Serum Amyloid P-component as a therapeutic agent or biomarker

Field of the Invention

The present invention relates to the treatment of inflammatory diseases and in particular to the provision of therapeutic agents which may halt or slow the progression of inflammatory diseases or disorders. Such diseases include fibrosis, hypertrophy and cardiovascular inflammatory conditions. In particular the agent may be used to halt or slow the progression of hypertensive heart disease into heart failure with preserved ejection fraction. A suitable therapeutic agent is Serum Amyloid P-component. The invention also relates to the use of Serum Amyloid P-component as a biomarker in diagnosing or monitoring the progression of such diseases. The marker may find use in monitoring the of remodelling of the left ventricle of the heart.

Background to the Invention

Fibrosis is the end point of many chronic inflammatory diseases and involves the formation of excess fibrous connective tissue in an organ or tissue. Fibrosis causes renal, lung, gastrointestinal and myocardial disorders, examples of which include pulmonary fibrosis, cystic fibrosis, cirrhosis of the liver, Crohn's disease and myocardial fibrosis. Hypertrophy is the increase in the volume of an organ or tissue due to the enlargement of its component cells. Examples include myocardial hypertrophy. Cardiovascular inflammatory diseases such as heart attack, stroke, myocarditis, atherosclerosis.

There is growing awareness of the importance of the syndrome of heart failure with preserved ejection fraction (HFPEF) which contributes to approximately 20-50% of the heart failure (HF) population. The basic pathophysiological abnormalities responsible for HFPEF are impaired ventricular relaxation and reduced compliance which lead to an alteration in the pressure-volume relationship within the ventricle. Resultant high filling pressure predisposes to diastolic dysfunction (DD) and eventual HFPEF. While the principal cause of these abnormalities remains unclear, initial data from several laboratories indicate that myocardial hypertrophy and excessive fibrosis in the cardiac interstitium lead to reduced compliance, or increased stiffness, of the left ventricle (LV) and hence, DD. A primary cause of HFPEF is hypertensive heart disease (HHD). Experimental and clinical data have demonstrated serological and

morphometric evidence of increased myocardial fibrosis and myocyte size in HHD. These observations have been directly linked to abnormalities in diastolic function and myocardial stiffness.

Serum amyloid P-component (SAP) is a member of the pentraxin family with 66% homology to C-reactive protein (CRP). SAP functions as an innate immune system recognition molecule by binding to pathogen and damage associated molecular patterns. It is cleared by macrophage-like cells through FcTRs. In humans and rats, the levels of SAP in the serum are maintained at relatively constant levels, with CRP acting as an acute-phase response protein. This relationship is reversed in mice with SAP behaving as an acute-phase protein, and CRP remaining at low steady-state levels. However, human, rat, and mouse SAP bind to the same molecules, although with differing affinities, indicating a functional similarity.

A role for SAP in cardiovascular injury has been proposed due to observed correlations between serum levels of SAP and angina and myocardial infarction in elderly patients. Results from the Cardiovascular Health Study suggest that SAP correlates with cardiovascular disease risk factors such as obesity, blood pressure and dyslipidaemia. It has been shown that SAP is specifically accumulated and expressed in atherosclerotic lesions. However, patients with more severe kidney disease have significantly lower concentrations of SAP in plasma relative to that of patients with mild kidney disease and normal function, and there was a correlation between plasma SAP concentration and loss of kidney function. It has therefore been suggested that SAP may share similarities with the complement proteins that can be "consumed" at sites of inflammation, resulting in depressed plasma concentrations.

