Field of the invention

The present disclosure relates to methods and biomarkers of predicting or monitoring responsiveness to treatment with drugs modulating neprilysin activity (e.g. Entresto™).

Background of the invention

Heart failure (HF) is a complex clinical syndrome and health problem affecting at least 26 million people worldwide and is increasing in prevalence.

According to the medical dictionary definition, HF is an inability of the heart to keep up with the demands on it and, specifically, failure of the heart to pump blood with normal efficiency. HF may be due to failure of the right or left or both ventricles. The signs and symptoms can include shortness of breath (dyspnea), asthma due to the heart (cardiac asthma), edema, blueness or duskiness (cyanosis), and hypertrophy of the heart. HF is categorized into three groups based on ejection fraction (EF):

Those with HF and a reduced EF (HFrEF where left ventricular EF [LVEF] is <40%).

Those with HF and a mid-range EF (HFmrEF where LVEF is 40-50%). Those with HF and a preserved or normal EF (HFpEF where LVEF is

³50%).

HF may also be classified as acute or chronic. Chronic HF is a long-term condition, usually kept stable by the treatment of symptoms. Acute decompensated HF is a worsening of chronic heart failure symptoms which can result in acute respiratory distress.

HF has a heterogeneous pathophysiology. The main aim of HF therapy is to improve the pumping function of the heart. The common strategy for HF therapy is renin-angiotensin-aldosterone system (RAAS) inhibition by either suppressing an angiotensin converting enzyme (by inhibitors of angiotensin converting enzyme, ACEi) or blocking angiotensin II receptor (by angiotensin II receptor blockers, ARB).

Additionally, beta-blockers and mineralocorticoid receptor agonists have been incorporated into the care of patients with HFrEF.

In 2014 by the introduction of a new drug - LCZ696 (the trade name is Entresto™) - Novartis implemented a fundamentally new approach for HF management. The drug was approved under the FDA's priority review process on July 7, 2015. Entresto™ is a combination of angiotensin II receptor inhibitor (valsartan) and neprilysin inhibitor sacubitril, which makes it a first-in-class of ARNi drugs (Angiotensin Receptor - Neprilysin Inhibitor). Pharmacological action of this drug includes inhibition of neprilysin (neutral endopeptidase, NEP) - an endopeptidase that degrades vasoactive peptides, including natriuretic peptides (NPs). Thus, sacubitril increases the levels of these peptides, causing blood vessel dilation and reduction of extracellular fluid volume via sodium excretion. This makes Entresto™ the first medication able to target simultaneously the renin-angiotensin-aldosterone system as well as the NP system. Entresto™ has been shown to reduce risks associated with HF in both chronic and acute HF patients (PARADIGM-HF and PIONEER-HF trials) (1 , 2).

NEP is a zinc-dependent neutral endopeptidase present in many tissues and particularly abundant in kidneys. NEP is a type II membrane protein that functions as a membrane-bound endopeptidase, but a circulating enzymatically active form was also described. It has been identified in various body tissues, such as kidneys (mostly), brain, heart, lungs, adrenal glands, gastrointestinal mucosa, thyroid gland, male reproductive system ducts, and placenta, and on the surface of fibroblasts and neutrophils. The variety of NEP substrates is quite large, including NPs (A-type NP (ANP), B-type NP (BNP) and C-type NP (CNP)), bradykinin, adrenomedullin, enkephalines, substance P, oxytocin, b-amyloid, fibrinogen, and other blood plasma proteins. The relative affinity of NEP varies among substrates. The primary amino acid sequences of human ANP and human BNP are provided herein as SEQ ID NO: 1 and SEQ ID NO: 2 respectively.

