herein above (in the presence of an added (ex vivo) PKa inhibitor), from (2) the total amidolytic activity of the plasma sample; to provide the sPKa activity of the sample.

[0122] The methods described herein (e.g. in section I) relating to determining a level of amidolytic activity of a plasma sample (in the presence of an added (ex vivo) PKa inhibitor) can be used in the methods for determining sPKa activity. The methods for determining the sPKa activity of a plasma sample involve determining the total amidolytic activity of a plasma sample (i.e. the level of amidolytic activity of a plasma sample when a PKa inhibitor has not been added (ex vivo)) and determining the level of amidolytic activity of a plasma sample that is not attributable to PKa (i.e. the level of amidolytic activity of a plasma sample when a PKa inhibitor has been added (ex vivo)).

[0123] In some aspects, the total amidolytic activity of the plasma sample (i.e. the total amidolytic activity of the sample / substrate mixture) and the level of amidolytic activity of the plasma sample attributable to PKa (i.e. the level of amidolytic activity of the inhibitor / sample / substrate mixture) are determined substantially concomitantly or simultaneously.

[0124] The methods for determining the total amidolytic activity of a plasma sample and the level of amidolytic activity of a plasma sample that is not attributable to PKa can be substantially the same, except that determining the level of amidolytic activity of a plasma sample that is not attributable to PKa requires the (ex vivo) addition of a PKa inhibitor. Analogous conditions (or substantially analogous conditions) can be used when determining the total amidolytic activity of a plasma sample and the level of amidolytic activity of a plasma sample that is not attributable to PKa. The only difference between the methods for determining total amidolytic activity of a plasma sample and the methods for determining the level of amidolytic activity of a plasma sample that is not attributable to PKa, can be the addition of a PKa inhibitor in the methods for determining the level of amidolytic activity that is not attributable to PKa.

[0125] Part (i) of the method for determining the sPKa activity of a plasma sample is a method for determining the total amidolytic activity of a plasma sample. Therefore, in part (i) of the method, the level of amidolytic activity is determined without adding an inhibitor of the KKS. More specifically, in part (i), no inhibitors of any of the enzymes of the KKS are added. For example, no exogenous inhibitors of any of the enzymes of the KKS are added to the plasma sample ex vivo.

[0126] As noted above, a key difference between the method in part (i) of the method for determining the sPKa activity of a plasma sample and the methods described in section I is that the method in part (i) is conducted without adding any inhibitor of the KKS. Apart from this, similar, substantially the same, or the same conditions can be used in both sets of methods. Therefore, the features describe above in section I unrelated to the PKa inhibitor can be used (and are intended to be used) in the methods of part (i) under section II.

[0127] In particular, in part (i) of the method for determining the sPKa activity of a plasma sample, step a can be the same as step a set out above under section I. Specifically, the features described above for step a under section I can apply to step a under section II. The incubation conditions used in the methods for determining total amidolytic activity of a plasma sample can be substantially the same as the incubation conditions used in the methods for determining the level of amidolytic activity that is not attributable to PKa i.e. the methods described in section I. The total amidolytic activity of the plasma sample and the level of amidolytic activity not attributable to PKa can be determined following about the same duration of incubation of the plasma sample in step a of both methods. For example, the total amidolytic activity of the plasma sample and the level of amidolytic activity not attributable to PKa can be determined following about 6 hours of incubation of the plasma sample in step a of both methods. In some aspects, the total amidolytic activity of the plasma sample and the level of amidolytic activity not attributable to PKa can be determined following incubation of the plasma sample at a temperature of about 4°C for about 6 hours in step a of both methods.

[0128] In part (i) of the method for determining the sPKa activity of a plasma sample, step b can be the same as step c set out above under section I. Therefore, features relating to the inhibitor / sample mixture in step c under section I can be used, and are intended to be used, in the context of step b of part (i) under section II.

[0129] In the methods for determining the sPKa activity of a plasma sample, the substrate used in the method for determining total amidolytic activity of a plasma sample can be the same as the substrate used in the method for determining the level of amidolytic activity that is not attributable to PKa.

[0130] In part (i) of the method for determining the sPKa activity of a plasma sample, step c can be the same as step d set out above under section I. Therefore, features relating to the inhibitor / sample / substrate mixture in step d under section I can be used, and are intended to be used, in the context of the sample / substrate mixture in step c of part (i) under section II.

[0131] The invention therefore provides a method for determining the sPKa activity of a plasma sample comprising: i. determining the total amidolytic activity of a plasma sample without adding any inhibitor of the kallikrein-kinin system by: a. incubating the plasma sample at a temperature of between about 0°C and about 5°C for between about 5 hours and about 7 hours to induce kallikrein-kinin system activation in the sample; b. after step a, adding a chromogenic substrate for determining amidolytic activity to at least a portion of the sample to form a sample / substrate mixture, wherein the substrate is incubated at a temperature of between about 25°C and about 35°C for between about 5 minutes and about 15 minutes and then added to the sample at a temperature of between about 25°C and about 35°C; and c. after step b, determining the amidolytic activity of the sample / substrate mixture at a temperature of between about 25°C and about 35°C for between about 10 minutes and about 20 minutes, which is the total amidolytic activity of the plasma sample; and, ii. subtracting: (1) the level of amidolytic activity as determined by the method for determining a level of amidolytic activity of a plasma sample (when PKa activity is inhibited) as described herein, from (2) the total amidolytic activity of the plasma sample; to provide the sPKa activity of the sample.

[0132] The invention also provides a method for determining the sPKa activity of a plasma sample comprising: i. determining the total amidolytic activity of a plasma sample without adding any inhibitor of the kallikrein-kinin system by: a. incubating the plasma sample at a temperature of about 4°C for about 6 hours to induce kallikrein-kinin system activation in the sample; b. after step a, adding a chromogenic substrate for determining amidolytic activity to at least a portion of the sample to form a sample / substrate mixture, wherein the substrate is incubated at a temperature of about 30°C for about 10 minutes and then added to the sample at a temperature of about 30°C; and c. after step b, determining the amidolytic activity of the sample / substrate mixture at a temperature of about 30°C for about 15 minutes, which is the total amidolytic activity of the plasma sample; and, ii. subtracting: (1) the level of amidolytic activity as determined by the method for determining a level of amidolytic activity of a plasma sample (when PKa activity is inhibited) as described herein, from (2) the total amidolytic activity of the plasma sample; to provide the sPKa activity of the sample.

[0133] The invention also provides a method for determining the sPKa activity of a plasma sample comprising: i. determining the total amidolytic activity of a plasma sample without adding any inhibitor of the kallikrein-kinin system by: a. incubating the plasma sample at a temperature of about 4°C for about 6 hours to induce kallikrein-kinin system activation in the sample; b. after step a, adding a chromogenic substrate for determining amidolytic activity to at least a portion of the sample to form a sample / substrate mixture, wherein the substrate is incubated at a temperature of about 30°C for about 10 minutes and then added to the sample at a temperature of about 30°C; and c. after step b, determining the amidolytic activity of the sample / substrate mixture at a temperature of about 30°C for about 15 minutes, which is the total amidolytic activity of the plasma sample; wherein the PKa inhibitor is KV999272, or a salt and / or stereoisomer thereof, and the chromogenic substrate is H-D-Pro-Phe-Arg-pNA; and, ii. subtracting: (1) the level of amidolytic activity as determined by the method for determining a level of amidolytic activity of a plasma sample (when PKa activity is inhibited) as described herein, from (2) the total amidolytic activity of the plasma sample; to provide the sPKa activity of the sample.

[0134] The invention also provides a method for determining the sPKa activity of a plasma sample comprising: i. determining the total amidolytic activity of a plasma sample without adding any inhibitor of the kallikrein-kinin system by: a. incubating the plasma sample at a temperature of about 4°C for about 6 hours to induce kallikrein-kinin system activation in the sample; b. after step a, adding a chromogenic substrate for determining amidolytic activity to at least a portion of the sample to form a sample / substrate mixture, wherein the substrate is incubated at a temperature of about 30°C for about 10 minutes and then added to the sample at a temperature of about 30°C; and c. after step b, determining the amidolytic activity of the sample / substrate mixture, which is the total amidolytic activity of the plasma sample; wherein the PKa inhibitor is KV999272, or a salt and / or stereoisomer thereof, and the chromogenic substrate is H-D-Pro-Phe-Arg-pNA; and, ii. subtracting: (1) the level of amidolytic activity as determined by the method for determining a level of amidolytic activity of a plasma sample (when PKa activity is inhibited) as described herein, from (2) the total amidolytic activity of the plasma sample; to provide the sPKa activity of the sample.

[0135] The invention also provides a method for determining the sPKa activity of a plasma sample comprising: i. determining the total amidolytic activity of a plasma sample without adding any inhibitor of the kallikrein-kinin system by: al. incubating the plasma sample at a temperature of between about 0°C and about 5°C for between about 5 hours and about 7 hours to induce kallikrein-kinin system activation in the sample; bl. after step al, adding a chromogenic substrate for determining amidolytic activity to at least a portion of the sample to form a sample / substrate mixture, wherein the substrate is incubated at a temperature of between about 25°C and about 35°C for between about 5 minutes and about 15 minutes and then added to the sample at a temperature of between about 25°C and about 35°C; and cl. after step bl, determining the amidolytic activity of the sample / substrate mixture at a temperature of between about 25°C and about 35°C for between about 10 minutes and about 20 minutes, which is the total amidolytic activity of the plasma sample; and, ii. subtracting: (1) the level of amidolytic activity as determined by the method for determining a level of amidolytic activity of a plasma sample (when PKa activity is inhibited), from (2) the total amidolytic activity of the plasma sample; to provide the sPKa activity of the sample, wherein the method for determining a level of amidolytic activity of a plasma sample comprises: a2. incubating the plasma sample at a temperature of between about 0°C and about 5°C for between about 5 hours and about 7 hours to induce kallikrein-kinin system activation in the sample; b2. after step a2, adding a PKa inhibitor to at least a portion of the incubated plasma sample to form an inhibitor / sample mixture and incubating the inhibitor / sample mixture at room temperature (e.g. a temperature of between about 18°C and about 23°C) for between about 10 minutes and about 20 minutes; c2. after step b2, adding a chromogenic substrate for determining the amidolytic activity to at least a portion of the inhibitor / sample mixture to form an inhibitor / sample / substrate mixture, wherein the substrate is incubated at a temperature of between about 25°C and about 35°C for between about 5 minutes and about 15 minutes and then added to the inhibitor / sample mixture at a temperature of between about 25°C and about 35°C; and d2. after step c2, determining the amidolytic activity of the inhibitor / sample / substrate mixture at a temperature of between about 25°C and about 35°C for between about 10 minutes and about 20 minutes, which provides the level of amidolytic activity of the plasma sample. The invention also provides a method for determining the sPKa activity of a plasma sample comprising: i. determining the total amidolytic activity of a plasma sample without adding any inhibitor of the kallikrein-kinin system by: al. incubating the plasma sample at a temperature of between about 0°C and about 5°C for between about 5 hours and about 7 hours to induce kallikrein-kinin system activation in the sample; bl. after step al, adding a chromogenic substrate for determining amidolytic activity to at least a portion of the sample to form a sample / substrate mixture, wherein the substrate is incubated at a temperature of between about 25°C and about 35°C for between about 5 minutes and about 15 minutes and then added to the sample at a temperature of between about 25°C and about 35°C; and cl. after step bl, determining the amidolytic activity of the sample / substrate mixture at a temperature of between about 25°C and about 35°C for between about 10 minutes and about 20 minutes, which is the total amidolytic activity of the plasma sample, wherein the PKa inhibitor is KV999272, or a salt and / or stereoisomer thereof, and the chromogenic substrate is H-D-Pro-Phe-Arg-pNA; and, ii. subtracting: (1) the level of amidolytic activity as determined by the method for determining a level of amidolytic activity of a plasma sample (when PKa activity is inhibited), from (2) the total amidolytic activity of the plasma sample; to provide the sPKa activity of the sample, wherein the method for determining a level of amidolytic activity of a plasma sample comprises: a2. incubating the plasma sample at a temperature of between about 0°C and about 5°C for between about 5 hours and about 7 hours to induce kallikrein-kinin system activation in the sample; b2. after step a2, adding a PKa inhibitor to at least a portion of the incubated plasma sample to form an inhibitor / sample mixture and incubating the inhibitor / sample mixture at room temperature (e.g. a temperature of between about 18°C and about 23°C) for between about 10 minutes and about 20 minutes; c2. after step b2, adding a chromogenic substrate for determining the amidolytic activity to at least a portion of the inhibitor / sample mixture to form an inhibitor / sample / substrate mixture, wherein the substrate is incubated at a temperature of between about 25°C and about 35°C for between about 5 minutes and about 15 minutes and then added to the inhibitor / sample mixture at a temperature of between about 25°C and about 35°C; and d2. after step c2, determining the amidolytic activity of the inhibitor / sample / substrate mixture at a temperature of between about 25°C and about 35°C for between about 10 minutes and about 20 minutes, which provides the level of amidolytic activity of the plasma sample, wherein the PKa inhibitor is KV999272, or a salt and / or stereoisomer thereof, and the chromogenic substrate is H-D-Pro-Phe-Arg-pNA.

[0136] The invention also provides a method for determining the sPKa activity of a plasma sample comprising: i. determining the total amidolytic activity of a plasma sample without adding any inhibitor of the kallikrein-kinin system by: al. incubating the plasma sample at a temperature of about 4°C for about 6 hours to induce kallikrein-kinin system activation in the sample; bl. after step al, adding a chromogenic substrate for determining amidolytic activity to at least a portion of the sample to form a sample / substrate mixture, wherein the substrate is incubated at a temperature of about 30°C for about 10 minutes and then added to the sample at a temperature of about 30°C; and cl. after step bl, determining the amidolytic activity of the sample / substrate mixture at a temperature of about 30°C for about 15 minutes, which is the total amidolytic activity of the plasma sample; and, ii. subtracting: (1) the level of amidolytic activity as determined by the method for determining a level of amidolytic activity of a plasma sample (when PKa activity is inhibited), from (2) the total amidolytic activity of the plasma sample; to provide the sPKa activity of the sample, wherein the method for determining a level of amidolytic activity of a plasma sample comprises: a2. incubating the plasma sample at a temperature of about 4°C for about 6 hours to induce kallikrein-kinin system activation in the sample; b2. after step a2, adding a PKa inhibitor to at least a portion of the incubated plasma sample to form an inhibitor / sample mixture and incubating the inhibitor / sample mixture at room temperature (e.g. a temperature of between about 21°C and about 23°C) for about 15 minutes; c2. after step b2, adding a chromogenic substrate for determining the amidolytic activity to at least a portion of the inhibitor / sample mixture to form an inhibitor / sample / substrate mixture, wherein the substrate is incubated at a temperature of about 30°C for about 10 minutes and then added to the inhibitor / sample mixture at a temperature of about 30°C; and d2. after step c2, determining the amidolytic activity of the inhibitor / sample / substrate mixture at a temperature of about 30°C for about 15 minutes, which provides the level of amidolytic activity of the plasma sample.

[0137] The invention also provides a method for determining the sPKa activity of a plasma sample comprising: i. determining the total amidolytic activity of a plasma sample without adding any inhibitor of the kallikrein-kinin system by: al. incubating the plasma sample at a temperature of about 4°C for about 6 hours to induce kallikrein-kinin system activation in the sample; bl. after step al, adding a chromogenic substrate for determining amidolytic activity to at least a portion of the sample to form a sample / substrate mixture, wherein the substrate is incubated at a temperature of about 30°C for about 10 minutes and then added to the sample at a temperature of about 30°C; and cl. after step bl, determining the amidolytic activity of the sample / substrate mixture at a temperature of about 30°C for about 15 minutes, which is the total amidolytic activity of the plasma sample wherein the PKa inhibitor is KV999272, or a salt and / or stereoisomer thereof, and the chromogenic substrate is H-D-Pro-Phe-Arg-pNA; and, ii. subtracting: (1) the level of amidolytic activity as determined by the method for determining a level of amidolytic activity of a plasma sample (when PKa activity is inhibited), from (2) the total amidolytic activity of the plasma sample; to provide the sPKa activity of the sample, wherein the method for determining a level of amidolytic activity of a plasma sample as described herein comprises: a2. incubating the plasma sample at a temperature of about 4°C for about 6 hours to induce kallikrein-kinin system activation in the sample; b2. after step a2, adding a PKa inhibitor to at least a portion of the incubated plasma sample to form an inhibitor / sample mixture and incubating the inhibitor / sample mixture at room temperature (e.g. a temperature of between about 21°C and about 23°C) for about 15 minutes; c2. after step b2, adding a chromogenic substrate for determining the amidolytic activity to at least a portion of the inhibitor / sample mixture to form an inhibitor / sample / substrate mixture, wherein the substrate is incubated at a temperature of about 30°C for about 10 minutes and then added to the inhibitor / sample mixture at a temperature of about 30°C; and d2. after step c2, determining the amidolytic activity of the inhibitor / sample / substrate mixture at a temperature of about 30°C for about 15 minutes, which provides the level of amidolytic activity of the plasma sample, wherein the PKa inhibitor is KV999272, or a salt and / or stereoisomer thereof, and the chromogenic substrate is H-D-Pro-Phe-Arg-pNA.

[0138] III. Methods for determining a reference value of sPKa activity

[0139] Another aspect of the present invention relates to methods for determining a reference value of sPKa activity above which indicates elevated PKa activity comprising: conducting the method of determining sPKa activity described herein on multiple different plasma samples to identify multiple sPKa activity values, wherein the reference value of sPKa activity corresponds to the 95th percentile of the multiple sPKa activity values, and wherein the multiple different plasma samples are collected from at least one healthy subject.

[0140] As used herein, the term "reference value" is the value above which indicates elevated PKa activity. Therefore, a value of specific kallikrein activity of a plasma sample which is greater than the reference value indicates an elevated PKa activity of said plasma sample.

[0141] The reference value is determined from at least one healthy subject. That is, a plasma sample used in the methods for determining a reference value of specific kallikrein activity can be obtained from a subject who is apparently free of the target disease at the time that the blood sample is obtained. The reference value can be a level of sPKa activity in a population of healthy subjects (i.e. more than one subject that does not have or is not suspected of having a disease or disorder relating to dysfunctional KKS e.g. elevated PKa activity). The reference value can be determined in a particular population of healthy subjects. For example, the reference value can be a level of sPKa activity in a population of healthy female subjects. The reference value can be a level of sPKa activity in a population of healthy male subjects.

[0142] The reference value can be a single cut-off value. Typically, the 95th percentile is used. Methods of calculating the 95th percentile from a series of data are well known in the art. The 95th percentile is the value at which 95% of all measurements are below the value and 5% of measurements are above the value. Alternatively, the reference value can be the 75th, 80th, 85th, 90th, 98th or 99th percentile.

[0143] The reference value can be less than 5.00 nmol / min / mL. The reference value can be greater than 0.00 nmol / min / mL. The reference value can be in the range of from about 0.01 nmol / min / mL to about 5.00 nmol / min / mL. For example, the reference value can be in the range of from about 0.50 nmol / min / mL to about 4.50 nmol / min / mL, from about 1.00 nmol / min / mL to about 4.00 nmol / min / mL, from about 1.50 nmol / min / mL to about 3.50 nmol / min / mL, from about 2.00 nmol / min / mL to about 3.50 nmol / min / mL, or from about 2.50 nmol / min / mL to about 3.50 nmol / min / mL.

[0144] In a population of healthy female subjects, the reference value can be in the range of from about 2.50 nmol / min / mL to about 3.50 nmol / min / mL. In a population of healthy male subjects, the reference value can be in the range of from about 1.50 nmol / min / mL to about 2.50 nmol / min / mL.

[0145] To determine the reference value, the sPKa activity is determined for multiple different plasma samples, which can be obtained from multiple different healthy subjects. This provides the multiple sPKa activity values. The multiple sPKa activity values can be pooled into one dataset. From the collated data, the reference value (e.g. the 95th percentile) can be calculated.

[0146] The methods for determining sPKa activity of a plasma sample are discussed herein under section II. Specifically, the features described above for determining the sPKa activity of a plasma sample can be used, and are intended to be used, in the method for determining a reference value under section III.