It is unclear; however, what role SAP plays in the early myocardial response to hypertension, but of particular interest is the observation that treatment of mice with SAP can block ischaemic/reperfusion-induced cardiomyopathy. SAP, via FcTRs, can block the recruitment of bone marrow-derived fibroblast precursors to the injured myocardium and the differentiation of fibroblast precursors. SAP can also attenuate bleomycin-induced pulmonary fibrosis in mice. A reduction in aberrant collagen deposition is associated with a reduction in M2 macrophages and SAP prevents monocyte differentiation into M2 macrophages via an interaction with FcTRs. A subset of inflammatory cells, which express the pro-fibrotic growth factor transforming growth factor-β (TGF- β), are present in DD/ HFPEF patients. Given that myocardial fibrosis and myocyte hypertrophy are the pathologies responsible for the progression of HHD to HFPEF, the present inventors propose that SAP may halt this disease process. They have identifieda significant decrease in SAP in the coronary sinus serum of hypertensive patients at higher risk (increased B-type natriuretic peptide [BNP]), and so propose that this may reflect a susceptibility to exaggerated fibrosis. Therefore, they examined the therapeutic effect of SAP on the prevention of myocardial remodelling in the spontaneously hypertensive rat (SHR), and investigated whether SAP is a candidate biomarker of cardiac dysfunction, and has the ability to predict progression of DD. WO2011/092219 discloses a method of treating cardiovascular disease (CVD) comprising administering an antagonist/silencer of Leucine-rich alpha-2-glycoprotein (LRG) in combination with Serum Amyloid P Component (SAP). However, the document neither discloses SAP as a biomarker in its own right, nor as a sole therapeutic agent for the treatment of CVD.

Object of the Invention

It is thus an object of the present invention to provide a therapeutic agent for the treatment of inflammatory diseases and in particular to the provision of therapeutic agents which may halt or slow the progression of inflammatory diseases or disorders. Such diseases include fibrosis, hypertrophy and cardiovascular inflammatory

conditions. Another object is to provide a therapeutic agent for the treatment of hypertensive heart disease and in particular to the provision of therapeutic agents which may halt or slow the progression of hypertensive heart disease into heart failure with preserved ejection fraction.

A further object of the invention is to provide a biomarker for use in diagnosing or monitoring inflammatory diseases. A further object is to provide a biomarker for use in monitoring the progression of remodelling of the left ventricle of the heart.

Summary of the Invention

According to the present invention there is provided use use of Serum Amyloid P-component in a method of treatment of inflammatory diseases. The inflammatory disease may be selected from fibrosis, hypertrophy and cardiovascular inflammatory conditions.

In particular the invention relates to use of Serum Amyloid P-component in a method of treatment of hypertensive heart disease. The use may be for halting or slowing the progression of hypertensive heart disease into heart failure with preserved ejection fraction or in the attenuation of hypertrophy. The invention also provides a pharmaceutical composition consisting of Serum Amyloid P-component together with a pharmaceutically acceptable carrier or excipient. In particular the pharmaceutical composition comprises no active pharmaceutical agent in addition to SAP.

In a still further aspect the invention provides use of Serum Amyloid P- component as a biomarker inflammatory diseases for use in diagnosing or monitoring the progression of inflammatory diseases. One particular application is in monitoring the progression of remodelling of the left ventricle of the heart.

Use of SAP as a biomarker may involve use of diagnostic kits for determining the presence or levels of SAP in a patient. These kits may be of any conventional type as is known to the skilled person. Suitable methods include immunological,

colourimetric, immunohistochemistry and R A/DNA analysis kits. Examples include an ELISA assay, a competitive/inhibition ELISA, a sandwich ELISA assay, a micro- array based assay, a functionalised nanoparticle assay, other rapid assay platform such as quantum dots, fluorescent tags and electrosensors, an immunohistochemistry assay, or a flow cytometry assay. Samples for diagnosis may include tissue or bodily fluid samples.

The finding that SAP blocks monocyte and macrophage infiltration in tissues indicates that the molecule could find use in the treatment of a wide range of diseases such as fibrotic, inflamaroty and hypertrophic ciseases.