Amino acid sequence of human ANP given in one letter amino acid residue code, SEQ ID NO:1 : SLRRSSCFGGRMDRIGAQSGLGCNSFRY

Amino acid sequence of human BNP given in one letter amino acid residue code, SEQ ID NO:2: SPKMVQGSGCFGRKMDRISSSSGLGCKVLRRH

In both the PARADIGM-HF and PIONEER-HF trials, Entresto™ was shown to cause a prominent reduction in cardiovascular death and rehospitalization in HF patients with reduced ejection fraction. However, strict trial inclusion criteria, difficulties with drug titration and adverse outcomes reported for some cases suggest that pretreatment discrimination of patients who could benefit the most from ARNi might be of interest. According to a new paradigm of precision medicine, the right treatment is delivered at the right time to the right person. However, presently, doctors are not able to identify individuals who are most likely to respond to NEP inhibition by the ARNI sacubitril/valsartan (e.g. Entresto™) and those who will not probably respond to the treatment. The efficacy of drugs modulating NEP activity is expected to be different in patients with different activity of NEP. Given this, the skilled artisan will appreciate a method of evaluation of NEP activity to select patients who will benefit from the treatment with drugs modulating NEP activity.

The major part of NEP exists as a membrane-bound enzyme; some amount of the enzyme is present as a circulating form. Soluble NEP is generated presumably by the ectodomain shedding or via exosome-mediated secretion (3).

Membrane-bound and soluble NEP enzymes exhibit similar affinity for inhibitors, optimal pH, and Km range; however, the maximum reaction rate (Vmax) of soluble NEP is markedly lower than that of the membrane-bound enzyme (4). Therefore, the in vivo enzymatic activity of NEP is believed to be attributed mostly to the tissue- associated isoform. Several assays that measure concentration (by immunoassays) or activity (assays based on the cleavage of synthetic fluorogenic substrates) of circulating form of NEP were described (5-8). However, the major drawback of these kind of“direct” assays either enzymatic or immunological is that they are capable of taking into account only NEP activity or amount in isolated blood samples, leaving the membrane-bound enzyme (which is believed to be a major part of enzyme activity) out of consideration.

Summary of the invention

The invention relates to a method for predicting responsiveness of a subject to the treatment with inhibitors of neprilysin (NEP), comprising measuring in a sample obtained from the subject prior to treatment the amount of products produced by NEP from its endogenous substrates or substrates injected prior to analysis; or measuring the ratio of these products to the intact substrates (or to the total amount of cleaved and intact substrates) and classifying the subject as a responsive or non- responsive subject based on the amount of said products generated by NEP-mediated cleavage; or based on the ratio of these products to the intact substrates (or to the total amount of cleaved and intact substrates).

Legend to the figures

Figure 1 : Schematic representation of the cleavage sites of ANP and BNP by NEP.

The neo-epitopes produced in the result of NEP-mediated cleavage are marked. Figure 2: Graph showing the kinetics of the in vitro BNP cleavage by NEP. The amount of three BNP neo-epitope forms (BNP-neo5, BNP-neo17 and BNP-neo18) was measured with immunoassays based on polyclonal antibodies specific to the corresponding neo-epitopes (capture antibodies) and anti-BNP antibody 50E1 (epitope 26-32, detection antibodies). BNP cleavage within the ring structure was measured with BNP immunoassay based on anti-BNP antibodies 50E1 (capture antibodies) and antibodies 130 specific to the region 15-22 (detection antibodies).

Figure 3: Graph showing the kinetics of the in vitro ANP cleavage by NEP. The amount of ANP-neo8 form was measured by the immunoassay based on polyclonal antibodies specific to the neo-epitope (capture antibodies) and PAb anti-ANP antibody (detection antibodies). ANP was measured by the immunoassay based on anti-ANP mAb 23/1 (from Bio-Rad, specific to an epitope within the ring structure of ANP) used as capture antibodies in pair with ANP-specific PAb used as detection antibodies

Figure 4: Graph showing the time course of generation and clearance of BNP-neo17 form in rats non-treated (“-”) and treated (“+”) with sacubitril after intravenous injection of human BNP (synthetic). Error bars represent standard error of the mean (SE).