[0147] The expected minimum number of different healthy patients required to calculate the reference value has been discussed by Bujand and Adnan (see Bujang MA, Adnan TH. "Requirements for Minimum Sample Size for Sensitivity and Specificity Analysis" J Clin Diagn Res. 2016 Oct;10(10):YE01-YE06. doi: 10.7860 / JCDR / 2016 / 18129.8744). In some aspects of the present invention, the sPKa activity is determined for at least 10 different plasma samples (from at least 10 different healthy subjects, i.e. subjects who do not have or are not suspected of having a disease or disorder relating to the KKS). The sPKa activity can be determined for at least 34 different plasma samples in the methods for determining the reference value (i.e. a sample size of at least 34 healthy subjects). The sPKa activity can be determined for at least 100 different plasma samples in the methods for determining the reference value. The sPKa activity can be determined for between 10 and 200 different healthy patients, such as between 10 and 150 or between 34 and 100 different healthy patents. Accordingly, the reference value can be determined by determining the sPKa activity of multiple different plasma samples collected from at least 1, at least 34, at least 50, at least 100 or at least 150 different healthy subjects. Preferably, the multiple different plasma samples are collected from at least 34 healthy subjects.

[0148] It is envisaged that each laboratory (or testing site more generally) will determine a reference value based on its own population of healthy subjects. Performance of the methods described herein may also vary per testing site, as is a known and comment facet of biological assays and methods. It is therefore envisaged that the specific reference value may differ in each testing site, although the method to determine may be consistent.

[0149] The invention therefore provides a method for determining a reference value of sPKa activity above which indicates elevated PKa activity comprising: conducting the method of determining sPKa activity described under secton II on multiple different plasma samples to identify multiple sPKa activity values; wherein the reference value of sPKa activity corresponds to the 95th percentile of the multiple sPKa activity values; and wherein the multiple different plasma samples are collected from at least 34 healthy subjects.

[0150] The invention also provides a method for determining a reference value of sPKa activity above which indicates elevated PKa activity comprising: conducting the method of determining sPKa activity described under section II on multiple different plasma samples (e.g. at least 34 different plasma samples) to identify multiple sPKa activity values; wherein the reference value of sPKa activity corresponds to the 95th percentile of the multiple sPKa activity values; and wherein the multiple different plasma samples are collected from at least 34 healthy subjects.

[0151] The invention also provides a method for determining a reference value of sPKa activity above which indicates elevated PKa activity comprising: conducting the method of determining sPKa activity described under section II on multiple different plasma samples to identify multiple sPKa activity values; wherein the reference value of sPKa activity corresponds to the 95th percentile of the multiple sPKa activity values; and wherein the multiple different plasma samples are collected from at least one healthy subject (e.g. at least 34 healthy subjects), wherein the method for determining the sPKa activity described under section II comprises: i. determining the total amidolytic activity of a plasma sample without adding any inhibitor of the kallikrein-kinin system by: a. incubating the plasma sample at a temperature of between about 0°C and about 5°C for between about 5 hours and about 7 hours to induce kallikrein-kinin system activation in the sample; b. after step a, adding a chromogenic substrate for determining amidolytic activity to at least a portion of the sample to form a sample / substrate mixture, wherein the substrate is incubated at a temperature of between about 25°C and about 35°C for between about 5 minutes and about 15 minutes and then added to the sample at a temperature of between about 25°C and about 35°C; and c. after step b, determining the amidolytic activity of the sample / substrate mixture at a temperature of between about 25°C and about 35°C for between about 10 minutes and about 20 minutes, which is the total amidolytic activity of the plasma sample; and, ii. subtracting: (1) the level of amidolytic activity as determined by the method for determining a level of amidolytic activity of a plasma sample (when PKa activity is inhibited) as described herein, from (2) the total amidolytic activity of the plasma sample; to provide the sPKa activity of the sample.

[0152] The invention also provides a method for determining a reference value of sPKa activity above which indicates elevated PKa activity comprising: conducting the method of determining sPKa activity described under section II on multiple different plasma samples to identify multiple sPKa activity values; wherein the reference value of sPKa activity corresponds to the 95th percentile of the multiple sPKa activity values; and wherein the multiple different plasma samples are collected from at least one healthy subject (e.g. at least 34 healthy subjects), wherein the method for determining the sPKa activity described under section II comprises: i. determining the total amidolytic activity of a plasma sample without adding any inhibitor of the kallikrein-kinin system by: al. incubating the plasma sample at a temperature of between about 0°C and about 5°C for between about 5 hours and about 7 hours to induce kallikrein-kinin system activation in the sample; bl. after step al, adding a chromogenic substrate for determining amidolytic activity to at least a portion of the sample to form a sample / substrate mixture, wherein the substrate is incubated at a temperature of between about 25°C and about 35°C for between about 5 minutes and about 15 minutes and then added to the sample at a temperature of between about 25°C and about 35°C; and cl. after step bl, determining the amidolytic activity of the sample / substrate mixture at a temperature of between about 25°C and about 35°C for between about 10 minutes and about 20 minutes, which is the total amidolytic activity of the plasma sample; and, ii. subtracting: (1) the level of amidolytic activity as determined by the method for determining a level of amidolytic activity of a plasma sample (when PKa activity is inhibited), from (2) the total amidolytic activity of the plasma sample; to provide the sPKa activity of the sample, wherein the method for determining a level of amidolytic activity of a plasma sample comprises: a2. incubating the plasma sample at a temperature of between about 0°C and about 5°C for between about 5 hours and about 7 hours to induce kallikrein- kinin system activation in the sample; b2. after step a2, adding a PKa inhibitor to at least a portion of the incubated plasma sample to form an inhibitor / sample mixture and incubating the inhibitor / sample mixture at room temperature (e.g. a temperature of between about 18°C and about 23°C) for between about 10 minutes and about 20 minutes; c2. after step b2, adding a chromogenic substrate for determining the amidolytic activity to at least a portion of the inhibitor / sample mixture to form an inhibitor / sample / substrate mixture, wherein the substrate is incubated at a temperature of between about 25°C and about 35°C for between about 5 minutes and about 15 minutes and then added to the inhibitor / sample mixture at a temperature of between about 25°C and about 35°C; and d2. after step c2, determining the amidolytic activity of the inhibitor / sample / substrate mixture at a temperature of between about 25°C and about 35°C for between about 10 minutes and about 20 minutes, which provides the level of amidolytic activity of the plasma sample.

[0153] The invention also provides a method for determining a reference value of sPKa activity above which indicates elevated PKa activity comprising: conducting the method of determining sPKa activity described under section II on multiple different plasma samples to identify multiple sPKa activity values; wherein the reference value of sPKa activity corresponds to the 95th percentile of the multiple sPKa activity values; and wherein the multiple different plasma samples are collected from at least one healthy subject (e.g. at least 34 healthy subjects), wherein the method for determining the sPKa activity described under section II comprises: i. determining the total amidolytic activity of a plasma sample without adding any inhibitor of the kallikrein-kinin system by: al. incubating the plasma sample at a temperature of between about 0°C and about 5°C for between about 5 hours and about 7 hours to induce kallikrein-kinin system activation in the sample; bl. after step al, adding a chromogenic substrate for determining amidolytic activity to at least a portion of the sample to form a sample / substrate mixture, wherein the substrate is incubated at a temperature of between about 25°C and about 35°C for between about 5 minutes and about 15 minutes and then added to the sample at a temperature of between about 25°C and about 35°C; and cl. after step bl, determining the amidolytic activity of the sample / substrate mixture at a temperature of between about 25°C and about 35°C for between about 10 minutes and about 20 minutes, which is the total amidolytic activity of the plasma sample wherein the PKa inhibitor is KV999272, or a salt and / or stereoisomer thereof, and the chromogenic substrate is H-D-Pro-Phe-Arg-pNA; and, ii. subtracting: (1) the level of amidolytic activity as determined by the method for determining a level of amidolytic activity of a plasma sample (when PKa activity is inhibited), from (2) the total amidolytic activity of the plasma sample; to provide the sPKa activity of the sample, wherein the method for determining a level of amidolytic activity of a plasma sample comprises: a2. incubating the plasma sample at a temperature of between about 0°C and about 5°C for between about 5 hours and about 7 hours to induce kallikrein- kinin system activation in the sample; b2. after step a2, adding a PKa inhibitor to at least a portion of the incubated plasma sample to form an inhibitor / sample mixture and incubating the inhibitor / sample mixture at room temperature (e.g. a temperature of between about 18°C and about 23°C) for between about 10 minutes and about 20 minutes; c2. after step b2, adding a chromogenic substrate for determining the amidolytic activity to at least a portion of the inhibitor / sample mixture to form an inhibitor / sample / substrate mixture, wherein the substrate is incubated at a temperature of between about 25°C and about 35°C for between about 5 minutes and about 15 minutes and then added to the inhibitor / sample mixture at a temperature of between about 25°C and about 35°C; and d2. after step c2, determining the amidolytic activity of the inhibitor / sample / substrate mixture at a temperature of between about 25°C and about 35°C for between about 10 minutes and about 20 minutes, which provides the level of amidolytic activity of the plasma sample, wherein the PKa inhibitor is KV999272, or a salt and / or stereoisomer thereof, and the chromogenic substrate is H-D-Pro-Phe-Arg-pNA.

[0154] The invention also provides a method for determining a reference value of sPKa activity above which indicates elevated PKa activity comprising: conducting the method of determining sPKa activity described under section II on multiple different plasma samples to identify multiple sPKa activity values; wherein the reference value of sPKa activity corresponds to the 95th percentile of the multiple sPKa activity values; and wherein the multiple different plasma samples are collected from at least one healthy subject (e.g. at least 34 healthy subjects), wherein the method for determining the sPKa activity described under section II comprises: i. determining the total amidolytic activity of a plasma sample without adding any inhibitor of the kallikrein-kinin system by: a. incubating the plasma sample at a temperature of about 4°C for about 6 hours to induce kallikrein-kinin system activation in the sample; b. after step a, adding a chromogenic substrate for determining amidolytic activity to at least a portion of the sample to form a sample / substrate mixture, wherein the substrate is incubated at a temperature of about 30°C for about 10 minutes and then added to the sample at a temperature of about 30°C; and c. after step b, determining the amidolytic activity of the sample / substrate mixture at a temperature of about 30°C for about 15 minutes, which is the total amidolytic activity of the plasma sample; and, ii. subtracting: (1) the level of amidolytic activity as determined by the method for determining a level of amidolytic activity of a plasma sample (when PKa activity is inhibited) as described herein, from (2) the total amidolytic activity of the plasma sample; to provide the sPKa activity of the sample.

[0155] The invention also provides a method for determining a reference value of sPKa activity above which indicates elevated PKa activity comprising: conducting the method of determining sPKa activity described under section II on multiple different plasma samples to identify multiple sPKa activity values; wherein the reference value of sPKa activity corresponds to the 95th percentile of the multiple sPKa activity values; and wherein the multiple different plasma samples are collected from at least one healthy subject (e.g. at least 34 healthy subjects), wherein the method for determining the sPKa activity described under section II comprises: i. determining the total amidolytic activity of a plasma sample without adding any inhibitor of the kallikrein-kinin system by: al. incubating the plasma sample at a temperature of about 4°C for about 6 hours to induce kallikrein-kinin system activation in the sample; bl. after step al, adding a chromogenic substrate for determining amidolytic activity to at least a portion of the sample to form a sample / substrate mixture, wherein the substrate is incubated at a temperature of about 30°C for about 10 minutes and then added to the sample at a temperature of about 30°C; and cl. after step bl, determining the amidolytic activity of the sample / substrate mixture at a temperature of about 30°C for about 15 minutes, which is the total amidolytic activity of the plasma sample; and, ii. subtracting: (1) the level of amidolytic activity as determined by the method for determining a level of amidolytic activity of a plasma sample (when PKa activity is inhibited), from (2) the total amidolytic activity of the plasma sample; to provide the sPKa activity of the sample, wherein the method for determining a level of amidolytic activity of a plasma sample comprises: a2. incubating the plasma sample at a temperature of about 4°C for about 6 hours to induce kallikrein-kinin system activation in the sample; b2. after step a2, adding a PKa inhibitor to at least a portion of the incubated plasma sample to form an inhibitor / sample mixture and incubating the inhibitor / sample mixture at room temperature (e.g. a temperature of between about 21°C and about 23°C) for about 15 minutes; c2. after step b2, adding a chromogenic substrate for determining the amidolytic activity to at least a portion of the inhibitor / sample mixture to form an inhibitor / sample / substrate mixture, wherein the substrate is incubated at a temperature of about 30°C for about 10 minutes and then added to the inhibitor / sample mixture at a temperature of about 30°C; and d2. after step c2, determining the amidolytic activity of the inhibitor / sample / substrate mixture at a temperature of about 30°C for about 15 minutes, which provides the level of amidolytic activity of the plasma sample.

[0156] The invention also provides a method for determining a reference value of sPKa activity above which indicates elevated PKa activity comprising: conducting the method of determining sPKa activity described under section II on multiple different plasma samples to identify multiple sPKa activity values; wherein the reference value of sPKa activity corresponds to the 95th percentile of the multiple sPKa activity values; and wherein the multiple different plasma samples are collected from at least one healthy subject (e.g. at least 34 healthy subjects), wherein the method for determining the sPKa activity described under section II comprises: i. determining the total amidolytic activity of a plasma sample without adding any inhibitor of the kallikrein-kinin system by: a. incubating the plasma sample at a temperature of between about 4°C for about 5 hours to induce kallikrein-kinin system activation in the sample; b. after step a, adding a chromogenic substrate for determining amidolytic activity to at least a portion of the sample to form a sample / substrate mixture, wherein the substrate is incubated at a temperature of about 30°C for about 10 minutes and then added to the sample at a temperature of about 30°C; and c. after step b, determining the amidolytic activity of the sample / substrate mixture at a temperature of about 30°C for about 15 minutes, which is the total amidolytic activity of the plasma sample; wherein the PKa inhibitor is KV999272, or a salt and / or stereoisomer thereof, and the chromogenic substrate is H-D-Pro-Phe-Arg-pNA; and, ii. subtracting: (1) the level of amidolytic activity as determined by the method for determining a level of amidolytic activity of a plasma sample (when PKa activity is inhibited) as described herein, from (2) the total amidolytic activity of the plasma sample; to provide the sPKa activity of the sample.

[0157] The invention also provides a method for determining a reference value of sPKa activity above which indicates elevated PKa activity comprising: conducting the method of determining sPKa activity described under section II on multiple different plasma samples to identify multiple sPKa activity values; wherein the reference value of sPKa activity corresponds to the 95th percentile of the multiple sPKa activity values; and wherein the multiple different plasma samples are collected from at least one healthy subject (e.g. at least 34 healthy subjects), wherein the method for determining the sPKa activity described under section II comprises: i. determining the total amidolytic activity of a plasma sample without adding any inhibitor of the kallikrein-kinin system by: al. incubating the plasma sample at a temperature of between about 4°C for about 6 hours to induce kallikrein-kinin system activation in the sample; bl. after step al, adding a chromogenic substrate for determining amidolytic activity to at least a portion of the sample to form a sample / substrate mixture, wherein the substrate is incubated at a temperature of about 30°C for about 10 minutes and then added to the sample at a temperature of about 30°C; and cl. after step bl, determining the amidolytic activity of the sample / substrate mixture, which is the total amidolytic activity of the plasma sample; wherein the PKa inhibitor is KV999272, or a salt and / or stereoisomer thereof, and the chromogenic substrate is H-D-Pro-Phe-Arg-pNA; and, ii. subtracting: (1) the level of amidolytic activity as determined by the method for determining a level of amidolytic activity of a plasma sample (when PKa activity is inhibited), from (2) the total amidolytic activity of the plasma sample; to provide the sPKa activity of the sample wherein the method for determining a level of amidolytic activity of a plasma sample comprises: a2. incubating the plasma sample at a temperature of about 4°C for about 6 hours to induce kallikrein-kinin system activation in the sample; b2. after step a2, adding a PKa inhibitor to at least a portion of the incubated plasma sample to form an inhibitor / sample mixture and incubating the inhibitor / sample mixture at room temperature (e.g. a temperature of between about 21°C and about 23°C) for about 15 minutes; c2. after step b2, adding a chromogenic substrate for determining the amidolytic activity to at least a portion of the inhibitor / sample mixture to form an inhibitor / sample / substrate mixture, wherein the substrate is incubated at a temperature of about 30°C for about 10 minutes and then added to the inhibitor / sample mixture at a temperature of about 30°C; and d2. after step c2, determining the amidolytic activity of the inhibitor / sample / substrate mixture at a temperature of about 30°C for about 15 minutes, which provides the level of amidolytic activity of the plasma sample, wherein the PKa inhibitor is KV999272, or a salt and / or stereoisomer thereof, and the chromogenic substrate is H-D-Pro-Phe-Arg-pNA.

[0158] IV. Methods for use in diagnosing a subject at risk of a disease, identifying a subject at risk of a disease, determining if a disease or disorder is susceptible to treatment with a PKa inhibitor, and / or treatment

[0159] Another aspect of the present invention relates to methods for use in diagnosing a subject with a disease or disorder relating to elevated PKa activity comprising: (a) determining the sPKa activity of a plasma sample collected from the subject, which is the subject's sPKa activity; (b) comparing the subject's sPKa activity with a reference value of sPKa activity above which indicates elevated PKa activity; and (c) diagnosing the subject with a disease or disorder relating to elevated PKa activity if the subject's sPKa activity is greater than the reference value. The clinician may take into account other factors in order to come to a diagnosis, e.g. an analysis of family history and / or relevant genetic testing, all of which is known in the art.

[0160] The present invention also provides a method for identifying a subject at risk of having a disease or disorder relating to elevated PKa activity comprising: (a) determining the sPKa activity of a plasma sample collected from the subject, which is the subject's sPKa activity; (b) comparing the subject's sPKa activity with a reference value of sPKa activity above which indicates elevated PKa activity; and (c) identifying a subject at risk of having a disease or disorder relating to elevated PKa activity if the subject's sPKa activity is greater than the reference value.

[0161] The present invention also provides a method for determining if a disease or disorder is susceptible to treatment with a PKa inhibitor comprising: (a) determining the sPKa activity of a plasma sample collected from a subject, which is the subject's sPKa activity; (b) comparing the subject's sPKa activity with a reference value of sPKa activity above which indicates elevated PKa activity; and (c) identifying the disease or disorder as being susceptible to treatment with a PKa inhibitor if the subject's sPKa activity is greater than the reference value.

[0162] In these methods, the sPKa activity of the subject can be determined by the methods described herein under section II. The sPKa activity of the plasma sample obtained from the subject is a measurement of the sPKa activity of the subject. Therefore, as used herein, the "subject's sPKa activity" is the sPKa activity of a plasma sample obtained from said subject.

[0163] In some aspects, the subject does not have a detectable and / or effective amount of any inhibitor of the KKS in their blood when providing the blood sample from which the plasma sample is obtained and then tested. For instance, the subject may not have been administered any exogenous inhibitors of the KKS prior to obtaining the plasma sample. The subject may not have been administered any exogenous inhibitors of the KKS prior for the previous at least 5 days, at least 10 days, at least 15 days, at least 30 days, or at least 50 days prior to obtaining the plasma sample from the subject.

[0164] Alternatively, the subject can be being treated with an inhibitor of the KKS (e.g. a PKa inhibitor) when providing a blood sample for testing, and these methods aim to determine whether further therapy is needed, e.g. whether a change of treatment is required and / or whether the subject may be suffering from a second disease or disorder, such that these methods identify a new comorbidity. The subject's level of sPKa activity can be compared with a reference value to determine whether said subject has or is at risk for developing a disease or disorder relating to elevated PKa activity. The reference value can be determined as described herein above under section III. If the level of sPKa activity deviates from the reference value, the subject can be identified as having or at risk for developing a disease or disorder relating to the KKS. If the level of sPKa activity is greater than to the reference value, the subject can be identified as having or at risk of developing a disease or disorder relating to elevated PKa activity.

[0165] In some aspects, the subject is a human subject suspected of having or at risk for a disease or disorder relating to the KKS. Preferably, the subject is a human subject suspected of having or at risk for a disease or disorder relating to elevated PKa activity. The subject may present with a disease or disorder that is not well understood. Utilising the methods described herein can determine if the subject's PKa system is elevated such that disease or disorder may be susceptible to treatment with a PKa inhibitor.

[0166] For example, the subject can be a human patient presenting with a symptom of a disease or disorder relating to an elevated PKa activity. For example, the subject may have swelling of tissues such as in the hands, feet, limbs, face, intestinal tract, and / or airway; fatigue; headache; muscle aches; skin tingling; abdominal pain; nausea; vomiting; diarrhoea; difficulty swallowing; hoarseness; shortness of breath; and / or mood changes. In other aspects, the subject may have no symptom of a disease or disorder relating to elevated PKa activity at the time of obtaining the blood sample, has no history of said symptom, or no history of a disease or disorder relating to elevated PKa activity.