Fibrotic diseases include pulmonary fibrosis, cystic fibrosis, cirrhosis of the liver, Crohn's disease and myocardial fibrosis. Inflammaroty diseases include myocardial hypertrophy, Acne, Asthma, Autoimmune disease, Prostatitis,

Glomerulonephritis, Hypersensitivities, Inflammatory bowel disease, Pelvic

inflammatory disease, Rheumatoid arthritis and Sarcoidosis. Cardiovascular

inflammatory diseases such as heart attack, stroke, myocarditis, atherosclerosis, Coronary heart disease, Ischaemic heart disease, Hypertensive heart disease, Heart Failure, Diastolic heart failure, Diastolic dysfunction, Atherosclerosis, Dilated cardiomyopathy, Diabetic cardiomyopathy, Endocarditis, Inflammatory Cardiomegaly, Myocarditis, Atrial Fibrilation, Amyloidosis, Corpulmonale, Pulmonary hypertension, Reperfusion injury and Vasculitis. Hypertrophic disease includes osteoarthropathy, pulmonary osteoarthropathy, myocardial hypertrophy, hypertrophic cardiomyopathy and Hypertrophic Facet Disease. Brief Description of the Drawings

Figure 1. Effect of SAP treatment on echocardiography parameters. Panel A shows representative M-mode echocardiographic images. Panel B demonstrates measures of LVH. Results represent mean and standard error from the mean. * p<0.05 vs. SHR control group, ** p<0.01 vs. SHR control group, *** p<0.001 vs. SHR control group. Figure 2. Effect of SAP treatment on perivascular collagen. Representative images of picrosirius red stained tissue from one animal in each group are shown. Results represent mean and standard deviation. ** p<0.01 vs. SHR control group.

Figure 3. Effect of SAP on serum MCP-1 and CXCL-1. * p<0.05 vs. SHR control group, ** p<0.01 vs. SHR control group, *** p<0.001 vs. SHR control group.

Figure 4. SAP concentrations in (A) the AH v HFPEF groups and (B) the progressor and non-progressor cohorts at time point 1 & 2. Results represent median ± interquartile range. * p<0.05, ** p<0.01, *** p<0.001.

Detailed Description of the Drawings

Materials and methods

In vivo study

Animals

An exaggerated interstitial and perivascular deposition of fibrillar collagen in the absence of necrosis has been shown in the hypertrophied left ventricle of the SHR. This has also been demonstrated in humans with essential hypertension. Ten week old male SHRs and age/sex matched Wistar Kyoto rats (WKY) were supplied by Charles River Laboratories. All rats were housed in the animal facility under identical conditions, with a 12 hour light-dark cycle. Body weight was recorded on a weekly basis. The experimental procedures were designed in accordance with institutional guidelines, and approved by the Animal Research Ethics Committee at University College Dublin, Ireland.

Experimental design

Serum amyloid P-component (SAP) (Merck Pharmaceuticals) was suspended in phosphate buffered saline (PBS) (Invitrogen/Gibco). Injections of SAP into humans, mice, and rats appear to have no toxic effects [23, 24]. The animals received alternate day intraperitoneal injection of either PBS (vehicle) or SAP (1.6mg/Kg) for 12 weeks. SHR and WKY rats were divided into three groups: one group of 10-week-old normotensive WKY rats treated with vehicle (WKY-V), one group of 10-week-old SHRs treated with vehicle (SHR-V), and one group of 10-week-old SHRs treated with SAP (SHR-SAP).

Systolic blood pressure measurement

Systolic blood pressure was measured while the animals were under anaesthesia (isoflurane 2%) by using the non-invasive tail-cuff method (Letica Scientific

Instruments LE 5001). The mean of three consecutive measurements was obtained for each animal at 6 and 12 weeks.