Figure 5: Graph showing the time course of generation and clearance of BNP-neo18 form in rats non-treated (“-”) and treated (“+”) with sacubitril after intravenous injection of human BNP (synthetic). Error bars represent standard error of the mean (SE).

Figure 6: Graph showing the time course of generation and clearance of BNP-neo5 form in rats non-treated (“-”) and treated (“+”) with sacubitril after intravenous injection of human BNP (synthetic). Error bars represent standard error of the mean (SE).

Figure 7: Graph showing the time course of generation and clearance of ANP-neo8 form in rats non-treated (“-”) and treated (“+”) with sacubitril after intravenous injection of human BNP (synthetic). Error bars represent standard deviation (SD).

Figure 8: graph showing the time course of clearance of ANP in rats non-treated (“-”) and treated (“+”) with sacubitril after intravenous injection of human ANP (synthetic). Error bars represent standard error of the mean (SE). Figure 9: Diagram showing BNP-neo17/total BNP ratio in HF plasma.

Detailed description of the invention

The present invention is intended to provide methods and biomarkers for predicting efficacy and evaluation of treatment with drugs modulating neprilysin (NEP) activity, including methods for predicting responsiveness and monitoring

responsiveness to NEP inhibitors. More specifically, this invention provides methods of measuring the products produced by NEP from natriuretic peptides (NPs) or other substrates to use as biomarkers for a selection of heart failure (HF) patients for treatment and/or monitoring the efficacy of treatment with NEP inhibitor(s). The present invention provides antibodies as well as epitopes of antibodies, specific to the forms of NPs produced by NEP-mediated cleavage. Antibodies specific to particular epitopes are suitable for the precise immunodetection of specific forms of natriuretic peptides generated by NEP-mediated cleavage in the presence of other (e.g. intact) forms of corresponding substrates. The method of measuring the products produced by NEP- mediated cleavage from NPs or other substrates of the present invention can be used for selection of target HF patients for a safe and efficacious treatment and/or monitoring the efficacy of treatment with NEP inhibitors including Entresto™. The described here forms of NPs generated in the result of NEP-mediated cleavage are also suggested as blood biomarkers to be used as diagnostic and prognostic biomarkers for a cardiovascular disease.

In addition, various embodiments of this invention include measuring the amount of products produced by NEP from bradykinin, adrenomedullin, enkephalines, substance P, oxytocin, b-amyloid, fibrinogen, or other blood plasma proteins, or substrates injected prior to analysis, to predicting efficacy and evaluation of treatment with drugs modulating NEP activity, and evaluating the activity of NEP, including both soluble and membrane forms of NEP.

The invention as described herein and claimed in the appending claims at least partially solves one or more of the problems known in the art.

To overcome all the limitations of methods evaluating only the activity or amount of soluble NEP representing a minor part of enzyme activity in the organism, in the present invention we describe a method based on the measurements of the amount of product(s), or their ratio to intact form(s) (or to the total amount of cleaved and intact substrates), produced by NEP from its endogenous substrates or substrates injected prior to analysis. Potential substrates of NEP to be used for this purpose include NPs (ANP, BNP, CNP), bradykinin, adrenomedullin, enkephalines, substance P, oxytocin, b-amyloid, fibrinogen, or other blood plasma protein substrates of NEP. In contrast to any method of evaluating the activity of soluble NEP, measuring the amount of products (or the ratio to intact forms (or to the total amount of cleaved and intact substrates)) produced by NEP-mediated cleavage from its endogenous substrates or substrates injected into the organism prior to analysis will take into account the inherent NEP of the organism (tissue and circulating enzyme).

Among different known substrates of NEP, NPs, ANP and BNP, are of particular importance: these molecules are circulating physiologically active peptides that promote many effects, such as vasodilation, natriuresis and diuresis, as well as inhibition of RAAS. NPs are crucial in HF due to their cardioprotective effects. The use of NEP inhibitors in patients with HF is aimed to increase the levels of active NPs and as a consequence to enhance the compensation mechanisms mediated by NPs.