[0167] Examples of diseases or disorders relating to elevated PKa activity include angioedema; HAE, including : (i) HAE type 1, (ii) HAE type 2, and (iii) HAE-nClINH, including HAE-nClINH where the genetic cause is unknown; BK-AEnH, including AE-nCl Inh, ACE and tPA induced angioedema; vascular hyperpermeability; stroke including ischemic stroke and haemorrhagic accidents; retinal edema; diabetic retinopathy; impaired visual acuity; DME; retinal vein occlusion; AMD; neuroinflammation; neuroinflammatory / neurodegenerative disorders such as MS (multiple sclerosis); other neurodegenerative diseases such as Alzheimer's disease, epilepsy and migraine; sepsis; bacterial sepsis; inflammation; anaphylaxis; thrombosis; thromboembolism caused by increased propensity of medical devices that come into contact with blood to clot blood; prothrombotic conditions including disseminated intravascular coagulation (DIC), venous thromboembolism (VTE), cancer associated thrombosis, complications caused by mechanical and bioprosthetic heart valves, complications caused by catheters, complications caused by ECMO, complications caused by LVAD, complications caused by dialysis, complications caused by CPB, sickle cell disease, joint arthroplasty, thrombosis induced to tPA, Paget- Schroetter syndrome and Budd-Chari syndrome; atherosclerosis; COVID-19; acute respiratory distress syndrome (ARDS); idiopathic pulmonary fibrosis (IPF); rheumatoid arthritis (RA); cold-induced urticarial autoinflammatory syndrome; obesity; diabetes; kidney disease; renal fibrosis; glomerulosclerosis; renal scarring; ischemia / reperfusion injury in native or transplant kidneys; glioblastoma and acute kidney injury.

[0168] Further examples of diseases or disorders relating to elevated PKa activity include pancreatitis, cerebral haemorrhage, nephropathy, cardiomyopathy, neuropathy, inflammatory bowel disease, arthritis, septic shock, hypotension, cancer, adult respiratory distress syndrome, cardiopulmonary bypass surgery, bleeding from post operative surgery, and ophthalmic diseases or disorders. Ophthalmic diseases or disorders can affect the front or back of the eye. Front-of-eye ophthalmic diseases or disorders include, for example, corneal oedema, anterior uveitis, pterygium, corneal diseases or opacifications with an exudative or inflammatory component, conjunctivitis, allergy and laser induced exudation. Back-of-eye ophthalmic diseases or disorders include, for example, impaired visual acuity, exudative eye diseases and more particularly exudative retinopathies, exudative macular degeneration, macular edema (e.g. diabetic macular edema), diabetic retinopathy, retinal vein occlusion, age-related macular degeneration or retinopathy of prematurity.

[0169] Preferably, the disease or disorder identified / diagnosed using the methods described herein can be bradykinin-mediated angioedema. In other words, the methods can be used to identify a subject as having bradykinin-mediated angioedema. The bradykinin-mediated angioedema can be HAE including: (i) HAE type 1, (ii) HAE type 2, and (iii) HAE-nClINH. Thus, the methods for use in diagnosis described herein can be used to diagnose a subject with HAE-nClINH. It is particularly advantageous to provide new methods for use in identifying / diagnosing HAE-nClINH, as discussed above, therefore identifying / diagnosing HAE-nClINH is a preferred aspect of the present invention.

[0170] If the subject is diagnosed with / identified as having a disease or disorder relating to elevated PKa activity as set out above, the method can further comprise administering to the subject an effective amount of a therapeutic agent. Suitable therapeutic agents are discussed below.

[0171] The methods described herein can also be applied to evaluate the efficacy of treatment for a disease or disorder relating elevated PKa activity. The methods described herein can be used to monitor the progress of a disease or disorder relating to elevated PKa activity, assess the efficacy of a treatment for the disease or disorder, and / or predict disease status (e.g. attack versus quiescence) in a subject.

[0172] For instance, the invention can provide a method for evaluating treatment of a disease or disorder relating to elevated PKa in a subject comprising: (a) determining the sPKa activity of a plasma sample collected from the subject, which is the subject's sPKa activity; (b) comparing the subject's sPKa activity with a reference value of sPKa activity above which indicates elevated PKa activity; and (c) evaluating effectiveness of the treatment based on whether the subject's sPKa activity is greater than the reference value.

[0173] As above, the subject's sPKa activity can be determined as set out in section II. The reference value can be determined as set out in section III.

[0174] If the subject's sPKa activity is not greater than the reference value, the conclusion may be that the course of treatment that the subject is taking is sufficient. Therefore, if the subject's sPKa activity is not greater than the reference value, the subject may be instructed to continue their existing treatment and / or return for a further evaluation in the future (e.g. in a month's, 2 months', 3 months', 4 months', 6 months', or a year's, time).

[0175] Alternatively, if the subject's sPKa activity is greater than the reference value, the conclusion may be that the course of treatment that the subject is taking is not sufficient. Therefore, if the subject's sPKa activity is greater than the reference value, the subject may be at risk of experiencing (or susceptible to) a disease or disorder relating to elevated PKa activity. The subject may therefore to change their course of treatment. For instance, the subject may increase the dose of their existing treatment. Alternatively, the subject may change their medication.

[0176] As described above, HAE is a disease or disorder relating to elevated PKa activity. A subject may be taking a prophylactic HAE treatment such as lanadelumab, serpin Cl esterase inhibitor, berotralstat, or deucrictibant. Over time, the effectiveness of the treatment may wane. For instance, the subject's PKa activity may increase to greater than a reference value (determined in at least one healthy subject) such that the subject becomes susceptible to suffering an HAE attack. The subject may therefore be evaluated on a regular basis to ensure that the prophylactic treatment is still effective (i.e. effective at preventing the subject's sPKa activity from being greater than a reference value of a healthy subject). For instance, the subject may be evaluated every 1 month, every 2 months, every 3 months, every 4 months, every 6 months, or every year. If evaluation according to the present invention finds that the subject’s sPKa activity is greater than a reference value, the subject may be administered an on-demand treatment of HAE. The on-demand treatment may be sebetralstat, which is discussed further below in section V. More specifically, the subject may be administered sebetralstat to treat any attacks of HAE, which the patient has been determined as being at risk of, because of the methods described herein. Alternatively, the subject may be being treated for is bradykinin-mediated angioedema non-hereditary (BK-AEnH), which as described above, can be difficult to diagnose and treat. Patients being treated for BK-AEnH may therefore benefit from their treatment being evaluated by the methods described herein. Specifically, a subject suffering from BK-AEnH and being treated with tranexamic acid may be evaluated in accordance with the invention.

[0177] The invention therefore provides a method for use in diagnosing a subject with a disease or disorder relating to elevated PKa activity comprising: a. determining the sPKa activity of a plasma sample collected from the subject, which is the subject's sPKa activity; b. comparing the subject's sPKa activity with a reference value of sPKa activity above which indicates elevated PKa activity; and c. diagnosing the subject with a disease or disorder relating to elevated PKa activity if the subject's sPKa activity is greater than the reference value, wherein the disease or disorder relating to elevated PKa activity is bradykinin-mediated angioedema.

[0178] The invention also provides a method for use in diagnosing a subject with a disease or disorder relating to elevated PKa activity comprising: a. determining the sPKa activity of a plasma sample collected from the subject, which is the subject's sPKa activity; b. comparing the subject's sPKa activity with a reference value of sPKa activity above which indicates elevated PKa activity; and c. diagnosing the subject with a disease or disorder relating to elevated PKa activity if the subject's sPKa activity is greater than the reference value, wherein the disease or disorder relating to elevated PKa activity is bradykinin-mediated angioedema, wherein the bradykinin-mediated angioedema is HAE (e.g. HAE-nClINH).

[0179] The invention also provides a method for use in diagnosing a subject with a disease or disorder relating to elevated PKa activity comprising: a. determining the sPKa activity of a plasma sample collected from the subject, which is the subject's sPKa activity; b. comparing the subject's sPKa activity with a reference value of sPKa activity above which indicates elevated PKa activity; and c. diagnosing the subject with a disease or disorder relating to elevated PKa activity if the subject's sPKa activity is greater than the reference value, wherein the subject is a human patient presenting with a symptom of a disease or disorder relating to an elevated PKa activity.

[0180] The invention also provides a method for use in diagnosing a subject with a disease or disorder relating to elevated PKa activity comprising: a. determining the sPKa activity of a plasma sample collected from the subject, which is the subject's sPKa activity; b. comparing the subject's sPKa activity with a reference value of sPKa activity above which indicates elevated PKa activity; and c. diagnosing the subject with a disease or disorder relating to elevated PKa activity if the subject's sPKa activity is greater than the reference value, wherein the subject is a human patient presenting with a symptom of a disease or disorder relating to an elevated PKa activity, wherein the symptom is swelling of tissues such as in the hands, feet, limbs, face, intestinal tract, and / or airway; fatigue; headache; muscle aches; skin tingling; abdominal pain; nausea; vomiting; diarrhoea; difficulty swallowing; hoarseness; shortness of breath; and / or mood changes.

[0181] The invention also provides a method for use in diagnosing a subject with a disease or disorder relating to elevated PKa activity comprising: a. determining the sPKa activity of a plasma sample collected from the subject, which is the subject's sPKa activity; b. comparing the subject's sPKa activity with a reference value of sPKa activity above which indicates elevated PKa activity; and c. diagnosing the subject with a disease or disorder relating to elevated PKa activity if the subject's sPKa activity is greater than the reference value, wherein the method for determining sPKa activity in step a is as described under section II herein; and wherein the reference value of sPKa activity in step b is determined according to the methods for determining a reference value as described under section III herein.

[0182] The invention also provides a method for use in diagnosing a subject with a disease or disorder relating to elevated PKa activity comprising: al. determining the sPKa activity of a plasma sample collected from the subject, which is the subject's sPKa activity; bl. comparing the subject's sPKa activity with a reference value of sPKa activity above which indicates elevated PKa activity; and cl. diagnosing the subject with a disease or disorder relating to elevated PKa activity (in particular HAE, more specifically HAE-nClINH) if the subject's sPKa activity is greater than the reference value, wherein the method for determining sPKa activity in step al comprises: i. determining the total amidolytic activity of a plasma sample without adding any inhibitor of the kallikrein-kinin system by: a2. incubating the plasma sample at a temperature of about 4°C for about 6 hours to induce kallikrein-kinin system activation in the sample; b2. after step a2, adding a chromogenic substrate for determining amidolytic activity to at least a portion of the sample to form a sample / substrate mixture, wherein the substrate is incubated at a temperature of about 30°C for about 10 minutes and then added to the sample at a temperature of about 30°C; and c2. after step b2, determining the amidolytic activity of the sample / substrate mixture at a temperature of about 30°C for about 15 minutes, which is the total amidolytic activity of the plasma sample wherein the PKa inhibitor is KV999272, or a salt and / or stereoisomer thereof, and the chromogenic substrate is H-D-Pro-Phe-Arg-pNA; and, ii. subtracting: (1) the level of amidolytic activity as determined by the method for determining a level of amidolytic activity of a plasma sample (when PKa activity is inhibited), from (2) the total amidolytic activity of the plasma sample; to provide the sPKa activity of the sample, wherein the method for determining a level of amidolytic activity of a plasma sample comprises: a3. incubating the plasma sample at a temperature of about 4°C for about 6 hours to induce kallikrein-kinin system activation in the sample; b3. after step a3, adding a PKa inhibitor to at least a portion of the incubated plasma sample to form an inhibitor / sample mixture and incubating the inhibitor / sample mixture at room temperature (e.g. a temperature of between about 21°C and about 23°C) for about 15 minutes; c3. after step b3, adding a chromogenic substrate for determining the amidolytic activity to at least a portion of the inhibitor / sample mixture to form an inhibitor / sample / substrate mixture, wherein the substrate is incubated at a temperature of about 30°C for about 10 minutes and then added to the inhibitor / sample mixture at a temperature of about 30°C; and d3. after step c3, determining the amidolytic activity of the inhibitor / sample / substrate mixture at a temperature of about 30°C for about 15 minutes, which provides the level of amidolytic activity of the plasma sample wherein the PKa inhibitor is KV999272, or a salt and / or stereoisomer thereof, and the chromogenic substrate is H-D-Pro-Phe-Arg-pNA; and wherein the reference value of sPKa activity in step bl is determined according to a method for determining a reference value of sPKa activity above which indicates elevated PKa activity, said method comprising: conducting the method of determining sPKa activity (as set out above) on multiple different plasma samples to identify multiple sPKa activity values; wherein the reference value of sPKa activity corresponds to the 95th percentile of the multiple sPKa activity values; and wherein the multiple different plasma samples are collected from at least one healthy subject (e.g. at least 34 healthy subjects).

[0183] The invention also provides a method for identifying a subject at risk of having a disease or disorder relating to elevated PKa activity comprising: a. determining the sPKa activity of a plasma sample collected from the subject, which is the subject's sPKa activity; b. comparing the subject's sPKa activity with a reference value of sPKa activity above which indicates elevated PKa activity; and c. identifying a subject at risk of having a disease or disorder relating to elevated PKa activity if the subject's sPKa activity is greater than the reference value wherein the disease or disorder relating to elevated PKa activity is bradykinin-mediated angioedema.

[0184] The invention also provides a method for identifying a subject at risk of having a disease or disorder relating to elevated PKa activity comprising: a. determining the sPKa activity of a plasma sample collected from the subject, which is the subject's sPKa activity; b. comparing the subject's sPKa activity with a reference value of sPKa activity above which indicates elevated PKa activity; and c. identifying a subject at risk of having a disease or disorder relating to elevated PKa activity if the subject's sPKa activity is greater than the reference value wherein the disease or disorder relating to elevated PKa activity is bradykinin-mediated angioedema, wherein the bradykinin-mediated angioedema is HAE (e.g. HAE-nClINH).

[0185] The invention also provides a method for identifying a subject at risk of having a disease or disorder relating to elevated PKa activity comprising: a. determining the sPKa activity of a plasma sample collected from the subject, which is the subject's sPKa activity; b. comparing the subject's sPKa activity with a reference value of sPKa activity above which indicates elevated PKa activity; and c. identifying a subject at risk of having a disease or disorder relating to elevated PKa activity if the subject's sPKa activity is greater than the reference value wherein the subject is a human patient presenting with a symptom of a disease or disorder relating to an elevated PKa activity.

[0186] The invention also provides a method for identifying a subject at risk of having a disease or disorder relating to elevated PKa activity comprising: a. determining the sPKa activity of a plasma sample collected from the subject, which is the subject's sPKa activity; b. comparing the subject's sPKa activity with a reference value of sPKa activity above which indicates elevated PKa activity; and c. identifying a subject at risk of having a disease or disorder relating to elevated PKa activity if the subject's sPKa activity is greater than the reference value wherein the subject is a human patient presenting with a symptom of a disease or disorder relating to an elevated PKa activity, wherein the symptom is swelling of tissues such as in the hands, feet, limbs, face, intestinal tract, and / or airway; fatigue; headache; muscle aches; skin tingling; abdominal pain; nausea; vomiting; diarrhoea; difficulty swallowing; hoarseness; shortness of breath; and / or mood changes. The invention also provides a method for identifying a subject at risk of having a disease or disorder relating to elevated PKa activity comprising: al. determining the sPKa activity of a plasma sample collected from the subject, which is the subject's sPKa activity; bl. comparing the subject's sPKa activity with a reference value of sPKa activity above which indicates elevated PKa activity; and cl. diagnosing the subject with a disease or disorder relating to elevated PKa activity (in particular HAE, more specifically HAE-nClINH) if the subject's sPKa activity is greater than the reference value, wherein the method for determining sPKa activity in step al comprises: i. determining the total amidolytic activity of a plasma sample without adding any inhibitor of the kallikrein-kinin system by: a2. incubating the plasma sample at a temperature of about 4°C for about 6 hours to induce kallikrein-kinin system activation in the sample; b2. after step a2, adding a chromogenic substrate for determining amidolytic activity to at least a portion of the sample to form a sample / substrate mixture, wherein the substrate is incubated at a temperature of about 30°C for about 10 minutes and then added to the sample at a temperature of about 30°C; and c2. after step b2, determining the amidolytic activity of the sample / substrate mixture at a temperature of about 30°C for about 15 minutes, which is the total amidolytic activity of the plasma sample wherein the PKa inhibitor is KV999272, or a salt and / or stereoisomer thereof, and the chromogenic substrate is H-D-Pro-Phe-Arg-pNA; and, ii. subtracting: (1) the level of amidolytic activity as determined by the method for determining a level of amidolytic activity of a plasma sample (when PKa activity is inhibited), from (2) the total amidolytic activity of the plasma sample; to provide the sPKa activity of the sample, wherein the method for determining a level of amidolytic activity of a plasma sample comprises: a3. incubating the plasma sample at a temperature of about 4°C for about 6 hours to induce kallikrein-kinin system activation in the sample; b3. after step a3, adding a PKa inhibitor to at least a portion of the incubated plasma sample to form an inhibitor / sample mixture and incubating the inhibitor / sa mple mixture at room temperature (e.g. a temperature of between about 21°C and about 23°C) for about 15 minutes; c3. after step b3, adding a chromogenic substrate for determining the amidolytic activity to at least a portion of the inhibitor / sample mixture to form an inhibitor / sample / substrate mixture, wherein the substrate is incubated at a temperature of about 30°C for about 10 minutes and then added to the inhibitor / sample mixture at a temperature of about 30°C; and d3. after step c3, determining the amidolytic activity of the inhibitor / sample / substrate mixture at a temperature of about 30°C for about 15 minutes, which provides the level of amidolytic activity of the plasma sample wherein the PKa inhibitor is KV999272, or a salt and / or stereoisomer thereof, and the chromogenic substrate is H-D-Pro-Phe-Arg-pNA; and wherein the reference value of sPKa activity in step bl is determined according to a method for determining a reference value of sPKa activity above which indicates elevated PKa activity, said method comprising: conducting the method of determining sPKa activity (as set out above) on multiple different plasma samples to identify multiple sPKa activity values; wherein the reference value of sPKa activity corresponds to the 95th percentile of the multiple sPKa activity values; and wherein the multiple different plasma samples are collected from at least one healthy subject (e.g. at least 34 healthy subjects).

[0187] The invention also provides a method for determining if a disease or disorder is susceptible to treatment with a PKa inhibitor comprising: a. determining the sPKa activity of a plasma sample collected from a subject, which is the subject's sPKa activity; b. comparing the subject's sPKa activity with a reference value of sPKa activity above which indicates elevated PKa activity; and c. identifying the disease or disorder as being susceptible to treatment with a PKa inhibitor if the subject's sPKa activity is greater than the reference value wherein the disease or disorder is bradykinin-mediated angioedema.

[0188] The invention also provides a method for determining if a disease or disorder is susceptible to treatment with a PKa inhibitor comprising: a. determining the sPKa activity of a plasma sample collected from a subject, which is the subject's sPKa activity; b. comparing the subject's sPKa activity with a reference value of sPKa activity above which indicates elevated PKa activity; and c. identifying the disease or disorder as being susceptible to treatment with a PKa inhibitor if the subject's sPKa activity is greater than the reference value wherein the disease or disorder is bradykinin-mediated angioedema, wherein the bradykinin-mediated angioedema is HAE (e.g. HAE-nClINH).

[0189] The invention also provides a method for determining if a disease or disorder is susceptible to treatment with a PKa inhibitor comprising: a. determining the sPKa activity of a plasma sample collected from a subject, which is the subject's sPKa activity; b. comparing the subject's sPKa activity with a reference value of sPKa activity above which indicates elevated PKa activity; and c. identifying the disease or disorder as being susceptible to treatment with a PKa inhibitor if the subject's sPKa activity is greater than the reference value wherein the disease or disorder is bradykinin-mediated angioedema, wherein the bradykinin-mediated angioedema is HAE (e.g. HAE-nClINH), and the PKa inhibitor is sebetralstat.

[0190] The invention also provides a method for determining if a disease or disorder is susceptible to treatment with a PKa inhibitor comprising: a. determining the sPKa activity of a plasma sample collected from a subject, which is the subject's sPKa activity; b. comparing the subject's sPKa activity with a reference value of sPKa activity above which indicates elevated PKa activity; and c. identifying the disease or disorder as being susceptible to treatment with a PKa inhibitor if the subject's sPKa activity is greater than the reference value, wherein the subject is a human patient presenting with a symptom of a disease or disorder relating to an elevated PKa activity.