Blood sampling

Blood was collected at baseline and 6 weeks via the tail vein method and at 12 weeks during terminal bleed (abdominal aorta). Whole blood at baseline and 6 weeks was collected in 300ul Microvette serum tubes (Sarstedt) and centrifuged at lOOOOrpm at 20°C for 5 mins. Whole blood during terminal bleed was collected in 5ml serum separating clot activator tubes (Greiner Vacuette) and centrifuged at lOOOg at 20°C for 10 mins. All centrifugation was performed within 30mins of collection.

Echocardiography

Echocardiography was performed at baseline and 12 weeks with a Vevo 770 High- Resolution In Vivo Micro -Imaging System (Visualsonics) equipped with a lOmHz transducer while the rats were under inhaled anaesthesia (isoflurane 2%). M-mode and 2-dimensional (2D) images were obtained in the parasternal long- and short-axis views. The interventricular septal thickness, posterior wall thickness, and LV diameter were measured in systole and diastole at the tips of the papillary muscle. Measurements were taken over three consecutive cardiac cycles and averaged. Left ventricular mass (LVM) was calculated according to Devereux's formula and indexed to tibial length (LVMi). Blinded analysis was performed by two separate independent observers.

Heart sample preparation

The animals were sacrificed by terminal bleed while under inhaled anaesthetic

(isoflurane 4%). The hearts were removed en-bloc, rinsed in PBS, weighed and dissected into 3 coronal sections. The LV mid-sections (papillary level) were fixed with 10% neutral buffered formalin (Sigma), processed and 6^m-thick, paraffin-embedded sections produced for histomorphologic and immunohistochemical (IHC) analysis. The LV base was snap frozen for later protein extraction and hydroxyproline analysis. The apex was stored in RNAlater (Qiagen) for RNA/gDNA extraction.

Myocardial collagen volume fraction measurement The slides were stained with picrosirius red stain. Quantification of staining was estimated using a digital image analysis system (Aperio). The slides were imaged at high magnification and a positive pixel count algorithm was applied to analyse the area and intensity of a given stain. A colour deconvolution algorithm was subsequently applied to separate the image into 4 channels corresponding to different colours representing negative, weakly positive, moderately positive and strongly positive pixels. Interstitial collagen volume fraction (CVF) and perivascular CVF were generated. Perivascular CVF was indexed to vessel lumen area (PVI) as described in the literature [25-27].

Serum MCP-1 and CXCL-1 measurement

Serum MCP-1 and CXCL-1 were quantified by ELISA using commercial kits (Meso Scale Discovery, Multi- Array Assay System, Rat Cytokine Assay Ultra-Sensitive Kit). In brief, 25uls of blocking agent was added to each well and incubated for 30mins with vigorous shaking (300rpm) at room temperature. Subsequently, 25uls of standard or sample was dispensed into each well and incubated for 2 hours with vigorous shaking (300rpm) at room temperature. After washing, 25uls of detection antibody was added to each well and incubated for 2 hours with vigorous shaking (300rpm) at room

temperature. Following the final wash, 150uls of read buffer was dispensed into each well and the plate analysed on a Sector Imager.

Biomarker Study

Patient groups

In order to examine the role of SAP as a diagnostic marker, previously collected sera from patients with asymptomatic hypertension and HFPEF were compared. To examine the role of SAP as a prognostic marker, patients with evidence of progressive LVDD were identified from the STOP-HF (St. Vincent's Screening to Prevent Heart Failure) programme. The echocardiographic measurement left atrial volume index (LA VI) was used to identify patients with progression. A change in LA VI of greater than 3.5mls/m ² was considered significant. 'Progressors' were age and sex matched with 'non- progressors'.

SAP measurement

Serum SAP was quantified by ELISA using a commercial kit (Hycult Biotech, HK331 Human SAP ELISA). In brief, lOOuls of sample or standard was incubated for one hour at room temperature in microtiter wells coated with antibodies recognising human SAP. After washing, lOOuls of biotinylated tracer antibody was dispensed into each well and incubated for one hour at room temperature. Washing was repeated and then lOOuls of streptavidin-peroxidase conjugate was added to each well and incubated for one hour at room temperature. Following the final wash, lOOuls of tetramethylbenzidine substrate was dispensed into each well and incubated for 30mins at room temperature. The reaction was stopped by adding lOOuls of oxalic acid. The absorbance at 450nm was measured with a spectrophotometer.