Notably, the level of active NPs in the circulation depends not only on the degradation rate, but also on the production level and the efficiency of processing of inactive precursor molecules, not susceptible to NEP cleavage. As both production and degradation of active NPs may vary among individuals, this might cause different response to the treatment by NEP inhibitors in different patients.

The basis for discrimination between patients who will benefit from the treatment with NEP inhibitors including Entresto™ and those who will not likely respond to the treatment could be a companion diagnostic test, which evaluates both the activity of NEP and the level of NPs that can be potentially restored by inhibition of NEP. This integral parameter we call NP restoration potential (NP-RP). Patients with active NEP and high level of production of active forms of NPs (high NP-RP) are expected to benefit from the treatment with NEP inhibitors, whereas patients with low NEP activity and/or low levels of active forms of NPs (low NP-RP) may have only a modest if any effect from the treatment with drugs inhibiting NEP. Given this, the evaluation of NP-RP could be very useful for the safe and efficacious use of NEP inhibitors for the treatment of HF.

The suggested above approach have several potential advantages. First, ANP and BNP are present in the circulation in concentrations, which are high enough for their reliable immunodetection. Second, there are sites of ANP and BNP cleavage, which are uniquely specific for NEP. Third, the amount of the product(s) generated by NEP may indicate the potential for improvement of HF patients, as NPs exhibit compensatory physiological effects. Cleavage of ANP and BNP by NEP results in the formation of novel proteolytic epitopes, which are not present in intact molecules. These novel forms (so called neo-epitope forms) are expected to be present in the circulation of HF patients and concentrations of such forms are expected to vary depending on the activity of NEP and the levels of active NPs, reflecting differences in NP-RPs in different individuals. The amount of neo-epitope forms can be measured by means of immunoassays based on antibodies specific to neo-epitopes generated by NEP- mediated cleavage.

In general, the present invention features methods and biomarkers for predicting efficacy and evaluation of treatment with drugs modulating NEP activity, including methods for predicting responsiveness and monitoring responsiveness to NEP inhibitors. Such a method could be useful for pre-treatment discrimination of patients who might benefit from NEP inhibition-based therapy (e.g. Entresto™). This implementation falls into the realm of a companion diagnostic approach, which is rapidly moving to the cardiovascular disease area.

The invention also features immunoassay methods based on antibodies specific to neo-epitopes of ANP and BNP suggested for specific measurements of the products of ANP and BNP cleavage by NEP. The described method can be used for selection of the target HF patients for the safe and efficacious treatment and/or monitoring the efficacy of treatment with NEP inhibitors. The described here forms of NPs generated in the result of NEP-mediated cleavage are also suggested as blood biomarkers to be used as diagnostic and prognostic biomarkers for a cardiovascular disease.

In addition, various embodiments of this invention include measuring the amount of products produced by NEP from other substrates of NEP, e.g. bradykinin, adrenomedullin, enkephalines, substance P, oxytocin, b-amyloid, fibrinogen, or other blood plasma protein substrates of NEP, or substrates injected prior to analysis, to predicting efficacy and evaluation of treatment with drugs modulating NEP activity and evaluating the activity of NEP, including both soluble and membrane forms of NEP.

The present invention also relates to immunoassay methods based on antibodies specific to neo-epitopes of NEP substrates suggested for specific measurements of the products of their cleavage by NEP.

Examples Example 1 : NEP-mediated cleavage of ANP and BNP is expected to generate proteolytic epitopes that are absent in the intact peptides.

Human ANP is known to be primarily cleaved by NEP at two sites - between Cys7-Phe8 and between Gly16-Ala17. Given this, there are four proteolytic epitopes that are absent in the intact ANP (neo-epitopes), which can be generated by the action of NEP - ANP-neo7 (comprising Cys-7 at the C-terminus), ANP-neo8 (comprising Phe-8 at the N-terminus), ANP-neo16 (comprising Gly-16 at the C- terminus) and ANP-neo17 (comprising Ala-17 at the N-terminus) (neo - stands for neo epitope).