[0191] The invention also provides a method for determining if a disease or disorder is susceptible to treatment with a PKa inhibitor comprising: a. determining the sPKa activity of a plasma sample collected from a subject, which is the subject's sPKa activity; b. comparing the subject's sPKa activity with a reference value of sPKa activity above which indicates elevated PKa activity; and c. identifying the disease or disorder as being susceptible to treatment with a PKa inhibitor if the subject's sPKa activity is greater than the reference value, wherein the subject is a human patient presenting with a symptom of a disease or disorder relating to an elevated PKa activity, wherein the symptom is swelling of tissues such as in the hands, feet, limbs, face, intestinal tract, and / or airway; fatigue; headache; muscle aches; skin tingling; abdominal pain; nausea; vomiting; diarrhoea; difficulty swallowing; hoarseness; shortness of breath; and / or mood changes.

[0192] The invention also provides a method for determining if a disease or disorder is susceptible to treatment with a PKa inhibitor comprising: a. determining the sPKa activity of a plasma sample collected from a subject, which is the subject's sPKa activity; b. comparing the subject's sPKa activity with a reference value of sPKa activity above which indicates elevated PKa activity; and c. identifying the disease or disorder as being susceptible to treatment with a PKa inhibitor if the subject's sPKa activity is greater than the reference value, wherein the subject is a human patient presenting with a symptom of a disease or disorder relating to an elevated PKa activity, wherein the symptom is swelling of tissues such as in the hands, feet, limbs, face, intestinal tract, and / or airway; fatigue; headache; muscle aches; skin tingling; abdominal pain; nausea; vomiting; diarrhoea; difficulty swallowing; hoarseness; shortness of breath; and / or mood changes, and wherein the PKa inhibitor is sebetralstat.

[0193] The invention also provides a method for determining if a disease or disorder is susceptible to treatment with a PKa inhibitor comprising: al. determining the sPKa activity of a plasma sample collected from the subject, which is the subject's sPKa activity; bl. comparing the subject's sPKa activity with a reference value of sPKa activity above which indicates elevated PKa activity; and cl. diagnosing the subject with a disease or disorder relating to elevated PKa activity (in particular HAE, more specifically HAE-nClINH) if the subject's sPKa activity is greater than the reference value, wherein the method for determining sPKa activity in step al comprises: i. determining the total amidolytic activity of a plasma sample without adding any inhibitor of the kallikrein-kinin system by: a2. incubating the plasma sample at a temperature of about 4°C for about 6 hours to induce kallikrein-kinin system activation in the sample; b2. after step a2, adding a chromogenic substrate for determining amidolytic activity to at least a portion of the sample to form a sample / substrate mixture, wherein the substrate is incubated at a temperature of about 30°C for about 10 minutes and then added to the sample at a temperature of about 30°C; and c2. after step b2, determining the amidolytic activity of the sample / substrate mixture at a temperature of about 30°C for about 15 minutes, which is the total amidolytic activity of the plasma sample wherein the PKa inhibitor is KV999272, or a salt and / or stereoisomer thereof, and the chromogenic substrate is H-D-Pro-Phe-Arg-pNA; and, ii. subtracting: (1) the level of amidolytic activity as determined by the method for determining a level of amidolytic activity of a plasma sample (when PKa activity is inhibited), from (2) the total amidolytic activity of the plasma sample; to provide the sPKa activity of the sample, wherein the method for determining a level of amidolytic activity of a plasma sample comprises: a3. incubating the plasma sample at a temperature of about 4°C for about 6 hours to induce kallikrein-kinin system activation in the sample; b3. after step a3, adding a PKa inhibitor to at least a portion of the incubated plasma sample to form an inhibitor / sample mixture and incubating the inhibitor / sample mixture at room temperature (e.g. a temperature of between about 21°C and about 23°C) for about 15 minutes; c3. after step b3, adding a chromogenic substrate for determining the amidolytic activity to at least a portion of the inhibitor / sample mixture to form an inhibitor / sample / substrate mixture, wherein the substrate is incubated at a temperature of about 30°C for about 10 minutes and then added to the inhibitor / sample mixture at a temperature of about 30°C; and d3. after step c3, determining the amidolytic activity of the inhibitor / sample / substrate mixture at a temperature of about 30°C for about 15 minutes, which provides the level of amidolytic activity of the plasma sample wherein the PKa inhibitor is KV999272, or a salt and / or stereoisomer thereof, and the chromogenic substrate is H-D-Pro-Phe-Arg-pNA; and wherein the reference value of sPKa activity in step bl is determined according to a method for determining a reference value of sPKa activity above which indicates elevated PKa activity, said method comprising: conducting the method of determining sPKa activity (as set out above) on multiple different plasma samples to identify multiple sPKa activity values; wherein the reference value of sPKa activity corresponds to the 95th percentile of the multiple sPKa activity values; and wherein the multiple different plasma samples are collected from at least one healthy subject (e.g. at least 34 healthy subjects).

[0194] The inventors have also discovered that it can be advantageous to use a reference value determined in a particular population of healthy subjects in the methods described herein, e.g. those above in section IV relating to methods which involve comparing a subject's sPKa activity with a reference value of sPKa activity.

[0195] For instance, the inventors have discovered that sPKa activity can vary depending on a subject's demographic (e.g. sex). Therefore, it can be beneficial to compare a subject's sPKa activity with a reference value determined in a corresponding population of healthy subjects i.e. a population of healthy subjects with matching demographics (e.g. a population of healthy subjects with the same sex as the subject). This can provide improved methods for use in diagnosing a subject with a disease or disorder relating to elevated PKa activity, identifying a subject at risk of having a disease or disorder relating to elevated PKa activity, determining if a disease or disorder is susceptible to treatment with a PKa inhibitor and evaluating treatment of a disease or disorder relating to elevated PKa in a subject.

[0196] In Example 6, plasma from healthy female subjects trended towards higher sPKa activity than plasma from healthy male subjects. Therefore, when seeking to diagnose / identify a subject with a disease or disorder relating to elevated PKa activity, it can be advantageous to compare the subject's sPKa activity to a reference value determined in a population of healthy subjects of the same sex as the subject. For instance, when the subject being diagnosed / identified is male, it can be beneficial to compare the male subject's sPKa activity with a reference value determined in a population of healthy male subjects. When the subject being diagnosed is female, it can be beneficial to compare the female subject's sPKa activity with a reference value determined in a population of healthy female subjects.

[0197] A population of healthy male subjects can comprise substantially all male subjects. A population of healthy male subjects can comprise some female subjects, provided that the majority of the population is male. For instance, a population of healthy male subjects can comprise at least 70% male subjects, at least 80% male subjects, at least 90% male subjects, at least 95% male subjects, or at least 99% male subjects. Preferably, a population of healthy male subjects consists entirely of male subjects.

[0198] A population of healthy female subjects can comprise substantially all female subjects. A population of healthy female subjects can comprise some male subjects, provided that the majority of the population is female. For instance, a population of healthy female subjects can comprise at least 70% female subjects, at least 80% female subjects, at least 90% female subjects, at least 95% female subjects, or at least 99% female subjects. Preferably, a population of healthy female subjects consists entirely of female subjects.

[0199] V. Methods of treating a subject

[0200] Another aspect of the present invention relates to methods for treating a disease or disorder relating to elevated PKa activity in a subject, comprising administering to the subject a therapeutic agent for treating the disease or disorder, wherein the subject has a sPKa activity that is greater than a reference value, the reference value being a sPKa activity above which indicates elevated PKa activity; and wherein the sPKa activity of the subject is as determined as discussed herein.

[0201] The invention also provides a therapeutic agent for use in the treatment of a disease or disorder relating to elevated PKa activity in a subject, comprising administering to the subject a therapeutic agent for treating the disease or disorder, wherein the subject has a sPKa activity that is greater than a reference value, the reference value being a sPKa activity above which indicates elevated PKa activity; and wherein the sPKa activity of the subject is as determined as discussed herein.

[0202] The level of sPKa activity in a plasma sample can indicate that the disease or disorder relating to elevated PKa activity may be treatable, for example with a PKa inhibitor. A subject identified as having or at risk for developing a disease or disorder relating to elevated PKa activity can be a subject who susceptible to treatment. A subject identified as having or at risk for developing a disease or disorder relating to elevated PKa activity can be treated with any appropriate therapeutic agent. In the methods for treating a disease or disorder relating to elevated PKa activity in a subject, a subject having a sPKa activity that is greater than to a reference value can be treated with a therapeutic agent for treating the disease or disorder. In some aspects, the subject can be identified as having or diagnosed with a disease or disorder relating to elevated PKa activity. The methods described in section IV can be used in this regard. The methods described in section II can be used to determine the subject's sPKa activity. The methods described in section III can be used to determine the reference value.

[0203] Examples of diseases or disorders relating to elevated PKa activity are described herein above. Each of these diseases can be treated according to the present invention. Preferably, the disease or disorder relating to elevated PKa activity to be treated is bradykinin-mediated angioedema. The bradykinin-mediated angioedema can be HAE including: (i) HAE type 1, (ii) HAE type 2, and (iii) HAE-nClINH. Thus, the methods of treatment described herein can relate to treatment of HAE-nClINH, which as noted above, is a preferred aspect of the present invention. Preferably, the methods of treatment described herein can relate to treatment HAE-nClINH where the genetic cause is unknown.

[0204] The therapeutic agent for treating the diseases or disorders relating to elevated PKa activity can be any suitable therapeutic agent. A suitable therapeutic agent is an agent that is suitable for treating a disease or disorder relating to elevated PKa activity e.g. a known marketed treatment. A suitable therapeutic agent can be a peptide, a small molecule inhibitor, or an antibody or antigen-binding fragment thereof. The therapeutic agent can be a kal likrein binding agent, a bradykinin B2 receptor antagonist, a FXIIa binding agent, or a C1INH replacement agent. As used herein, a FXIIa binding agent or FXIIa inhibitor may also be a Factor XII ( FXI I) binding agents or inhibitors. The terms "FXIIa inhibitor" and "FXI I inhibitor" may be used interchangeably. In some aspects, the therapeutic agent is lanadelumab, garadacimab, sebetralstat, berotralstat, icatibant, ecallantide, RZ-402 (ASP 440), STAR 0215, avoralstat, C1INH replacement therapy (such as Cinryze®, Ruconest®, Berinert®, or Haegarda®), donidalorsen, or deucrictibant. In some aspects, the therapeutic agent is lanadelumab, garadacimab, sebetralstat, berotralstat, ecallantide, avoralstat, icatibant, deucrictibant, or C1INH replacement therapy (such as Cinryze®, Ruconest®, Berinert®, or Haegarda®). In some aspects, the therapeutic agent is sebetralstat.

[0205] For example, the substrate used in the methods described herein may be Z-Phe-Arg-AMC (e.g. Z-Phe-Arg- AMC.HCI) when the therapeutic agent administered to the subject is berotralstat. Alternatively, the substrate used in the methods described herein may be Z-Phe-Arg-AMC (e.g. Z-Phe-Arg-AMC. HCI) when the therapeutic agent administered to the subject is donidalorsen. In some aspects, the therapeutic agent is administered one or more times to the subject.

[0206] The therapeutic agent can be provided as a pharmaceutically acceptable salt or solvate thereof. The therapeutic agent can also be provided in a pharmaceutical composition additionally comprising one or more pharmaceutically acceptable excipients.

[0207] If sebetralstat is used as the therapeutic agent, sebetralstat can be orally administered in a therapeutically effective amount. Sebetralstat can be administered at an amount of between about 100 mg and about 1500 mg, about 300 mg to about 1800 mg, about 100 mg and about 1400 mg, about 200 mg and about 1200 mg, about 300 mg and about 1200 mg, about 600 mg and about 1200 mg, about 450 mg and about 900 mg, about 500 mg and about 1000 mg, about 450 mg and about 600 mg, about 500 mg and about 700 mg (more specifically, 600 mg), about 800 mg and about 1000 mg per day, about 900 mg and about 1400 mg (more specifically 1200 mg), or about 900 mg and about 1200 mg. The dosage amount can be 300 mg. The dosage amount can be 600 mg. The daily dosage amount can be 900 mg. In another specific embodiment, the daily dosage amount can be 1200 mg. The dosage amount can be 1800 mg.

[0208] Preferably, the dosage amount is 600 mg.

[0209] The safety and efficacy of sebetralstat has been confirmed in a phase 2 clinical trial. See e.g. Aygbren-Pursun et al. (The Lancet, Volume 401, Issue 10375, P458-469, February 11, 2023) or the clinical trial results under NCT05259917. Sebetralstat is currently in phase 3 studies (Cohn DM, Aygbren-Pursun E, Bernstein JA, et al. Evaluation of patient-reported outcome measures for on-demand treatment of hereditary angioedema attacks and design of KONFIDENT, a phase 3 trial of sebetralstat. Clin Transl Allergy 2023; 13: el2288).

[0210] Sebetralstat is preferably used as an "on-demand" treatment. "On-demand" treatment, in the context of HAE, means that sebetralstat is administered upon need of therapy in connection with one specific HAE attack. The attack may be an HAE attack that arises even though the patent is concomitantly taking a prophylactic treatment for HAE (e.g. lanadelumab, berotralstat, or deucrictibant). "On-demand treatment" is also referred to in the art as "acute treatment" "On-demand" does not require the administration of sebetralstat continuously at regular intervals (e.g. once a week, twice a week, etc.) irrespective of an instance of an HAE attack. This is in contrast to some other known treatments of HAE (e.g. treatments with C1INH replacement therapy (such as Cinryze®, Ruconest®, Berinert®, or Haegarda®, as described above) that require continuous and regular dosing for therapy. Instead, sebetralstat is taken when the patient requires fast-acting therapeutic effects. Therefore, preferably, the dosage amount is administered within 1 hour of an HAE attack being recognised.

[0211] Sebetralstat can be used to treat patent-recognised attacks of HAE, preferably within 1 hour of an HAE attack being recognised. Sebetralstat can be used to treat adults and adolescents aged 12 years and older. Sebetralstat can be administered as a 300mg dose. Sebetralstat can be administered as a 600mg dose. A second dose of sebetralstat can be administered after at least 3 hours.

[0212] Each dosage amount administered to the patient can be sub-divided into small unit dosage amounts. For example, the preferred 600 mg dosage amount of the sebetralstat can be sub-divided into two unit dosage amounts (e.g. two tablets), each comprising 300 mg of the sebetralstat.

[0213] Sebetralstat can be administered as an oral dosage form comprising sebetralstat and pharmaceutically acceptable excipients. The oral dosage form can be in the form of a tablet or a capsule. The oral dosage form can be a tablet. The oral dosage form can be a capsule. The oral dosage form can be an orodispersible tablet.

[0214] The dosage form can be a tablet comprising microcrystalline cellulose as a diluent, croscarmellose sodium as a disintegrant, polyvinyl pyrrolidone as a binder, and optionally magnesium stearate as a lubricant. In a preferred tablet, sebetralstat comprises: (i) at least about 40 wt% of the tablet (more specifically about 40 wt% to about 60 wt%), compared to the total mass of the tablet; (ii) about 25 wt% to about 60 wt% of the diluent (more specifically about 25 wt% to about 40 wt%, compared to the total mass of the tablet; (iii) about 1 wt% to about 15 wt% of the disintegrant (more specifically about 2 wt% to about 6 wt%), compared to the total mass of the tablet; (iv) about 1 wt% to about 20 wt% of the binder (more specifically about 2 wt% to about 5 wt%), compared to the total mass of the tablet; and when present, (v) about 0.1 to about 5 wt% lubricant (more specifically about 0.1 wt% to about 1.5 wt%), compared to the total mass of the tablet. The dosage form can be a tablet containing 300 mg of the sebetralstat.

[0215] The tablet can further comprise extragranular excipients comprising: microcrystalline cellulose as an extragranular diluent, croscarmellose sodium as an extragranular disintegrant, polyvinyl pyrrolidone as an extragranular binder, and / or magnesium stearate as an extragranular lubricant.

[0216] The dosage forms described herein (e.g. the tablets) can be film coated, wherein the film coating can comprise one or more of hypromellose, lactose monohydrate, titanium dioxide and triacetin. The invention therefore provides a method for treating a disease or disorder relating to elevated PKa activity in a subject, comprising administering to the subject a therapeutic agent for treating the disease or disorder; wherein the subject has a sPKa activity that is greater than a reference value, the reference value being a sPKa activity above which indicates elevated PKa activity; wherein the sPKa activity is as defined under section II herein, wherein the reference value is as defined under section III herein, wherein the disease or disorder relating to elevated PKa activity is bradykinin-mediated angioedema, and wherein the therapeutic agent is lanadelumab, garadacimab, sebetralstat, berotralstat, icatibant, ecallantide, RZ-402 (ASP 440), STAR 0215, avoralstat, C1INH replacement therapy (such as Cinryze®, Ruconest®, Berinert®, or Haegarda®), donidalorsen, or deucrictibant.

[0217] The invention also provides a method for treating a disease or disorder relating to elevated PKa activity in a subject, comprising administering to the subject a therapeutic agent for treating the disease or disorder; wherein the subject has a sPKa activity that is greater than a reference value, the reference value being a sPKa activity above which indicates elevated PKa activity; wherein the sPKa activity is as defined under section II herein, wherein the reference value is as defined under section III herein, wherein the disease or disorder relating to elevated PKa activity is bradykinin-mediated angioedema, wherein the bradykinin-mediated angioedema is HAE (e.g. HAE-nClINH), and wherein the therapeutic agent is lanadelumab, garadacimab, sebetralstat, berotralstat, icatibant, ecallantide, RZ-402 (ASP 440), STAR 0215, avoralstat, C1INH replacement therapy (such as Cinryze®, Ruconest®, Berinert®, or Haegarda®), donidalorsen, or deucrictibant.

[0218] The invention also provides a method for treating a disease or disorder relating to elevated PKa activity in a subject, comprising administering to the subject a therapeutic agent for treating the disease or disorder; wherein the subject has a sPKa activity that is greater than a reference value, the reference value being a sPKa activity above which indicates elevated PKa activity; wherein the sPKa activity is as defined under section II herein, wherein the reference value is as defined under section III herein, wherein the disease or disorder relating to elevated PKa activity is bradykinin-mediated angioedema, wherein the bradykinin-mediated angioedema is HAE (e.g. HAE-nClINH), and wherein the therapeutic agent is sebetralstat. The invention also provides a method for treating a disease or disorder relating to elevated PKa activity in a subject, comprising administering to the subject a therapeutic agent for treating the disease or disorder; wherein the subject has a sPKa activity that is greater than a reference value, the reference value being a sPKa activity above which indicates elevated PKa activity; wherein the sPKa activity of the subject is determined according to the method for determining sPKa activity, said method comprising: i. determining the total amidolytic activity of a plasma sample without adding any inhibitor of the kallikrein-kinin system by: al. incubating the plasma sample at a temperature of about 4°C for about 6 hours to induce kallikrein-kinin system activation in the sample; bl. after step al, adding a chromogenic substrate for determining amidolytic activity to at least a portion of the sample to form a sample / substrate mixture, wherein the substrate is incubated at a temperature of about 30°C for about 10 minutes and then added to the sample at a temperature of about 30°C; and cl. after step bl, determining the amidolytic activity of the sample / substrate mixture at a temperature of about 30°C for about 15 minutes, which is the total amidolytic activity of the plasma sample wherein the PKa inhibitor is KV999272, or a salt and / or stereoisomer thereof, and the chromogenic substrate is H-D-Pro-Phe-Arg-pNA; and, ii. subtracting: (1) the level of amidolytic activity as determined by the method for determining a level of amidolytic activity of a plasma sample (when PKa activity is inhibited), from (2) the total amidolytic activity of the plasma sample; to provide the sPKa activity of the sample, wherein the method for determining a level of amidolytic activity of a plasma sample comprises: a2. incubating the plasma sample at a temperature of about 4°C for about 6 hours to induce kallikrein-kinin system activation in the sample; b2. after step a2, adding a PKa inhibitor to at least a portion of the incubated plasma sample to form an inhibitor / sample mixture and incubating the inhibitor / sample mixture at room temperature (e.g. a temperature of between about 21°C and about 23°C) for about 15 minutes; c2. after step b2, adding a chromogenic substrate for determining the amidolytic activity to at least a portion of the inhibitor / sample mixture to form an inhibitor / sample / substrate mixture, wherein the substrate is incubated at a temperature of about 30°C for about 10 minutes and then added to the inhibitor / sample mixture at a temperature of about 30°C; and d2. after step c2, determining the amidolytic activity of the inhibitor / sample / substrate mixture at a temperature of about 30°C for about 15 minutes, which provides the level of amidolytic activity of the plasma sample, wherein the PKa inhibitor is KV999272, or a salt and / or stereoisomer thereof, and the chromogenic substrate is H-D-Pro-Phe-Arg-pNA; wherein the reference value is determined according to a method for determining a reference value of sPKa activity above which indicates elevated PKa activity, said method comprising: conducting the method of determining sPKa activity (as set out above) on multiple different plasma samples to identify multiple sPKa activity values; wherein the reference value of sPKa activity corresponds to the 95th percentile of the multiple sPKa activity values; and wherein the multiple different plasma samples are collected from at least one healthy subject (e.g. at least 34 healthy subjects); wherein the disease or disorder relating to elevated PKa activity is bradykinin-mediated angioedema, wherein the bradykinin-mediated angioedema is HAE (e.g. HAE-nClINH), and wherein the therapeutic agent is sebetralstat.

[0219] The invention also provides a therapeutic agent for use in the treatment of a disease or disorder relating to elevated PKa activity in a subject, comprising administering to the subject the therapeutic agent for treating the disease or disorder; wherein the subject has a sPKa activity that is greater than a reference value, the reference value being a sPKa activity above which indicates elevated PKa activity; wherein the sPKa activity of the subject is determined according to the method for determining sPKa activity, said method comprising: i. determining the total amidolytic activity of a plasma sample without adding any inhibitor of the kallikrein-kinin system by: al. incubating the plasma sample at a temperature of about 4°C for about 6 hours to induce kallikrein-kinin system activation in the sample; bl. after step al, adding a chromogenic substrate for determining amidolytic activity to at least a portion of the sample to form a sample / substrate mixture, wherein the substrate is incubated at a temperature of about 30°C for about 10 minutes and then added to the sample at a temperature of about 30°C; and cl. after step bl, determining the amidolytic activity of the sample / substrate mixture at a temperature of about 30°C for about 15 minutes, which is the total amidolytic activity of the plasma sample wherein the PKa inhibitor is KV999272, or a salt and / or stereoisomer thereof, and the chromogenic substrate is H-D-Pro-Phe-Arg-pNA; and, ii. subtracting: (1) the level of amidolytic activity as determined by the method for determining a level of amidolytic activity of a plasma sample (when PKa activity is inhibited), from (2) the total amidolytic activity of the plasma sample; to provide the sPKa activity of the sample, wherein the method for determining a level of amidolytic activity of a plasma sample comprises: a2. incubating the plasma sample at a temperature of about 4°C for about 6 hours to induce kallikrein-kinin system activation in the sample; b2. after step a2, adding a PKa inhibitor to at least a portion of the incubated plasma sample to form an inhibitor / sample mixture and incubating the inhibitor / sample mixture at room temperature (e.g. a temperature of between about 21°C and about 23°C) for about 15 minutes; c2. after step b2, adding a chromogenic substrate for determining the amidolytic activity to at least a portion of the inhibitor / sample mixture to form an inhibitor / sample / substrate mixture, wherein the substrate is incubated at a temperature of about 30°C for about 10 minutes and then added to the inhibitor / sample mixture at a temperature of about 30°C; and d2. after step c2, determining the amidolytic activity of the inhibitor / sample / substrate mixture at a temperature of about 30°C for about 15 minutes, which provides the level of amidolytic activity of the plasma sample, wherein the PKa inhibitor is KV999272, or a salt and / or stereoisomer thereof, and the chromogenic substrate is H-D-Pro-Phe-Arg-pNA; wherein the reference value is determined according to a method for determining a reference value of sPKa activity above which indicates elevated PKa activity, said method comprising: conducting the method of determining sPKa activity (as set out above) on multiple different plasma samples to identify multiple sPKa activity values; wherein the reference value of sPKa activity corresponds to the 95th percentile of the multiple sPKa activity values; and wherein the multiple different plasma samples are collected from at least one healthy subject (e.g. at least 34 healthy subjects); wherein the disease or disorder relating to elevated PKa activity is bradykinin-mediated angioedema, wherein the bradykinin-mediated angioedema is HAE (e.g. HAE-nClINH); and wherein the therapeutic agent is sebetralstat.

[0220] VI. Kits

[0221] The present invention also provides kits for use in determining a level of amidolytic activity of a plasma sample. In particular, the kits of the present invention can be used in the methods for determining a level of amidolytic activity of a plasma sample as described herein i.e. for determining the level of amidolytic activity of a plasma sample that is not attributable to PKa. In other aspects, the kits of the present invention can be used in the methods for determining the sPKa activity of a plasma sample described herein. In other aspects, the kits of the present invention can be diagnostic kits. For example, the kits can be used as part of methods of diagnosing a subject with a disease or disorder relating to elevated PKa activity. The kits can be used in any of the methods described herein.

[0222] Such kits can comprise a PKa inhibitor and a substrate for determining the amidolytic activity. The PKa inhibitor can be selected from sebetralstat, berotralstat, avoralstat, KVD001, feniralstat, lanadelumab, STAR-0215, RZ-402 (ASP-440), ATN-249, and KV999272, or a salt and / or stereoisomer thereof. The substrate for determining the amidolytic activity can be H-D-Pro-Phe-Arg-pNA. The kits may also comprise one or more buffers.

[0223] In some aspects, the kits can comprise instructions for use in accordance with any of the methods described herein. The included instructions can comprise a description of how to use the components contained in the kit for measuring the level of amidolytic activity of a plasma sample obtained from a human subject. The instructions may further comprise a description of how to use the components contained in the kit for determining the sPKa activity of a plasma sample obtained from a human subject. The instructions relating to use of the kit typically include information as to the amount of each component and suitable conditions for performing the methods described herein. Instructions supplied in the kits are typically written instructions on a label or package insert (e.g. a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable. The label or package insert may indicate that the kit is used for determining a level of amidolytic activity of a plasma sample. The label or package insert may indicate that the kit is used for determining a level of amidolytic activity of a plasma sample. The label or package insert may indicate that the kit is used for determining the sPKa activity in a plasma sample. The label or package insert may indicate that the kit is used in methods that diagnose a subject with a disease or disorder relating to elevated PKa activity.

[0224] The kits of the present invention are in suitable packaging. Suitable packaging includes vials, bottles, jars, flexible packing (e.g. plastic bags) and the like.

[0225] In some aspects, the invention provide articles of manufacture comprising contents of the kits described herein.

[0226] Numbered embodiments

[0227] 1. A method for determining a level of amidolytic activity of a plasma sample comprising: a. incubating the plasma sample to induce kallikrein-kinin system activation in the sample; b. after step a, adding a plasma kallikrein (PKa) inhibitor to at least a portion of the incubated plasma sample to form an inhibitor / sample mixture; c. after step b, adding a substrate for determining the amidolytic activity to at least a portion of the inhibitor / sample mixture to form an inhibitor / sample / substrate mixture; and d. after step c, determining the amidolytic activity of the inhibitor / sample / substrate mixture, which provides the level of amidolytic activity of the plasma sample.

[0228] 2. The method according to numbered embodiment 1, wherein the plasma sample has been separated from whole blood within 48 hours of collecting the whole blood from a subject.

[0229] 3. The method according to any preceding numbered embodiment, wherein the plasma sample has been separated from whole blood within 1 hour of collecting the whole blood from a subject.

[0230] 4. The method according to any preceding numbered embodiment, wherein the plasma sample is obtained from a blood sample collected from the subject. 5. The method according to any of numbered embodiments 1-4, wherein the plasma sample is incubated at a temperature of less than about 15°C in step a.

[0231] 6. The method according to any of numbered embodiments 1-5, wherein the plasma sample is incubated at a temperature of less than about 10°C in step a.

[0232] 7. The method according to any of numbered embodiments 1-6, wherein the plasma sample is incubated at a temperature of less than about 5°C in step a.

[0233] 8. The method according to any of numbered embodiments 1-7, wherein the plasma sample is incubated at a temperature of between about 0°C and about 5°C.

[0234] 9. The method according to any of numbered embodiments 1-8, wherein the plasma sample is incubated at a temperature of about 4°C in step a.

[0235] 10. The method according to any of numbered embodiments 1-9, wherein the plasma sample is incubated on wet ice in step a.

[0236] 11. The method according to any of numbered embodiments 1-10, wherein the plasma sample is frozen prior to carrying out the method.

[0237] 12. The method according to numbered embodiment 11, wherein the frozen plasma sample is thawed on ice (frozen water) prior to carrying out the method.

[0238] 13. The method according to any of numbered embodiments 1-12, wherein the plasma sample is in liquid form when it is incubated in step a.

[0239] 14. The method according to any of numbered embodiments 1-13, wherein the plasma sample is incubated for at least about 4 hours in step a.

[0240] 15. The method according to any of numbered embodiments 1-14, wherein the plasma sample is incubated for less than about 18 hours in step a. 16. The method according to any of numbered embodiments 1-15, wherein the plasma sample is incubated for less than about 12 hours in step a.

[0241] 17. The method according to any of numbered embodiments 1-16, wherein the plasma sample is incubated for between about 4 hours and about 18 hours in step a.

[0242] 18. The method according to any of numbered embodiments 1-17, wherein the plasma sample is incubated for between about 4 hours and about 12 hours in step a.

[0243] 19. The method according to any of numbered embodiments 1-18, wherein the plasma sample is incubated for between about 5 hours and about 7 hours in step a.

[0244] 20. The method according to any of numbered embodiments 1-19, wherein the plasma sample is incubated for about 6 hours in step a.

[0245] 21. The method according to any of numbered embodiments 1-20, wherein cold incubation of the plasma sample in step a induces activation of the kallikrein-kinin system in the plasma sample.

[0246] 22. The method according to any of numbered embodiments 1-21, wherein step a is carried out in the absence of any external kallikrein-kinin system-activating agent.

[0247] 23. The method according to any of numbered embodiments 1-22, wherein no inhibitor of the kallikrein-kinin system activation is added in in step a.

[0248] 24. The method according to any of numbered embodiments 1-23, wherein the inhibitor / sample mixture is incubated at a temperature of between about 15°C and about 25°C in step b.

[0249] 25. The method according to any of numbered embodiments 1-24, wherein the inhibitor / sample mixture is incubated at a temperature of between about 21°C and about 23°C in step b.

[0250] 26. The method according to any of numbered embodiments 1-25, wherein the inhibitor / sample mixture is incubated at room temperature in step b.

[0251] 27. The method according to any of numbered embodiments 1-26, wherein the inhibitor / sample mixture is incubated for between about 5 mins and about 30 mins in step b. 28. The method according to any of numbered embodiments 1-27, wherein the inhibitor / sample mixture is incubated for between about 5 mins and about 20 mins in step b.

[0252] 29. The method according to any of numbered embodiments 1-28, wherein the inhibitor / sample mixture is incubated for between about 10 mins and about 20 mins in step b.

[0253] 30. The method according to any of numbered embodiments 1-29, wherein the inhibitor / sample mixture is incubated for about 15 mins in step b.

[0254] 31. The method according to any of numbered embodiments 1-30, wherein step b is carried out in the absence of any external kal likrein-kinin system-activating agent.

[0255] 32. The method according to any of numbered embodiments 1-31, wherein the substrate for determining amidolytic activity is incubated at a temperature of between about 15°C and about 40°C, and then added in step c at a temperature of between about 15°C and about 40°C.

[0256] 33. The method according to any of numbered embodiments 1-32, wherein the substrate for determining amidolytic activity is incubated at a temperature of between about 25°C and about 35°C, and then added in step c at a temperature of between about 25°C and about 35°C.

[0257] 34. The method according to any of numbered embodiments 1-33, wherein the substrate for determining amidolytic activity is incubated at a temperature of about 30°C, and then added in step c at a temperature of about 30°C.

[0258] 35. The method according to any of numbered embodiments 29-34, wherein the substrate for determining amidolytic activity is incubated for between about 5 mins and about 20 mins.

[0259] 36. The method according to any of numbered embodiments 29-35, wherein the substrate for determining amidolytic activity is incubated for about 10 mins.

[0260] 37. The method according to any preceding numbered embodiment, wherein the inhibitor / sample / substrate mixture is incubated at a temperature of between about 20°C and about 40°C in step d. 38. The method according to any preceding numbered embodiment, wherein the inhibitor / sample / substrate mixture is incubated at a temperature of about 30°C in step d.

[0261] 39. The method according to any of numbered embodiments 37-38, wherein the inhibitor / sample / substrate mixture is incubated when the level of amidolytic activity is being determined.

[0262] 40. The method according to any preceding numbered embodiment, wherein the PKa inhibitor is added in an amount sufficient to inhibit at least about 90% of PKa activity in the sample.

[0263] 41. The method according to any preceding numbered embodiment, wherein the PKa inhibitor is added in an amount sufficient to inhibit at least about 95% of PKa activity in the sample.

[0264] 42. The method according to any preceding numbered embodiment, wherein the PKa inhibitor is a selective PKa inhibitor.

[0265] 43. The method according to any preceding numbered embodiment, wherein the PKa inhibitor is selected from the group consisting of sebetralstat, berotralstat, avoralstat, KVD001, feniralstat, lanadelumab, STAR-0215, RZ-402 (ASP-440), ATN-249, and KV999272, or a salt and / or stereoisomer thereof.

[0266] 44. The method according to any preceding numbered embodiment, wherein the PKa inhibitor is KV999272, or a salt and / or stereoisomer thereof.

[0267] 45. The method according to numbered embodiment 44, wherein the PKa inhibitor is KV999272.

[0268] 46. The method according to any preceding numbered embodiment, wherein the PKa inhibitor is the only kallikrein-kinin system inhibitor added.

[0269] 47. The method according to any preceding numbered embodiment, wherein the method is carried out in the absence of any external kallikrein-kinin system-activating agent.

[0270] 48. The method according to any preceding numbered embodiment, wherein the substrate that is capable of detecting amidolytic activity is a chromogenic substrate. The method according to numbered embodiment 48, wherein the chromogenic substrate is H-D- Pro-Phe-Arg-pNA-(hydrochloride). The method according to numbered embodiment 48, wherein the chromogenic substrate is H-D- Pro-Phe-Arg-pNA-2HCI. A method for determining the specific plasma kal likrein (sPKa) activity of a plasma sample comprising: i. determining the total amidolytic activity of a plasma sample without adding any inhibitor of the kallikrein-kinin system by: a. incubating the plasma sample to induce kallikrein-kinin system activation in the sample; b. after step a, adding a substrate for determining amidolytic activity to at least a portion of the sample to form a sample / substrate mixture; and c. after step b, determining the amidolytic activity of the sample / substrate mixture, which is the total amidolytic activity of the plasma sample; and, ii. subtracting: (1) the level of amidolytic activity as determined by the method according to any one of numbered embodiments 1 to 50, from (2) the total amidolytic activity of the plasma sample; to provide the sPKa activity of the sample. The method according to numbered embodiment 51, wherein the level of amidolytic activity determined by the method according to any one of numbered embodiments 1 to 50 and the total amidolytic activity of the plasma sample are determined substantially concomitantly. The method according to any of numbered embodiments 51-52, wherein the level of amidolytic activity determined by the method according to any one of numbered embodiments 1 to 50 and the total amidolytic activity of the plasma sample are determined following about the same duration of incubation of the plasma sample in step a of both methods. The method according to any of numbered embodiments 51-53, wherein the level of amidolytic activity determined by the method according to any one of numbered embodiments 1 to 50 and the total amidolytic activity of the plasma sample are determined following about 6 hours of incubation of the plasma sample in step a of both methods. 55. The method according to any of numbered embodiments 51-54, wherein the level of amidolytic activity determined by the method according to any one of numbered embodiments 1 to 50 and the total amidolytic activity of the plasma sample are determined following incubation of the plasma sample at a temperature of about 4°C in step a of both methods.

[0271] 56. The method according to any of numbered embodiments 51-55, wherein analogous conditions are used when determining the level of amidolytic activity by the method according to numbered embodiments any one of 1 to 50 and the total amidolytic activity, except that determining the level of amidolytic activity by the method according to any one of numbered embodiments 1 to 50 requires the use of an added PKa inhibitor.

[0272] 57. A method for determining a reference value of specific plasma kallikrein (sPKa) activity above which indicates elevated plasma kallikrein (PKa) activity comprising: conducting the method of determining sPKa activity according to any of numbered embodiments 51 to 56 on multiple different plasma samples to identify multiple sPKa activity values; wherein the reference value of sPKa activity corresponds to the 95th percentile of the multiple sPKa activity values; and wherein the multiple different plasma samples are collected from at least one healthy subject.

[0273] 58. The method according to numbered embodiment 57, wherein "multiple different plasma samples" means at least 10 different plasma samples.

[0274] 59. The method according to any of numbered embodiments 57-58, wherein "multiple different plasma samples" means at least 34 different plasma samples.

[0275] 60. The method according to any of numbered embodiments 57-59, wherein "multiple different plasma samples" means at least 100 different plasma samples.

[0276] 61. The method according to any of numbered embodiments 57-60, wherein "multiple different plasma samples" means plasma samples collected from multiple different healthy subjects.

[0277] 62. The method according to any of numbered embodiments 57-61, wherein the multiple different plasma samples are collected from at least 34 different healthy subjects. 63. The method according to any of numbered embodiments 57-62, wherein the multiple different plasma samples are collected from at least 50 different healthy subjects.

[0278] 64. The method according to any of numbered embodiments 57-63, wherein a healthy subject is a subject that does not have or is not suspected of having a disease or disorder relating to dysfunctional kallikrein-kinin system (e.g. elevated PKa activity).

[0279] 65. A method for use in diagnosing a subject with a disease or disorder relating to elevated plasma kail ikrein (PKa) activity comprising: a. determining the specific plasma kal likrein (sPKa) activity of a plasma sample collected from the subject, which is the subject's sPKa activity; b. comparing the subject's sPKa activity with a reference value of sPKa activity above which indicates elevated PKa activity; and c. diagnosing the subject with a disease or disorder relating to elevated PKa activity if the subject's sPKa activity is greater than the reference value.

[0280] 66. The method according to numbered embodiment 65, wherein the subject's sPKa activity is determined by the method according to any of numbered embodiments 51-56.

[0281] 67. The method according to any of numbered embodiments 65-66, wherein the reference value of sPKa activity above which indicates elevated PKa activity is determined by the method according to any of numbered embodiments 57-64.

[0282] 68. The method according to any of numbered embodiments 65-67, wherein the subject is a human subject suspected of having or at risk for a disease or disorder relating to elevated PKa activity.

[0283] 69. The method according to any of numbered embodiments 65-68, wherein the human subject is a human patient presenting with a symptom of a disease or disorder relating to elevated PKa activity.

[0284] 70. The method according to numbered embodiment 69, wherein the symptom is swelling of tissues such as in the hands, feet, limbs, face, intestinal tract, and / or airway; fatigue; headache; muscle aches; skin tingling; abdominal pain; nausea; vomiting; diarrhoea; difficulty swallowing; hoarseness; shortness of breath; and / or mood changes. 71. The method according to any of numbered embodiments 65-68, wherein the disease or disorder relating to elevated PKa activity is bradykinin-mediated angioedema.

[0285] 72. The method according to numbered embodiment 71, wherein the bradykinin-mediated angioedema is hereditary angioedema (HAE); or wherein the bradykinin mediated angioedema is bradykinin-mediated angioedema non-hereditary (BK-AEnH).

[0286] 73. The method according to numbered embodiment 72, wherein the bradykinin-mediated angioedema is HAE.

[0287] 74. The method according to numbered embodiment 73, wherein the bradykinin-mediated angioedema is HAE type 1.

[0288] 75. The method according to numbered embodiment 73, wherein the bradykinin-mediated angioedema is HAE type 2.

[0289] 76. The method according to numbered embodiment 73, wherein the bradykinin-mediated angioedema is HAE with normal Cl inhibitor levels and function (HAE-nClINH).

[0290] 77. A method for treating a disease or disorder relating to elevated plasma kail ikrein (PKa) activity in a subject, comprising administering to the subject a therapeutic agent for treating the disease or disorder; wherein the subject has a specific plasma kail ikrein (sPKa) activity that is greater than a reference value, the reference value being a sPKa activity above which indicates elevated PKa activity; and wherein the sPKa activity of the subject is as defined in any one of numbered embodiments 51 to 56.