Human BNP is known to be primarily cleaved by NEP at two sites - between Met4-Val5 and between Arg17-lle18. Given this, there are three proteolytic epitopes that are absent in the intact peptide (neo-epitopes), which can be generated by the action of NEP - BNP-neo5 (comprising Val-5 at the N-terminus), BNP-neo17 (comprising Arg-17 at the C-terminus) and BNP-neo18 (comprising lle-18 at the N- terminus). The schematic representation of neo-epitope ANP and BNP forms generated by the action of NEP are shown in Figure 1.

Example 2: Production of polyclonal antibodies specific to neo-epitope forms of ANP and BNP

To measure the amount of ANP-neoA8, BNP-neo5, BNP-neo17 and BNP- neo18 forms by means of immunoassays, we developed rabbit polyclonal antibodies (PAbs) specific to these neo-epitopes. We immunized rabbits with a set of synthetic peptides containing the ANP-neoA8, BNP-neo5, BNP-neo17 and BNP-neo18 epitopes coupled to carrier proteins (bovine serum albumin and human transferrin).

PAbs specific to AN P-neo8, BNP-neo5, BNP-neo17 and BNP-neo18 were purified from rabbit serum by affinity chromatography on a matrix with immobilized neo- epitope-containing peptides. This step was preceded by negative affinity

chromatography on resin coupled to either ANP (for ANP-neo8-specific antibodies) or the BNP4-25 fragment (for BNP-neo5, BNP-neo17 and BNP-neo18 antibodies) to remove all PAbs specific to epitopes other than the neo-epitopes.

Example 3: Detection of neo-epitope forms produced from BNP and ANP by NEP- mediated cleavage in vitro.

The produced polyclonal antibodies against three BNP neo-epitopes were used to develop specific sandwich-type immunoassays to detect neo-epitope forms of BNP. We tested PAbs as coat antibodies in pairs with anti-BNP monoclonal antibody (mAb) 50E1 (from HyTest, epitope 26-32) used as detection antibody. The developed assays were shown to have a good performance.

For in vitro BNP cleavage reaction we used 1000 ng/mL human BNP (synthetic, from Bachem) in 50 mmol/L Tris-HCI, pH 7.4, 150 mmol/L NaCI, 0.1% Triton X-100. The final concentration of NEP (from R&D, recombinant expressed in CHO cells) was 6.3 nmol/L. The experimental probes and control sample without NEP were incubated at 37°C for 2 hours and analyzed by using sandwich-type immunoassays based on polyclonal antibodies against BNP-neo5, BNP-neo17 and BNP-neo18 in pairs with 50E1 antibody. BNP immunoassay based on anti-BNP mAb 50E1 (capture antibodies) and mAb 130 (from HyTest) specific to the region 15-22 (detection antibodies) was used to measure the total amount of BNP. The kinetic of production of BNP-neo5, BNP-neo17 and BNP-neo18 forms from BNP by the action of NEP is shown in Figure 2.

We also tested the generation of one of neo-epitope ANP forms, ANP- neoA8, by the action of NEP in vitro. For in vitro ANP cleavage reaction we used 40000 ng/mL ANP (synthetic, from Bachem) in 50 mmol/L Tris-HCI, pH 7.4, 150 mmol/L NaCI, 0.1 % Triton X-100. The final concentration of NEP (from R&D, recombinant expressed in CHO cells) was 1 nmol/L. A PAb against ANP-neoA8 was used as a coat antibody in pair with ANP-specific PAb used as detection antibodies in the assay for ANP-neo8 form. The total amount of ANP was measured by the immunoassay based on anti-ANP mAb 23/1 (from Bio-Rad, specific to an epitope within the ring structure of ANP) used as capture antibodies in pair with ANP-specific PAb used as detection antibodies. The kinetic of production of ANP-neoA8 form from ANP by the action of NEP is shown in Figure 3.