[0291] 78. A therapeutic agent for use in the treatment of a disease or disorder relating to elevated plasma kail ikrein (PKa) activity in a subject, comprising administering to the subject the therapeutic agent for treating the disease or disorder; wherein the subject has a specific plasma kail ikrein (sPKa) activity that is greater than a reference value, the reference value being a sPKa activity above which indicates elevated PKa activity; and wherein the sPKa activity of the subject is as defined in any one of numbered embodiments 51 to 56. The method according to numbered embodiment 77, or the therapeutic agent for use according to numbered embodiment 78, wherein the reference value is as defined in any of numbered embodiments 57-64. The method according numbered embodiment 77, or the therapeutic agent for use according to numbered embodiment 78, wherein the subject's sPKa activity is determined by the method according to any of numbered embodiments 51-56. The method according to any of numbered embodiments 77 or 79-80, or the therapeutic agent for use according to any of numbered embodiments 78-80, wherein the disease or disorder relating to elevated PKa activity has been diagnosed using the method according to any of numbered embodiments 65-76. The method according to any of numbered embodiments 77 or 79-81, or the therapeutic agent for use according to any of numbered embodiments 78-81, wherein the disease or disorder relating to elevated PKa activity is bradykinin-mediated angioedema. The method according to numbered embodiment 82, or the therapeutic agent for use according to numbered embodiment 82, wherein the bradykinin-mediated angioedema is hereditary angioedema (HAE); or wherein the bradykinin-mediated angioedema is bradykinin-mediated angioedema non-hereditary (BK-AEnH). The method according to numbered embodiment 83, or the therapeutic agent for use according to numbered embodiment 83, wherein the bradykinin-mediated angioedema is HAE. The method according to numbered embodiment 84, or the therapeutic agent for use according to numbered embodiment 84, wherein the bradykinin-mediated angioedema is HAE type 1. The method according to numbered embodiment 84, or the therapeutic agent for use according to numbered embodiment 84, wherein the bradykinin-mediated angioedema is HAE type 2. 87. The method according to numbered embodiment 84, or the therapeutic agent for use according to numbered embodiment 84, wherein the bradykinin-mediated angioedema is HAE with normal Cl inhibitor levels and function (HAE-nClINH).

[0292] 88. The method according to any of numbered embodiments 77 and 79-87, or the therapeutic agent for use according to any of numbered embodiments 78-87, wherein the therapeutic agent is selected from a kallikrein binding agent, a bradykinin B2 receptor antagonist, a Factor XI la (FXIIa) binding agent, or a C1INH replacement agent.

[0293] 89. The method according to numbered embodiment 88, or the therapeutic agent for use according to numbered embodiment 88, wherein the therapeutic agent is a PKa inhibitor or a FXIIa inhibitor.

[0294] 90. The method according to any of numbered embodiments 77 and 79-87, or the therapeutic agent for use according to any of numbered embodiments 78-87, wherein the therapeutic agent is selected from lanadelumab, garadacimab, sebetralstat, berotralstat, icatibant, ecallantide, RZ-402 (ASP 440), STAR 0215, avoralstat, C1INH replacement therapy (such as Cinryze®, Ruconest®, Berinert®, or Haegarda®), donidalorsen, and deucrictibant.

[0295] 91. The method according to numbered embodiment 90, or the therapeutic agent for use according to numbered embodiment 90, wherein the therapeutic agent is sebetralstat.

[0296] 92. A method for identifying a subject at risk of having a disease or disorder relating to elevated plasma kallikrein (PKa) activity comprising: a. determining the specific plasma kallikrein (sPKa) activity of a plasma sample collected from the subject, which is the subject's sPKa activity; b. comparing the subject's sPKa activity with a reference value of sPKa activity above which indicates elevated PKa activity; and c. identifying a subject at risk of having a disease or disorder relating to elevated PKa activity if the subject's sPKa activity is greater than the reference value.

[0297] 93. A method for determining if a disease or disorder is susceptible to treatment with a plasma kallikrein (PKa) inhibitor comprising: a. determining the specific plasma kallikrein (sPKa) activity of a plasma sample collected from a subject, which is the subject's sPKa activity; b. comparing the subject's sPKa activity with a reference value of sPKa activity above which indicates elevated PKa activity; and c. identifying the disease or disorder as being susceptible to treatment with a PKa inhibitor if the subject's sPKa activity is greater than the reference value. The method according to any of numbered embodiments 92-93, wherein the subject's sPKa activity is determined by the method according to any of numbered embodiments 51-56. The method according to any of numbered embodiments 92-94, wherein the reference value is determined according by the method according to any of numbered embodiments 57-64. The method according to any of numbered embodiments 92-95, wherein the disease or disorder relating to elevated PKa activity is bradykinin-mediated angioedema: wherein the bradykinin- mediated angioedema is hereditary angioedema (HAE); or wherein the bradykinin-mediated angioedema is bradykinin-mediated angioedema non-hereditary (BK-AEnH). The method according to any of numbered embodiments 92-96, wherein the bradykinin-mediated angioedema is HAE. The method according to numbered embodiment 97, wherein the bradykinin-mediated angioedema is HAE type 1. The method according to numbered embodiment 97, wherein the bradykinin-mediated angioedema is HAE type 2. . The method according to numbered embodiment 97, wherein the bradykinin-mediated angioedema is HAE with normal Cl inhibitor levels and function (HAE-nClINH). . The method according to any of numbered embodiments 92-96, wherein the bradykinin-mediated angioedema is bradykinin-mediated angioedema non-hereditary (BK-AEnH). . The method according to numbered embodiment 101, wherein the subject is being treated with an angiotensin converting enzyme (ACE) inhibitor, and the disease or disorder as being susceptible to treatment with a PKa inhibitor is environmental, hormonal, or drug induced; acquired angioedema; anaphylaxis associated angioedema; angiotensin converting enzyme (ACE or ace) inhibitor induced angioedema; dipeptidyl peptidase-4 inhibitor induced angioedema; or tPA induced angioedema (tissue plasminogen activator induced angioedema).

[0298] 103. The method according to any of numbered embodiments 65-76 or 92-102, wherein the subject is subsequently treated with a therapeutic agent for the treatment of a disease or disorder relating to elevated PKa activity.

[0299] 104. The method according to numbered embodiment 103, wherein the therapeutic agent is selected from a kallikrein binding agent, a bradykinin B2 receptor antagonist, a Factor XI la (FXIIa) binding agent, or a C1INH replacement agent.

[0300] 105. The method according to numbered embodiment 104, wherein the therapeutic agent is a PKa inhibitor or a FXIIa inhibitor.

[0301] 106. The method according to numbered embodiment 103, wherein the therapeutic agent is selected from lanadelumab, garadacimab, sebetralstat, berotralstat, icatibant, ecallantide, RZ- 402 (ASP 440), STAR 0215, avoralstat, C1INH replacement therapy (such as Cinryze®, Ruconest®, Berinert®, or Haegarda®), donidalorsen, and deucrictibant.

[0302] 107. The method according to numbered embodiment 106, wherein the therapeutic agent is sebetralstat.

[0303] 108. A method for evaluating treatment of a disease or disorder relating to elevated plasma kallikrein (PKa) in a subject comprising: a. determining the specific plasma kallikrein (sPKa) activity of a plasma sample collected from the subject, which is the subject's sPKa activity; b. comparing the subject's sPKa activity with a reference value of sPKa activity above which indicates elevated PKa activity; and c. evaluating effectiveness of the treatment based on whether the subject's sPKa activity is greater than the reference value.

[0304] 109. The method according to numbered embodiment 108, wherein the subject's sPKa activity is determined by the method according to any of numbered embodiments 51-56. 110. The method according to any of numbered embodiments 108-109, wherein the reference value is determined according by the method according to any of numbered embodiments 57-64.

[0305] 111. The method according to any of numbered embodiments 108-110, wherein the disease or disorder relating to elevated PKa activity is bradykinin-mediated angioedema: wherein the bradykinin-mediated angioedema is hereditary angioedema (HAE); or wherein the bradykinin- mediated angioedema is bradykinin-mediated angioedema non-hereditary (BK-AEnH).

[0306] 112. The method according to any of numbered embodiments 108-111, wherein the bradykinin-mediated angioedema is HAE.

[0307] 113. The method according to numbered embodiment 112, wherein the bradykinin-mediated angioedema is HAE type 1.

[0308] 114. The method according to numbered embodiment 112, wherein the bradykinin-mediated angioedema is HAE type 2.

[0309] 115. The method according to numbered embodiment 112, wherein the bradykinin-mediated angioedema is HAE with normal Cl inhibitor levels and funchon (HAE-nClINH).

[0310] 116. The method according to any of numbered embodiments 112-115, wherein the treatment being evaluated is a prophylactic treatment of HAE.

[0311] 117. The method according to numbered embodiment 116, wherein the prophylactic treatment is selected from lanadelumab, garadacimab, berotralstat, STAR 0215, C1INH replacement therapy (such as Cinryze®, Ruconest®, Berinert®, or Haegarda®), donidalorsen, and deucrictibant.

[0312] 118. The method according to numbered embodiment 117, wherein the prophylactic treatment is lanadelumab.

[0313] 119. The method according to numbered embodiment 117, wherein the prophylactic treatment is berotralstat. . The method according to numbered embodiment 117, wherein the prophylactic treatment is deucrictibant. . The method according to any of numbered embodiments 116-120, wherein if the subject's sPKa activity is greater than the reference value, the treatment is considered insufficient to prevent an HAE attack. . The method according to any of numbered embodiments 116-121, wherein if the subject's sPKa activity is greater than the reference value, the subject is at risk of an HAE attack. . The method according to any of numbered embodiments 108-122, wherein the subject is administered an on-demand treatment of HAE following evaluation. . The method according to any of numbered embodiments 123, wherein the on-demand treatment of HAE is sebetralstat. . The method according to any of numbered embodiments 108-111, wherein the treatment being evaluated is tranexamic acid. . A kit comprising: a plasma kail ikrein (PKa) inhibitor; a substrate for determining amidolytic activity; and instructions for use in determining specific plasma kail ikrein (sPKa) activity. . The kit according to numbered embodiment 126, wherein the instructions for use in determining sPKa activity are instructions for carrying out the method according to any of numbered embodiments 51-56. . The kit according to any of numbered embodiment 126-127, wherein the PKa inhibitor is selected from the group consisting of sebetralstat, berotralstat, avoralstat, KVD001, feniralstat, lanadelumab, STAR-0215, RZ-402 (ASP-440), ATN-249, and KV999272, or a salt and / or stereoisomer thereof. . The kit according to any of numbered embodiments 126-128, wherein the PKa inhibitor is KV999272, or a salt and / or stereoisomer thereof. 130. The kit according to numbered embodiment 129, wherein the PKa inhibitor is KV999272.

[0314] 131. The kit according to any of numbered embodiments 126-130, wherein the substrate for determining amidolytic activity is a chromogenic substrate.

[0315] 132. The kit according to numbered embodiment 131, wherein the chromogenic substrate is H-D-Pro-Phe-Arg-pNA'(hydrochloride).

[0316] 133. The kit according to numbered embodiment 131, wherein the chromogenic substrate is H-D-Pro-Phe-Arg-pNA'2HCL

[0317] 1A: Baseline-corrected Michaelis-Menton plots showing pNA cleavage from Pro-Phe-Arg-pNA by

[0318] PKa, Thrombin, FXIIa and Trypsin. igure IB: Baseline-corrected Michaelis-Menton plots showing AFC cleavage from Pro-Phe-Arg-AFC by

[0319] PKa, Thrombin, FXIIa and Trypsin. Total amidolytic activity and amidolytic activity in healthy individuals in the presence of (i) a selective PKa inhibitor or (ii) a broad spectrum protease inhibitor. igure 3A: sPKa activity in healthy individuals and patients with HAE-C1INH, as determined from the sPKa activity assay. igure 3B: Total amidolytic activity in healthy individuals and patients with HAE-C1INH, as determined from the amidolytic activity assay. Receiver operating characteristic (ROC) curve comparing diagnostic performance of the sPKa activity assay at baseline (0 hours) versus after 6 hours of cold incubation on ice, and versus total amidolytic activity. Total amidolytic activity and sPKa activity in healthy individuals and patients with HAE-C1INH, as determined from the amidolytic activity and sPKa activity assays. igure 5A: sPKa activity in healthy individuals and patients with HAE-C1INH, as determined from the sPKa activity assay, in female subjects. igure 5B: sPKa activity in healthy individuals and patients with HAE-C1INH, as determined from the sPKa activity assay, in male subjects. igure 6: Total amidolytic activity in healthy individuals at a first time point and a second time point four months later. activity in an HAE-nClINH patient during the intercritical period, as determined from the sPKa activity assay. igure 7B: sPKa activity in an HAE-nClINH patient during the intercritical period and during an attack, as determined from the sPKa activity assay. igure 7C: Values of sPKa activity determined in two HAE-nClINH patients. igure 8: sPKa activity in subjects taking estrogen oral contraception, as determined from the sPKa activity assay. Effect of prewarming of the substrate before adding to the sample on absorbance readings. igure 10A: Effect of incubating healthy plasma at 0 hours, 6 hours or 12 hours on amidolytic activity, as determined from the amidolytic activity assay. igure 10B: Effect of incubating healthy plasma at 0 hours, 6 hours or 18 hours, as determined from the amidolytic activity assay. Effect of incubating HAE-C1INH plasma at 0 hours, 4 hours or 6 hours, determined from the amidolytic activity assay.

[0320] 11: X-ray powder diffraction pattern of sebetralstat as generated in Example 12.

[0321] Embodiments provided herein may be more fully understood by reference to the following examples.

[0322] These examples are meant to be illustrative of the methods and treatments provided herein, but are not in any way limiting.

[0323] While examples of certain particular embodiments are provided herein, it will be apparent to those skilled in the art that various changes and modifications may be made. Such modifications are also intended to fall within the scope of the invention. as a PKa inhibitor and a desired i concentration

[0324] PKa inhibitory activity can be determined in vitro using standard published methods (see e.g. Johansen et al., Int. J. Tiss. Reac. 1986, 8, 185; Shori et al., Biochem. Pharmacol., 1992, 43, 1209; Sturzebecher et al.,

[0325] Biol. Chem. Hoppe-Seyler, 1992, 373, 1025). Human PKa (Protogen) was incubated at 25 °C with the fluorogenic substrate H-D-Pro-Phe-Arg-AFC or chromogenic substrate H-D-Pro-Phe-Arg-pNA and various concentrations of the test compound. Residual enzyme activity (initial rate of reaction) was determined by measuring the change in fluorescence or optical absorbance , and the desired inhibitory concentration of the test compound was determined. The PKa inhibitor was then confirmed as inhibiting the desired level of PKa activity in plasma collected from blood.

[0326] Clermont et al. (Investigative Ophthalmology & Visual Science May 2016, Vol.57, 2390-2399) demonstrates that 1 pM of KV999272 achieves >95% inhibition of PKa (see e.g. Figure 3B of Clermont et al.).

[0327] Example 2: Evaluating substrate promiscuity

[0328] Aim: To determine the cross-enzyme reactivity of two different substrates used in PKa activation assays. In particular, the ability of PKa, FXIIa, thrombin and trypsin to act on the chromogenic substrate H-D-Pro- Phe-Arg-pNA and the fluorogenic substrate H-D-Pro-Phe-Arg-AFC was determined.

[0329] Methods:

[0330] Using an in vitro isolated enzyme assay, substrate promiscuity of the chromogenic substrate H-D-Pro-Phe- Arg-pNA hydrochloride (Bachem) was determined by comparing Km and Vmax values generated for human PKa versus a number of other related and relevant proteases e.g. thrombin, plasmin, FXIIa, trypsin. Experiments were conducted at 25°C by incubating the enzyme (0.1-4 nM depending on the enzyme) with a serial dilution of the chromogenic substrate starting at an initial concentration of 1 mM. Protease activity was measured by monitoring the absorbance at 405 nm from cleaved substrate over 5 min enabling determination of the linear rate of increase in absorbance per minute. Using GraphPad Prism software, data were plotted as graphs of rate of reaction (v) against the concentration of substrate [S] and the Michaelis-Menton equation used to estimate Km and Vmax for the cleavage of the substrate by PKa and for each of the other enzymes.

[0331] Promiscuity of the substrate for each enzyme was confirmed by repeating the experimental conditions using the fluorescent substrate H-D-Pro-Phe-Arg-AFC.

[0332] Results:

[0333] FXIIa, thrombin and trypsin each turned over the substrate H-D-Pro-Phe-Arg-pNA (Figure 1A) and H-D- Pro-Phe-Arg-AFC (Figure IB). These data confirm that PKa is not the sole amidolytic enzyme responsible for cleaving the peptide substrates that are typically used in assays to assess PKa activity. Determining sPKa activity according to the invention can overcome substrate promiscuity. Example 3: Demonstrating contribution of PKa to total amidolytic activity

[0334] Aim: As noted above, proteases other than PKa can contribute to cleavage of H-D-Pro-Phe-Arg-pNA, such as FXIIa, thrombin, trypsin, tryptase, KLK5, LKL4 and KLK2. These proteases thus contribute to total amidolytic activity. Therefore, using a selective PKa inhibitor is useful for establishing assay specificity. The aim of this study was to demonstrate the contribution of PKa activity to total amidolytic activity using the selective PKa inhibitor KV999272.

[0335] Method: Amidolytic activity was measured in plasma samples from four healthy controls post 6 hours of cold incubation. Protease activity was measured by monitoring the absorbance at 405 nm from cleaved substrate over 5 min enabling determination of the linear rate of increase in absorbance per minute. KV999272 (a selective PKa inhibitor) or 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF; a broad-spectrum protease inhibitor) was added to the sample and the amidolytic activity was measured again. The percent reduction of the total amidolytic activity was determined.

[0336] Results: On average, AEBSF inhibited 93% of amidolytic activity in the samples, whereas KV999272 inhibited 22% of amidolytic activity in the samples i.e. AEBSF caused a significantly greater reduction in amidolytic activity than KV999272 (Figure 2). In other words, AEBSF inhibited amidolytic activity in the healthy samples that was not inhibited by KV999272. This additional amidolytic activity corresponds to the activity of proteases other than PKa to cleavage of H-D-Pro-Phe-Arg-pNA, such as FXIIa, thrombin, trypsin, tryptase, KLK5, LKL4 and KLK2. Therefore, these results demonstrate that PKa activity is a component of total amidolytic activity.

[0337] Example 4: Evaluation of a sPKa activity assay in healthy individuals and patients with HAE-C1INH

[0338] Aim: The objective of this analysis was to establish an assay to distinguish the biochemical characteristics of elevated PKa activity in plasma from HAE-C1INH individuals when compared to plasma from healthy control individuals. SPKa activity was measured in all samples at baseline and post 6 hours of cold incubation.

[0339] Method:

[0340] PKa activity was measured in plasma samples from healthy controls (n=42) and HAE-C1INH patients (n=25). HAE plasma was obtained during the intercritical attack free period. Plasma was obtained from HAE participants from part 1 of the KVD900-201 Clinical Trial (EudraCT number: 2018-004489-32) - "KVD900" is sebetralstat. This represents blood samples taken and processed for plasma at 12 different clinical sites. Only blood samples which were deemed of suitable sample quality on visual inspection by the designated scientist (e.g. non-haemolysed) were selected for analysis.

[0341] Healthy plasma was acquired, following informed consent, from two sources:

[0342] 1. Blood collection and processing for platelet poor plasma at the KalVista laboratory in Salisbury, UK (n=26 selected at random from n=50 donors).

[0343] 2. Normal healthy blood purchased from commercial supplier (Reprocell, USA) processed for platelet poor plasma (n=16).

[0344] Blood, once drawn into 3.2% sodium citrate (Na-citrate) blood collection tubes, was kept at room temperature until plasma was generated. Platelet poor plasma was generated by centrifugation at 2000 x g for 10 minutes at 20°C. Plasma upper layer was withdrawn with care not to take the buffy coat to avoid KKS activation by platelets, and aliquoted.

[0345] These healthy plasma samples were collected under the following criteria:

[0346] 1. Subject is generally healthy and feeling well.

[0347] 2. Subject has not been diagnosed with an infectious disease.

[0348] 3. If female, subject is not pregnant.

[0349] 4. Subject is between 18-70 years of age.

[0350] 5. Subject must provide clinical information on medication use, hormonal contraception, and tobacco use.

[0351] Samples were buried in wet ice and analysed at baseline and following 6 hours of cold incubation at 4°C. Frozen plasma samples from -80°C were thawed on ice.

[0352] Amidolytic activity (Vmax) was measured using chromogenic substrate H-D-Pro-Phe-Arg-pNA-2HCI (Bachem, USA). Prior to each measurement, samples were incubated with either the PKa inhibitor ((2'S,2"R)-4-(2'-(2"-amino-3"-(4"'-ethoxyphenyl)propanoylamino)-3'-phenylpropanoylamino)piperidine- 1-carboxamidine) or phosphate-buffered saline (PBS) for 15 minutes at room temperature.

[0353] The substrate was prepared as a 200uL solution diluted from 20mM stock to ImM in the assay buffer (Tris / NaCI, 50mM / 150mM, pH 7.8). The substrate was warmed to 30°C for 10 mins. Samples were then diluted 1:20 in assay buffer containing the chromogenic substrate (50 mM Trisl50mM NaCI, pH 7.8). PKa catalytic activity was determined using a Spark microplate reader (Tecan, UK). sPKa activity was quantified by the subtraction of amidolytic activity not inhibited by the inhibitor from the total measured activity.