These results from the in vitro cleavage reaction of BNP and ANP by NEP clearly demonstrate that suggested neo-epitope forms are generated by NEP-mediated cleavage of ANP and BNP and can be measured by means of immunoassays.

Example 4: In vivo studies of neo-epitope forms generation

To examine ANP and BNP cleavage by NEP in circulation, we injected a mix of ANP and BNP into rats with or without NEP inhibition by sacubitril and measured the neo-epitope forms of ANP and BNP as well as total ANP and BNP in plasma samples at several time points.

We anesthetized male Wistar rats weighing 250-350 g with zoletil-xylazine and cannulated the right femoral artery and vein. To obtain ANP and BNP kinetics without sacubitril, 2 ml of 0.9% NaCI was orally administered on the next day, and after 1 h the mixture of ANP and BNP at a final concentration of 12 nmol/kg (a dose reported to cause physiological responses in rats) in 0.9% NaCI was injected into the femoral vein.

For ANP and BNP kinetics with sacubitril treatment, the same rats were used 48 h after the control experiment. In in vivo experiment sacubitril is converted into an active sacubitrilat by endogenous esterase. A sacubitril suspension in 2 ml of 0.9% NaCI at a final dosage of 100 mg/kg was administered orally (at a concentration expected to provide effective NEP inhibition in rats the mixture of ANP and BNP was injected after 1 h, similarly to the control.

Blood sampling was performed identically with and without prior sacubitril treatment. Blood samples were collected from the femoral artery immediately before NP administration (0 min) and at time points of 1 , 2, 4, 8 and 20 min post injection using tubes containing 2.75 pi of 0.5 M EDTA, 2.75 mI of 100x protease inhibitor mixture and 2.75 mI of activated sacubitrilat (the final concentrations of inhibitors were 20 mM aprotinin, 5 mmol/L (4-(2-aminoethyl)benzenesulfonyl fluoride - AEBSF, 10 mmol/L benzamidine and 20 pmol/L sacubitrilat) to prevent possible NP degradation after sample collection. All of the samples were immediately centrifuged at 1300xg for 10 min at 4°C. 130 mI of EDTA plasma was then diluted 7.7 times in assay buffer (50 mmol/L Tris-HCI, 150 mmol/L NaCI, 0.01% Tween-40, 0.5% BSA) and frozen at -70°C.

By using the developed immunoassays for neo-epitope forms of BNP, we observed the production of BNP-neo5, BNP-neo17, BNP-neo18 forms in the circulation of rats. Inhibition of NEP by the specific inhibitor sacubitril considerably diminished the amount of produced BNP-neo17 and BNP-neo18 forms in the circulation of rats, suggesting that generation of these forms is NEP-dependent (Figure 4 for BNP-neo17 and Figure 5 for BNP-neo18). There was no significant difference in the amount of BNP-neo5 form generated in the presence of sacubitril, suggesting that generation of this form might be related not only to the activity of NEP, but may be dependent on the activity of other proteolytic enzymes (Figure 6).

We analyzed the generation of one of neo-epitope ANP forms, ANP-neo8, in the circulation of rats. Inhibition of NEP with sacubitril considerably diminished the amount of produced ANP-neo8 in the circulation of rats (Figure 7). Additionally, we showed that treatment of rats with sacubitril led to a prominent decrease in the clearance rate of ANP, analyzed with a competitive immunoassay, based on anti-ANP mAb 23/1 (from Bio-Rad, specific to an epitope within the ring structure of ANP), suggesting that the products of ANP degradation (e.g. neo-epitope forms) can also reflect the activity of NEP in the organism (Figure 8).

Example 5: BNP-neo17 detection in human circulation

We analyzed the presence of one of BNP neo-epitope forms, BNP-neo17, and its portion to total BNP pool in human circulation. To investigate this, we measured total BNP and BNP-neo17 in plasma samples obtained from HF patients. EDTA- plasma samples were obtained from 32 patients (aged between 60 and 84 years) who were hospitalized for acutely decompensated HF (NYHA class 11— IV). All patients were treated according to standard procedures based on current guidelines for HF measurement. None of them received ARNi or other NEP inhibition-based therapy.

Venous blood was collected into Vacuette®K3-EDTA blood collection tubes and centrifuged immediately at 2000xg and RT for 15 min. The supernatant was then transferred into tubes containing lyophilized protease inhibitor mixture; final

concentrations were: 20 pmol/L aprotinin, 5 mmol/L AEBSF, 10 mmol/L benzamidine and 20 pmol/L sacubitrilat to prevent NP degradation after blood sample collection. After thorough mixing, plasma samples were frozen and kept at -70°C until analysis.

The total BNP level (cleaved and noncleaved BNP) was measured by the automated single epitope sandwich BNP immunoassay (SES-BNP™ assay). The measurements were done by ET Healthcare Pylon BNP assay, performed at ET Healthcare, Palo Alto, CA.

Total BNP concentration varied within the range of 28.5-3384.5 ng/L (median 322.2, IQR 161.3-737.0). BNP-neo17 was detected in 19 of the plasma samples, while 13 patients (40.6%) exhibited concentrations of BNP-neo17 below LOD. BNP-neo17 concentration was considerably lower, at 0-37.3 ng/L (median 5.0, IQR 0- 9.1), and correlated with total BNP concentration (r= 0.784, P < 0.0001).

BNP-neo17/total BNP showed variability among individuals. Of 19 samples with detectable BNP-neo17 level, 5 samples exhibited the highest values of this parameter (5.6-11.7%); in 13 cases, the ratio was moderate (0.5-5.5%), and one sample exhibited very low BNP-neo17/total BNP (0.2%) (Figure 9). BNP-neo17/total BNP appeared to have no correlation with total BNP level, with r = -0.175, P=0.680.

This result indicates that BNP-neo17 is present in the human circulation and that its proportion of total BNP concentration varies independently of BNP level. The lack of correlation between BN P-neo17/total BNP and total BNP suggest that the BNP-neo17 portion of total BNP is the independent parameter reflecting BNP-RP and, potentially, can be used for classifying the subject as a responsive or non-responsive subject to the treatment with NEP inhibitors. Example 6: Identification of potential NEP substrates in plasma

To identify potential substrates of NEP in plasma, we incubated recombinant NEP with plasma proteins either immobilized on CNBr-activated sepharose or native plasma with subsequent analysis of the cleavage products by mass-spectrometry. For coupling of plasma proteins to CNBr-activated sepharose, we incubated 0.75 ml_ of heparin plasma of healthy donors with 2.5 ml_ of CNBr-activated sepharose. Recombinant human NEP (soluble domain Tyr52-Trp750, with an N- terminal 6-His tag) was produced in Expi293 cells and purified by means of nickel chelate affinity chromatography. For the cleavage reaction we incubated 75 pl_ of sepharose coupled to plasma proteins with 1-10 pg of recombinant NEP in 10 mM Tris- HCI, pH 7.5, 150 mM NaCI, 10 mM ZnCL for 2-18 hours at 37 °C. The trypsinized and not trypsinized samples were desalted with ZipTip C18 (Millipore) and then analyzed by LC-MS/MS with AmaZon Speed ETD (Bruker) LC-ESI-MS ⁿ analytic platform. In the other experimental setup, we incubated heparin plasma with recombinant NEP in a dialysis tube, then the produced peptides were extracted from the external buffer with solid-phase extraction method and analyzed by LC-MS/MS with AmaZon Speed ETD (Bruker) LC-ESI-MSn analytic platform. The list of potential plasma substrates of NEP identified by these methods is presented in Table 1.

Table 1. The list of potential NEP substrates identified by LC-MS/MS-based methods.

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