[0354] Each assay plate measured activity in plasma samples from 8 individuals (+ / - inhibitor), in duplicate. High and low controls were measured on each assay plate. High control values were considered to have met acceptance criteria if mean values had a coefficient of variation (CV) of less than 5% and low control values had a CV less than 20%. All plates analysed met the set acceptance criteria and therefore did not require re-analysis of samples.

[0355] Results:

[0356] At baseline and following 6 hours of cold incubation, sPKa activity for healthy plasma was 0.64+0.74 and 0.82+0.11 nmol / min / mL, respectively (mean+SEM, P not significant). The 95thpercentile of 6 hour cold incubated sPKa activity in healthy plasma was 2.73 nmol / min / mL. At baseline and after 6 hours cold incubation, sPKa activity for HAE-C1INH was 3.46+0.63 and 24.8+8.91 nmol / min / mL, respectively (P = 0.021). sPKa activity in HAE-C1INH plasma was above the 95thpercentile value for healthy plasma with assay sensitivity of 84% with specificity of 93%. 21 of the 25 HAE-C1INH samples were above the 95thpercentile value for healthy plasma. Figure 3A shows these results in graphical and tabular form. Receiver operating characteristic (ROC) analysis comparing healthy and HAE-C1INH displayed an AUC of 0.98 (P<0.0001).

[0357] At baseline and following 6 hours of cold incubation, total mean amidolytic activity for healthy plasma was 1.87 nmol / min / mL and 2.07 nmol / min / mL, respectively. The 95thpercentile of 6 hour cold incubated total mean amidolytic activity in healthy plasma was 4.50 nmol / min / mL. The assay sensitivity was 76% with specificity of 93%. 19 of the 25 HAE-C1INH samples were above the 95thpercentile value for healthy plasma. Figure 3B shows these results in graphical and tabular form. ROC analysis comparing healthy and HAE-C1INH displayed an AUC of 0.94 (P<0.0001).

[0358] Receiver operating characteristic (ROC) analysis

[0359] A ROC curve was generated (Figure 3C) to compare the diagnostic performance of the sPKa activity assay at baseline (0 hours) versus after 6 hours of cold incubation on ice. The 6 hour sPKa activity assay was also compared with the 6 hour total activity assay. SPKa activity measured at baseline, as well as post 6 hours of cold incubation, was used to compare the selectivity and sensitivity of the assays at each possible reference point.

[0360] The area under the curve (AUC) for the sPKa activity assay following 6 hours of cold incubation was 0.98. This was higher than the value obtained for the sPKa assay at baseline (0.93) and the total activity assay post 6 hours of cold incubation (0.94).

[0361] Conclusions

[0362] These data indicate that sPKa activity in HAE-C1INH is elevated during the intercritical period. An assay method that allows activation in a plasma sample followed by utilising a PKa inhibitor to determine sPKa activity leads to an improved assay having specificity of 93% and a sensitivity of 84% compared with healthy controls. Assays that consider only total amidolytic activity or do not allow activation in a plasma sample (i.e. where activity is measured at baseline) are inferior at detecting diseased plasma.

[0363] Example 4a: Evaluation of a sPKa activity assay in healthy individuals and patients with HAE-C1INH - further data

[0364] Further to the method described above in Example 4, 15 additional healthy subjects were added to the cohort of healthy controls. PKa activity was then measured in plasma samples from healthy controls (n=57, i.e. the 42 healthy controls from Example 4, plus the 15 additional healthy subjects) and HAE-C1INH patients (n=25). The patient demographics are summarised below:

[0365] Results:

[0366] In healthy plasma, sPKa activity measured at baseline and following 6 hours of cold incubation was 0.69+0.071 and 0.88+0.11 nmol / min / mL, respectively (mean±SEM, P not significant). The 95thpercentile of 6 hour cold-induced sPKa activity in healthy plasma was 3.07 nmol / min / mL (compared to 2.73 nmol / min / mL in Example 4). In HAE-C1INH plasma, sPKa activity measured at baseline and post 6 hours of cold incubation was 3.43+0.64 and 24.5+8.92 nmol / min / mL, respectively (P=0.022). The minor discrepancy between these values and those reported for the 25 HAE-C1INH patients in Example 4 is a result of routine data refinement (e.g. use of decimal places) that the inventors consider is inconsequential. sPKa activity in HAE-C1INH plasma was above the 95thpercentile value for healthy plasma with assay specificity of 95% and sensitivity of 84%.

[0367] ROC analysis comparing healthy and HAE-C1INH displayed an AUC of 0.97 (P<0.0001). This was higher than the value obtained for the sPKa assay at baseline (0.91) and the total activity assay post 6 hours of cold incubation (0.92).

[0368] Conclusions

[0369] These data support the conclusions derived in Example 4, and further demonstrate how the assay method can be used to determine sPKa activity with improved specificity and a sensitivity.

[0370] Total amidolytic activity and sPKa activity were measured in plasma samples from healthy controls (n=57) and HAE-C1INH patients (n=25) in accordance with the method described in Examples 4 and 4a. The results are shown in Figure 4.

[0371] In healthy plasma, sPKa activity accounted for 40% of the total of amidolytic activity. In HAE plasma, sPKa activity accounted for >90% of the total amidolytic activity. Analysis of sPKa activity, rather than total amidolytic activity, increased assay sensitivity to detection of HAE samples from 76% to 84%.

[0372] These results demonstrate that measuring sPKa activity reduces assay non-specific background amidolytic activity in healthy plasma and thereby improves assay selectivity and specificity. from female and male

[0373] Total amidolytic activity and sPKa activity was measured in plasma samples from healthy controls (n=57) and HAE-C1INH patients (n=25) in accordance with the method described in Examples 4 and 4a. The 57 healthy controls consisted of 22 females subjects and 35 male subjects. The 25 HAE-C1INH patients consisted of 16 female subjects and 9 male subjects. The data was analysed separately for females (Figure 5A) and males (Figure 5B).

[0374] The 95thpercentile reference value determined in the population of male subjects was 1.97 nmol / min / mL. The 95thpercentile reference value determined in the population of female subjects was 3.18 nmol / min / mL.

[0375] The results show that plasma from healthy females trended towards higher sPKa activity than plasma from healthy males.

[0376] Total amidolytic activity was measured in a first plasma sample (sample 1) from 20 healthy controls in accordance with the method described in Examples 4 and 4a. Four months later, a second plasma sample (sample 2) was taken from the same 20 healthy controls. No significant difference was observed between the activity values determined from sample 1 and sample 2 (Figure 6). Thus, the results demonstrate the repeatability of the assay in measuring amidolytic activity from multiple samples, taken at differing time points, from any single subject.

[0377] Example 8 - Use of the assay to identify HAE-nClINH

[0378] Plasma samples from two subjects with a presumptive diagnosis of HAE-nClINH were tested in the method described in Examples 4 and 4a to determine their sPKa activity. The first subject (Figure 7A) was a 22-year-old female with a history of subcutaneous and facial oedema, treated with tranexamic acid (prophylaxis) and Berinert (on-demand). The second subject (Figure 7B) was a 45-year-old female with a history of subcutaneous oedema, facial and abdominal attacks, responsive to on-demand treatment with tranexamic acid and Berinert. Both subjects were diagnosed with presumptive HAE-nClINH by a physician.

[0379] The plasma samples were obtained from the subjects during the intercritical period. The sPKa activity after 6 hours of cold incubation was 10.18 nmol / min / mL (Figure 7A) and 74.33 nmol / min / mL (Figure 7B), which were above the 95thpercentile reference value for healthy plasma, which as described in Example 4, was 2.73 nmol / min / mL. These sPKa activity values after 6 hours of cold incubation were also above the 95thpercentile reference value for healthy plasma of 3.07 nmol / min / mL, as described in Example 4a, as well as the 95thpercentile reference value for healthy plasma from female subjects of 3.18 nmol / min / mL, as described in Example 6. Data is also presented at baseline (0 hours) in Figures 7A and 7B. As shown, for one of the subjects, the sPKa activity of the plasma sample obtained during the intercritical period was below the reference value for healthy plasma at baseline.

[0380] This example demonstrates that individuals with HAE-nClINH display increased sPKa activity following 6h of cold incubation compared with healthy controls, and that the method of Examples 4 and 4a identified their plasma as diseased. More specifically, by incubating the plasma sample obtained during the intercritical period for 6 hours in the cold, a (diseased) subject was identified as having an elevated sPKa activity (i.e., an sPKa activity above the reference value) that was not detected at baseline (i.e. at 0 hours, without any incubation).

[0381] For one of the two subjects, a plasma sample was also obtained during an HAE attack. The sPKa activity after 6 hours of cold incubation was 390.82 nmol / min / mL (Figures 7B and 7C). sPKa activity was measured in plasma samples from healthy female subjects taking estrogen oral contraception with the method described in Examples 4 and 4a. As shown in Figure 8, following 6 hours of cold activation, sPKa activity increased in the samples. The results show that the sPKa activity assay is capable of detecting an increase in sPKa activity resulting from estrogen oral contraception. of the substrate before adding to the

[0382] Plasma samples were tested to determine the total amidolytic activity in accordance with the method described in Example 4 (i.e., without the PKa inhibitor), except that the temperature of the substrate prior to adding the substrate to the sample was varied. In particular, in one test, the substrate was warmed to 30°C for 10 mins prior to adding the substrate to the sample in accordance with Example 4 (Figure 9, left). In a separate test, the substrate was not warmed prior to adding the substrate to the sample so was at room temperature (Figure 9, right). This example demonstrates that prewarming the substrate to 30°C for 10 mins prior to adding the substrate to the sample improves absorbance readings. Specifically, the absorbance readings of the prewarmed substrate are a more consistent and linear rate as shown in Figure

[0383] 9. 11: Incubation to induce KKS activation is i to differentiate HAE-C1INH plasma from

[0384] Plasma samples were tested to determine the total amidolytic activity in accordance with the method described in Example 4 (i.e., without the PKa inhibitor), except that the length of time of cold incubation of the plasma samples was varied.

[0385] Plasma samples from 25 healthy subjects were incubated in the cold for 0 hours, 6 hours or 12 hours (Figure 10A). Separately, plasma samples from 19 healthy subjects were incubated in the cold for 0 hours, 6 hours or 18 hours (Figure 10B). The data show that incubating the plasma samples for 6 hours did not meaningfully affect total amidolytic activity of the healthy plasma.

[0386] Plasma samples from 12 HAE-C1INH patents were incubated in the cold for 0 hours, 4 hours or 6 hours (Figure 10C). The data show that there is increased total amidolytic activity over time.

[0387] Taken together, the data show that incubating the plasma sample to induce KKS activation (e.g. PKa activity) is important to differentiate HAE-C1INH plasma from healthy plasma.

[0388] Example 12: Preparation of sebetralstat

[0389] A. l-(4-Hydroxymethyl-benzyl)-lH-pyridi n-2-one

[0390] 4-(Chloromethyl)benzylalcohol (5.0 g, 31.93 mmol) was dissolved in acetone (150 mL). 2-hydroxypyridine (3.64 g, 38.3 mmol) and potassium carbonate (13.24 g, 95.78 mmol) were added and the reaction mixture was stirred at 50 °C for 3 hrs after which time the solvent was removed in vacuo and the residue taken up in chloroform (100 mL). This solution was washed with water (30 mL), brine (30 mL), dried (NazSC ) and evaporated in vacuo. The residue was purified by flash chromatography (silica), eluent 3% MeOH / 97% CHClg, to give a white solid identified as l-(4-hydroxymethyl-benzyl)-lH-pyridin-2-one (5.30g, 24.62mmol, 77% yield).

[0391] [M+Na]+= 238

[0392] B. l-(4-Chloromethyl-benzyl)-lH-pyridin-2-one l-(4-Hydroxymethyl-benzyl)-lH-pyridin-2-one (8.45 g, 39.3 mmol), dry DCM (80 mL) and triethylamine (7.66 ml, 55.0 mmol) were cooled in an ice bath. Methanesulfonyl chloride (3.95 ml, 51.0 mmol) was added and stirred in ice bath for 15 min. The ice bath was removed and stirring continued at rt temperature overnight. The reaction mixture was partitioned between DCM (100 mL) and saturated aqueous NH4CI solution (100 mL). The aqueous layer was extracted with further DCM (2 x 50 mL) and the combined organics washed with brine (50 mL), dried over Na2SO4, filtered and concentrated to give l-(4- chloromethyl-benzyl)-lH-pyridin-2-one (8.65 g, 36.6 mmol, 93 % yield) as a pale yellow solid.

[0393] [MH]+= 234.1

[0394] C. Methyl 3-(methoxymethyl)-l-(4-((2-oxopyridin-l{2H)-yl)methyl)benzyl)-lH-pyrazole-4-carboxylate

[0395] Potassium carbonate (519 mg, 3.76 mmol) was added to a solution of methyl 3-(methoxymethyl)-lH- pyrazole-4-carboxylate (320 mg, 1.88 mmol; CAS no. 318496-66-1 (synthesised according to the method described in WO 2012 / 009009, the contents of which are incorporated herein by reference)) and l-(4- (chloromethyl)benzyl)pyridin-2(lH)-one (527 mg, 2.26 mmol) in DMF (5 mL) and heated at 60 °C overnight. The reaction mixture was diluted with EtOAc (50 mL) and washed with brine (2 x 100 mL), dried over magnesium sulfate, filtered and reduced in vacuo. The crude product was purified by flash chromatography (40 g column, 0-100% EtOAc in isohexanes) to afford two regioisomers. The second isomer off the column was collected to afford methyl 3-(methoxymethyl)-l-(4-((2-oxopyridin-l(2H)- yl)methyl)benzyl)-lH-pyrazole-4-carboxylate (378 mg, 1.01 mmol, 53.7 % yield) as a colourless gum.

[0396] [MH]+= 368.2

[0397] D. 3-(Methoxymethyl)-l-(4-((2-oxopyridin-l(2H)-yl)methyl)benzyl)-lH-pyrazole-4-carboxylic acid

[0398] To methyl 3-(methoxymethyl)-l-(4-((2-oxopyridin-l(2H)-yl)methyl)benzyl)-lH-pyrazole-4-carboxylate (3.77 g, 10.26 mmol) in THF (5 mL) and MeOH (5 mL) was added 2M NaOH solution (15.39 ml, 30.8 mmol) and stirred at rt overnight. IM HCI (50 mL) was added and extracted with EtOAc (50 mL). The organic layer was washed with brine (50 mL), dried over magnesium sulfate, filtered and reduced in vacuo to give 3- (methoxymethyl)-l-(4-((2-oxopyridin-l(2H)-yl)methyl)benzyl)-lH-pyrazole-4-carboxylic acid (1.22 g, 3.45 mmol, 33.6 % yield) as a white powder.

[0399] [MH]+= 354.2

[0400] E. 3-Fluoro-4-methoxy-pyridine-2-carbonitrile

[0401] To a large microwave vial, copper (I) cyanide (1.304 g, 14.56 mmol) was added to a solution of 2-bromo- 3-fluoro-4-methoxypyridine (1 g, 4.85 mmol) in DMF (5 mL). The reaction vial was sealed and heated to 100 °C for 16 hrs. The reaction mixture was diluted with water (20 mL) and EtOAc (20 mL). The thick suspension was sonicated and required additional water (40 mL) and EtOAc (2 x 50 mL) with sonication to break-up the solid precipitated. The combined layers were filtered through a plug of celite and the organic layer isolated, washed with brine (50 mL), dried over magnesium sulfate, filtered and the solvent removed under reduced pressure to give a pale green solid identified as the desired compound 3-fluoro-4-methoxy- pyridine-2-carbonitrile (100 mg, 0.578 mmol, 12 % yield)

[0402] F. (3-Fluoro-4-methoxy-pyridin-2-ylmethyl)-carbamic acid tert-butyl ester

[0403] 3-Fluoro-4-methoxy-pyridine-2-carbonitrile (100 mg, 0.578 mmol) was dissolved in anhydrous methanol (10 mL, 247 mmol) and nickel chloride hexahydrate (14 mg, 0.058 mmol) was added followed by di-tert- butyl dicarbonate (255 mg, 1.157 mmol). The resulting pale green solution was cooled in an ice-salt bath to -5 °C and then sodium borohydride (153 mg, 4.05 mmol) was added portionwise maintaining the reaction temperature ~0 °C. The deep brown solution was left to stir at 0 °C and slowly allowed to warm to rt and then left to stir at rt for 3 hrs. The reaction mixture was evaporated to dryness at 40 °C to afford a black residue which was diluted with DCM (10 mL) and washed with sodium hydrogen carbonate (10 mL). An emulsion formed so the organics were separated via a phase separating cartridge and concentrated. The crude liquid was purified by chromatography eluting with EtOAc / iso-Hexane to afford the title compound, (3-fluoro-4-methoxy-pyridin-2-ylmethyl)-carbamic acid tert-butyl ester as a clear yellow oil (108 mg, 62 % yield) [MH]+= 257

[0404] G. C-(3-Fluoro-4-methoxy-pyridin-2-yl)-methylamine hydrochloride salt

[0405] (3-Fluoro-4-methoxy-pyridin-2-ylmethyl)-carbamic acid tert-butyl ester (108mg, 0.358mmol) was taken up in iso-propyl alcohol (1 mL) and then HCI (6N in iso-propyl alcohol) (1 mL, 0.578 mmol) was added at rt and left to stir at 40 °C for 2 hrs. The reaction mixture was concentrated under reduced pressure and then triturated with ether, sonicated and then decanted to give a cream coloured solid (75 mg, 55% yield) identified as C-(3-fluoro-4-methoxy-pyridin-2-yl)-methylamine hydrochloride salt.

[0406] [MH]+= 157 idin-2-vl)methyll-3-l i-l-({4-[(2-oxopyridin-l- oxamide

[0407] 3-(Methoxymethyl)-l-(4-((2-oxopyridin-l(2H)-yl)methyl)benzyl)-lH-pyrazole-4-carboxylic acid (825 mg, 2.34 mmol) and C-(3-fluoro-4-methoxy-pyridin-2-yl)-methylamine hydrochloride salt (450 mg, 2.34 mmol) were dissolved in DCM while cooling to 0°C. l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (627.0 mg, 3.27 mmol), HOBt (378.8 mg, 2.80 mmol) and triethylamine (1.63 mL, 1182 mmol) were added while stirring, the mixture allowed to warm to rt and stirring continued for 20 hrs. Chloroform (50 mL) was added, the mixture was washed with saturated NaHCO3(aq) and reduced in vacuo. The crude material was purified by chromatography eluting with methanol / DCM. The solvent was removed in vacuo and the resulting solid triturated with diethyl ether. The resulting solids were collected by filtration to afford the sebetralstat.

[0408] [MH]+= 492.0

[0409] NMR (CD3OD) 6: 3.41 (3H, s), 4.03 (3H, s), 4.65 (2 H, s), 4.72 (2H, d, J=2.3Hz), 5.24 (2H, s), 5.37 (2H, s), 6.44 (1H, td, J = 1.4, 6.8Hz), 6.62 (1H, d, J = 9.0Hz), 7.18-7.22 (1H, m), 7.31-7.38 (4H, m), 7.56-7.60 (1H, m), 7.75 (1H, dd, J = 1.9, 7.1Hz), 8.18 (1H, s), 8.27 (1H, d, J = 5.6Hz) ppm. An XRPD diffractogram of sebetralstat resultant from the above procedure is shown in Figure 11.

[0410] Peak position table: 13: Clinical trial of sebetralstat in with HAE-nClINH

[0411] The following is an extract from a clinical trial protocol. A key inclusion criteria of this trial protocol is that the subject has a sPKa activity greater than an "upper limit of normal" (i.e. a reference value above which indicates elevated PKa activity), which can be determined in accordance with the methods of the present invention. As described herein, better identification of diseased patients (i.e. those with a dysfunctional KKS, such as patents with HAE-nClINH) enables more effective treatment.

[0412] Running of the study would be consistent with current knowledge of the risks and benefits of the investigational medicinal product (which is sebetralstat), as well as with the moral, ethical, and scientific principles governing clinical research as set out in the Declaration of Helsinki (World Medical Association 2013) and the guidelines on Good Clinical Practice (GCP).

[0413] Primary Objective

[0414] • To demonstrate the clinical efficacy of sebetralstat compared with placebo for the on-demand treatment of PKa-mediated angioedema attacks in patients with hereditary angioedema (HAE) normal Cl inhibitor (C1INH) (HAE-nClINH).

[0415] Secondary Objective

[0416] • To investigate the safety and tolerability of sebetralstat in adolescent and adult patients with PKa-mediated angioedema attacks in patients with HAE-nClINH.

[0417] Screening Visit

[0418] Eligible patients >12 years old may undergo a screening assessment for trial inclusion. All patents may provide informed consent or assent, if applicable, prior to any trial related procedures being performed. Trial-related procedures for this visit are outlined in the Schedule of Events. Laboratory tests required to identify the proposed patient population are as follows:

[0419] • Complement testing: C1INH antigen, C1INH function, C4.

[0420] • Ex-vivo assay measuring sPKa activity. Patients may also be tested for at least the known genetic mutations associated with HAE-nClINH (also referred to as HAE Type III) (F12, KNG1, ANGPT1, PLG, MYOF, HS3ST6) and HAE Type I orType II (SERPING1); diagnostic genetic testing results may also be obtained from documented historical testing.

[0421] Patents who do not show sufficient PKa activity on the ex-vivo assay to qualify for the trial may be retested at any time within 12 weeks of the original screening visit. For best re-testing results, patients should be tested during an HAE attack or as soon as possible following an attack but prior to treatment of the attack.

[0422] Site personnel may train patients on the information they may be expected to provide in the patient diary and on the use of IM P.

[0423] Population

[0424] The trial population may include male and female patients 12 years of age and older with recurrent PKa- mediated angioedema attacks.

[0425] Inclusion Criteria

[0426] 1) Male or female patients 12 years of age and older.

[0427] 2) Diagnosis of HAE-nClINH (i.e. HAE Type III) with PKa-mediated angioedema defined as: a) Documented clinical history of recurrent angioedema (s.c. or mucosal, nonpruritic swelling episodes without accompanying urticaria) considered by the investigator to be consistent with HAE-nClINH;

[0428] AND b) PKa enzyme activity >lx upper limit of normal (ULN).

[0429] 3) Patient has had at least 2 documented angioedema attacks within 3 months prior to the Screening Visit.

[0430] 4) If a patient is receiving long-term prophylactic treatment with attenuated androgens or tranexamic acid, they must have been on a stable dose and regimen for at least 3 months prior to the Screening Visit and be willing to remain on a stable dose and regimen for the duration of the trial.

[0431] 5) Patients must meet one of the following contraception requirements as follows: a) Female patients who are fertile and heterosexually active must agree to use contraception from the Screening Visit until the Final or ET Visit. Acceptable methods of contraception include one or more of the following: i) Progestogen-only hormonal contraception associated with inhibition of ovulation: oral / injectable / implantable (hormonal contraception that contains estrogen including ethinylestradiol is excluded per Exclusion Criterion 7). ii) Intrauterine device. iii) Intrauterine hormone-releasing system. iv) Bilateral tubal occlusion. v) Vasectomized partner (provided that the partner is the sole heterosexual partner of the female patient of childbearing potential and that the vasectomized partner has received medical assessment of surgical success). vi) Male or female condom. vii) Cap, diaphragm, or sponge with spermicide. b) Patients who are not fertile or not heterosexually active, as defined below, do not require contraception. If the patient's status changes during the course of the trial, they may be required to meet the requirements specified in Inclusion Criterion 50. i) Female patients who refrain from heterosexual intercourse during the trial if the reliability of the heterosexual abstinence has been evaluated in relation to the duration of the clinical trial and is the preferred and usual lifestyle of the patient. ii) Female patients who are surgically sterile (e.g. status post hysterectomy, bilateral oophorectomy, or bilateral tubal ligation) or post-menopausal for at least 12 months. iii) Female patients who are premenarche and remain premenarcheal until the end of the trial. c) Male patients (including female partners) do not require contraception.

[0432] 6) Patients must be able to swallow trial tablets whole.

[0433] 7) Patients, as assessed by the Investigator, must be able to store IMP and be able to read, understand, and complete the diary.

[0434] 8) Patient is willing and able to adhere to all protocol requirements, as confirmed by the Investigator.

[0435] 9) Patient provides signed informed consent or assent (when applicable). A parent or legally authorized representative must also provide signed informed consent when required.

[0436] Exclusion Criteria

[0437] 1) Any concomitant diagnosis of another form of recurrent angioedema mediated by mechanisms other than PKa (e.g. mast cell mediated angioedema, angioedema associated with urticaria, bradykinin metabolism-mediated angioedema, idiopathic mast cell mediated angioedema that does not meet Inclusion Criterion.

[0438] 2) Confirmed diagnosis of HAE Type I or II at any time in the medical history: a) Clinical history consistent with HAE (s.c. or mucosal, nonpruritic swelling episodes without accompanying urticaria) AND EITHER i) Diagnostic testing results that confirm HAE Type I or II: C1INH functional level <40% of the normal level. Patients with functional C1INH level 40-50% of the normal level and a C4 level below the normal range OR ii) Documented genetic results that confirm known mutations for HAE Type I or II.

[0439] 3) A clinically significant history of poor response to bradykinin receptor 2 blocker, C1INH, FXIIa inhibitor, or PKa inhibitor in the opinion of the Investigator.

[0440] 4) Use of angiotensin-converting enzyme inhibitors within the previous 6 months prior to screening.

[0441] 5) Use of lanadelumab or berotralstat within the previous 30 days prior to the screening ex-vivo PKa assay.

[0442] 6) Use of ecallantide, icatibant, or C1INH within 5 days prior to the screening ex-vivo PKa assay.

[0443] 7) Use of any estrogen-containing medications with systemic absorption (such as oral contraceptives including ethinylestradiol or hormonal replacement therapy) within 90 days prior to screening.

[0444] 8) Patients who require sustained use of strong cytochrome P4503A4 (CYP3A4) inhibitors or inducers or moderate CYP3A4 inducers.

[0445] Note: These medications include but are not limited to the following:

[0446] Inhibitors: boceprevir, clarithromycin, cobicistat, dasabuvir, denoprevir, elvitegravir, idelalisib, indinavir, itraconazole, ketoconazole, lopinavir, nefazodone, nelfinavir ombitasvir, paritaprevir, posaconazole, ritonavir, saquinavir, telaprevir, telithromycin, tipranavir, troleandomycin, and voriconazole.

[0447] Inducers: Strong: apalutamide, carbamazepine, enzalutamide, mitotane, phenytoin, rifampin, St. John's Wort. Moderate: enobamate, efavirenz, bosentan, etravirine, phenobarbital, primidone, dabrafenib, lorlatinib, pexidartinib, sotorasib.

[0448] 9) Inadequate organ function, including but not limited to: a) Alanine aminotransferase >2x ULN b) Aspartate aminotransferase >2x ULN c) Bilirubin direct >1.25x ULN d) International normalized ratio >1.2 e) Clinically significant hepatic impairment defined as a Child-Pugh B or C.

[0449] 10) Any clinically significant comorbidity or systemic dysfunction, which in the opinion of the Investigator, would jeopardize the safety of the patient by participating in the trial.

[0450] 11) History of substance abuse or dependence that would interfere with the completion of the trial, as determined by the Investigator.

[0451] 12) Known hypersensitivity to sebetralstat or placebo or to any of the excipients.

[0452] 13) Participation in any interventional investigational clinical trial for HAE within 5 half-lives, or any other interventional investigational clinical trial within 4 weeks, of the last dosing of investigational drug prior to screening.

[0453] 14) Participation in any gene therapy treatment or trial for HAE.

[0454] 15) Any pregnant or breastfeeding patient.

Claims

Claims1. A method for determining a level of amidolytic activity of a plasma sample comprising: a. incubating the plasma sample to induce ka 11 ikrei n-ki n in system activation in the sample; b. after step a, adding a plasma kallikrein (PKa) inhibitor to at least a portion of the incubated plasma sample to form an inhibitor / sample mixture; c. after step b, adding a substrate for determining the amidolytic activity to at least a portion of the inhibitor / sample mixture to form an inhibitor / sample / substrate mixture; and d. after step c, determining the amidolytic activity of the inhibitor / sample / substrate mixture, which provides the level of amidolytic activity of the plasma sample.

2. The method according to claim 1, wherein the plasma sample is incubated at a temperature of less than about 15°C in step a.

3. The method according to any of claims 1-2, wherein the plasma sample is incubated at a temperature of about 4°C in step a.

4. The method according to any of claims 1-3, wherein the plasma sample is incubated for at least about 4 hours in step a.

5. The method according to any of claims 1-4, wherein the plasma sample is incubated for about 5 hours in step a.

6. The method according to any of claims 1-5, wherein the substrate for determining amidolytic activity is incubated at a temperature of between about 20°C and about 40°C, and then added in step c at a temperature of between about 20°C and about 40°C.

7. The method according to any of claims 1-6, wherein the substrate for determining amidolytic activity is incubated at a temperature of about 30°C, and then added in step c at a temperature of about 30°C.

8. The method according to any preceding claim, wherein the PKa inhibitor is added in an amount sufficient to inhibit at least about 95% of PKa activity in the sample.

9. The method according to any preceding claim, wherein the PKa inhibitor is KV999272, or a salt and / or stereoisomer thereof.

10. The method according to claim 9, wherein the PKa inhibitor is KV999272.

11. The method according to any preceding claim, wherein the PKa inhibitor is the only kallikrein-kinin system inhibitor added.

12. The method according to any preceding claim, wherein the substrate that is capable of detecting amidolytic activity is a chromogenic substrate.

13. The method according to claim 12, wherein the chromogenic substrate is H-D-Pro-Phe-Arg- pNA (hydrochloride).

14. A method for determining the specific plasma kail ikrein (sPKa) activity of a plasma sample comprising: i. determining the total amidolytic activity of a plasma sample without adding any inhibitor of the kallikrein-kinin system by: a. incubating the plasma sample to induce kallikrein-kinin system activation in the sample; b. after step a, adding a substrate for determining amidolytic activity to at least a portion of the sample to form a sample / substrate mixture; and c. after step b, determining the amidolytic activity of the sample / substrate mixture, which is the total amidolytic activity of the plasma sample; and, ii. subtracting: (1) the level of amidolytic activity as determined by the method according to any one of claims 1-13, from (2) the total amidolytic activity of the plasma sample; to provide the sPKa activity of the sample.

15. The method according to claim 14, wherein the level of amidolytic activity as determined by the method according to any one of claims 1-13 and the total amidolytic activity of theplasma sample are determined following about 6 hours of incubation of the plasma sample in step a of both methods.

16. The method according to any of claims 14-15, wherein analogous conditions are used when determining the level of amidolytic activity by the method according to claims any one of 1- 13 and the total amidolytic activity, except that determining the level of amidolytic activity by the method according to any one of claims 1-13 requires the use of an added plasma kallikrein (PKa) inhibitor.

17. A method for determining a reference value of specific plasma kallikrein (sPKa) activity above which indicates elevated plasma kallikrein (PKa) activity comprising: conducting the method of determining sPKa activity according to any of claims 14-16 on multiple different plasma samples to identify multiple sPKa activity values; wherein the reference value of sPKa activity corresponds to the 95th percentile of the multiple sPKa activity values; and wherein the multiple different plasma samples are collected from at least one healthy subject.

18. The method according to claim 17, wherein "multiple different plasma samples" means at least 34 different plasma samples.

19. The method according to any of claims 17-18, wherein "multiple different plasma samples" means plasma samples collected from multiple different healthy subjects.

20. A method for use in diagnosing a subject with a disease or disorder relating to elevated plasma kallikrein (PKa) activity comprising: a. determining the specific plasma kallikrein (sPKa) activity of a plasma sample collected from the subject, which is the subject's sPKa activity; b. comparing the subject’s sPKa activity with a reference value of sPKa activity above which indicates elevated PKa activity; and c. diagnosing the subject with a disease or disorder relating to elevated PKa activity if the subject's sPKa activity is greater than the reference value.

21. The method according to claim 20, wherein the subject's sPKa activity is determined by the method according to any of claims 14-16.

22. The method according to any of claims 20-21, wherein the reference value of sPKa activity above which indicates elevated PKa activity is determined by the method according to any of claims 17-19.

23. The method according to any of claims 20-22, wherein the disease or disorder relating to elevated PKa activity is bradykinin-mediated angioedema.

24. The method according to claim 23, wherein the bradykinin-mediated angioedema is hereditary angioedema (HAE); or wherein the bradykinin-mediated angioedema is bradykinin-mediated angioedema non-hereditary (BK-AEnH).

25. The method according to claim 24, wherein the bradykinin-mediated angioedema is HAE.

26. The method according to claim 25, wherein the bradykinin-mediated angioedema is HAE with normal Cl inhibitor levels and function (HAE-nClINH).Tl. A method for treating a disease or disorder relating to elevated plasma kallikrein (PKa) activity in a subject, comprising administering to the subject a therapeutic agent for treating the disease or disorder; wherein the subject has a specific plasma kallikrein (sPKa) activity that is greater than a reference value, the reference value being a sPKa activity above which indicates elevated PKa activity; and wherein the sPKa activity of the subject is as defined in any one of claims 14-16.

28. A therapeutic agent for use in the treatment of a disease or disorder relating to elevated plasma kallikrein (PKa) activity in a subject, comprising administering to the subject the therapeutic agent for treating the disease or disorder; wherein the subject has a specific plasma kallikrein (sPKa) activity that is greater than a reference value, the reference value being a sPKa activity above which indicates elevated PKa activity; and wherein the sPKa activity of the subject is as defined in any one of claims 14-16.

29. The method according to claim 27, or the therapeutic agent for use according to claim 28, wherein the reference value is as defined in any of claims 17-19.

30. The method according to any of claims T1 or 29, or the therapeutic agent for use according to any of claims 28-29, wherein the disease or disorder relating to elevated PKa activity has been diagnosed using the method according to any of claims 20-26.

31. The method according to any of claims 27 or 29-30, or the therapeutic agent for use according to any of claims 28-30, wherein the disease or disorder relating to elevated PKa activity is bradykinin-mediated angioedema.

32. The method according to claim 31, or the therapeutic agent for use according to claim 31, wherein the bradykinin-mediated angioedema is hereditary angioedema (HAE); or wherein the bradykinin mediated angioedema is bradykinin-mediated angioedema non-hereditary (BK-AEnH).

33. The method according to claim 32, or the therapeutic agent for use according to claim 32, wherein the bradykinin-mediated angioedema is HAE.

34. The method according to claim 33, or the therapeutic agent for use according to claim 33, wherein the bradykinin-mediated angioedema is HAE with normal Cl inhibitor levels and function (HAE-nClINH).

35. The method according to any of claims 27 and 29-34, or the therapeutic agent for use according to any of claims 28-34, wherein the therapeutic agent is selected from a kal likrein binding agent, a bradykinin B2 receptor antagonist, a Factor Xlla (FXIIa) binding agent, or a C1INH replacement agent.

36. The method according to any of claims l and 29-34, or the therapeutic agent for use according to any of claims 28-34, wherein the therapeutic agent is selected from lanadelumab, garadacimab, sebetralstat, berotralstat, icatibant, ecallantide, RZ-402 (ASP 440), STAR 0215, avoralstat, C1INH replacement therapy (such as Cinryze®, Ruconest®, Berinert®, or Haegarda®), donidalorsen, and deucrictibant.

31. The method according to claim 36, or the therapeutic agent for use according to claim 36, wherein the therapeutic agent is sebetralstat.

38. A method for identifying a subject at risk of having a disease or disorder relating to elevated plasma kallikrein (PKa) activity comprising: a. determining the specific plasma kallikrein (sPKa) activity of a plasma sample collected from the subject, which is the subject's sPKa activity; b. comparing the subject's sPKa activity with a reference value of sPKa activity above which indicates elevated PKa activity; and c. identifying a subject at risk of having a disease or disorder relating to elevated PKa activity if the subject's sPKa activity is greater than the reference value.

39. A method for determining if a disease or disorder is susceptible to treatment with a plasma kallikrein inhibitor comprising: a. determining the specific plasma kallikrein (sPKa) activity of a plasma sample collected from a subject, which is the subject's sPKa activity; b. comparing the subject's sPKa activity with a reference value of sPKa activity above which indicates elevated PKa activity; and c. identifying the disease or disorder as being susceptible to treatment with a PKa inhibitor if the subject's sPKa activity is greater than the reference value40. The method according to any of claims 38-39, wherein the subject's sPKa activity is determined by the method according to any of claims 14-16.

41. The method according to any of claims 38-40, wherein the reference value is determined according by the method according to any of claims 17-19.

42. The method according to any of claims 38-41, wherein the disease or disorder relating to elevated PKa activity is bradykinin-mediated angioedema: wherein the bradykinin-mediated angioedema is hereditary angioedema (HAE); or wherein the bradykinin-mediated angioedema is bradykinin-mediated angioedema non-hereditary (BK-AEnH).

43. The method according to any of claims 38-42, wherein the bradykinin-mediated angioedema is HAE.

44. The method according to claim 43, wherein the subject is bradykinin-mediated angioedema is HAE with normal Cl inhibitor levels and function (HAE-nClINH).

45. The method according to any of claims 20-26 or 38-44, wherein the subject is subsequently treated with a therapeutic agent for the treatment of a disease or disorder relating to elevated PKa activity.

46. The method according to claim 45, wherein the therapeutic agent is selected from a PKa binding agent, a bradykinin B2 receptor antagonist, a Factor Xlla (FXIIa) binding agent, or a C1INH replacement agent.

47. The method according to claim 45, wherein the therapeutic agent is selected from lanadelumab, garadacimab, sebetralstat, berotralstat, icatibant, ecallantide, RZ-402 (ASP 440), STAR 0215, avoralstat, C1INH replacement therapy (such as Cinryze®, Ruconest®, Berinert®, or Haegarda®), donidalorsen, or deucrictibant.

48. The method according to claim 47, wherein the therapeutic agent is sebetralstat.

49. A method for evaluating treatment of a disease or disorder relating to elevated plasma kallikrein (PKa) in a subject comprising: a. determining the specific plasma kallikrein (sPKa) activity of a plasma sample collected from the subject, which is the subject's sPKa activity; b. comparing the subject's sPKa activity with a reference value of sPKa activity above which indicates elevated PKa activity; and c. evaluating effectiveness of the treatment based on whether the subject's sPKa activity is greater than the reference value.

50. The method according to claim 49, wherein the subject's sPKa activity is determined by the method according to any of claims 14-16.

51. The method according to any of claims 49-50, wherein the reference value is determined according by the method according to any of claims 17-19.

52. The method according to any of claims 49-51, wherein the disease or disorder relating to elevated PKa activity is bradykinin-mediated angioedema: wherein the bradykinin-mediated angioedema is hereditary angioedema (HAE); or wherein the bradykinin-mediated angioedema is bradykinin-mediated angioedema non-hereditary (BK-AEnH).

53. The method according to any of claims 49-52, wherein the bradykinin-mediated angioedema is HAE.

54. The method according to claim 53, wherein the treatment being evaluated is a prophylactic treatment of HAE.

55. The method according to claim 54, wherein the prophylactic treatment is selected from lanadelumab, garadacimab, berotralstat, STAR 0215, C1INH replacement therapy (such as Cinryze®, Ruconest®, Berinert®, or Haegarda®), donidalorsen, and deucrictibant.

56. The method according to any of claims 54-55, wherein if the subject's sPKa activity is greater than the reference value, the subject is at risk of an HAE attack.

57. The method according to any of claims 49-56, wherein the subject is administered an on-demand treatment of HAE following evaluation.

58. The method according to any of claims 57, wherein the on-demand treatment of HAE is sebetralstat.

59. A kit comprising: a plasma kal likrein (PKa) inhibitor; a substrate for determining amidolytic activity; and instructions for use in determining specific plasma kal likrein (sPKa) activity.

60. The kit according to claim 59, wherein the instructions for use in determining sPKa activity are instructions for carrying out the method according to any of claims 14-16.

61. The kit according to any of claims 59-60, wherein the PKa inhibitor is selected from the group consisting of sebetralstat, berotralstat, avoralstat, KVD001, feniralstat, lanadelumab, STAR- 0215, RZ-402 (ASP-440), ATN-249, and KV999272, or a salt and / or stereoisomer thereof.

62. The kit according to any of claims 59-61, wherein the PKa inhibitor is KV999272, or a salt and / or stereoisomer thereof.

63. The kit according to claim 62, wherein the PKa inhibitor is KV999272.

64. The kit according to any of claims 59-62, wherein the substrate for determining amidolytic activity is a chromogenic substrate.

65. The kit according to claim 64, wherein the chromogenic substrate is H-D-Pro-Phe-Arg- pNA (hydrochloride).

66. The kit according to claim 64, wherein the chromogenic substrate is H-D-Pro-Phe-Arg- PNA-2HCI.

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