Statistical Analysis

Data are presented as either mean ± standard deviation or median ± interquartile range for normally and non-normally distributed continuous variables respectively. The animal data was normally distributed in relation to echocardiographic, PVI and the serum analysis. Therefore analysis of variance followed by Tukey's multiple

comparison tests were used to analyse the three groups of rats. The SAP data was non- parametric in both serum studies. Mann- Whitney tests were used to compare the AH with the HFPEF patients, and the progressors with the non-progressors at both time points. A Wilcoxon matched pairs test was used to compare SAP levels at time point 1

& 2 in the progressors.

Results

In vivo findings

Safety of SAP and effects on blood pressure

All animals survived until the end of the study period. Treatment was well tolerated and no difference in body weight was recorded between SAP and vehicle treated SHRs. Preliminary toxicological examination of liver, lung and kidney from SAP treated SHRs revealed no macroscopic signs of toxicity. There was no significant difference in systolic blood pressure measured at 6 and 12 weeks of treatment in the SHR-SAP and SHR-V groups. Systolic blood pressure was significantly lower in the WKY-V group (Table 1).

Effect of SAP on LVH

Fig. 1 shows representative echocardiographic images. Both independent observers found a significant reduction in posterior wall thickness and LVMi in the SHR-SAP group compared to the SHR-V group. The WKY-V group had significantly less LVH than both SHR groups. Table 1 demonstrates the averaged values of the two independent observers. Ejection fraction was significantly higher in the WKY-V group compared to the SHR-V group.

Table 1. Effect of SAP treatment on body weight, blood pressure and echocardiographic parameters.

Results represent mean ± S.E.M. of 10 rats in each group. EF, ejection fraction: FS, fractional shortening: IVSd, interventricular septum in diastole; LVPWd, left ventricular posterior wall in diastole: LVM, left ventricular mass: LVMi, LVM indexed to tibial length.

* p<0.05 vs. SHR control group.

** p<0.01 vs. SHR control group.

*** p<0.001 vs. SHR control group.

Effect of SAP on myocardial fibrosis

The picrosirius red stained slides were initially reviewed by 3 blinded observers (Fig. 2). Each observer identified more collagen in the SHR-V compared to the WKY-V and SHR-SAP groups. Digital analysis of the slides revealed a significant decrease in PVI in both WKY-V and SHR-SAP (p=0.0016) compared to SHR-V (Fig. 2). No significant difference in interstitial CVF was found between the groups. Figure 2 shows

representative images of collagen deposition in the myocardium of one animal from each group of rats. Increased perivascular staining of picrosirius red can be appreciated in the SHR-V image.

Effect of SAP on serum MCP-1 and CXCL-1

Various inflammatory markers were measured using ELISA including IL-Ιβ, CXCL-1, IL-4, IL-5, IL-6, TNF-a, IFN-γ and MCP-1. The neutrophil chemotactic protein CXCL- 1 was found to be significantly lower (p<0.05) in SHR-SAP and WKY-V compared to SHR-V. Likewise the monocyte chemotactic protein MCP-1 was significantly lower (p<0.001) in the SAP treated animals compared to SHR-V (Fig. 3).

Biomarker Study

SAP detects different disease stages

The concentration of SAP was found to be significantly lower (p = 0.0003) in the

HFPEF (n=36) group compared to the asymptomatic hypertensive group (n=40) shown in figure 4. SAP was also compared with a variety of fibro -inflammatory and echocardiographic markers. A positive correlation was observed with ejection fraction (r=0.3999, p=0.0006). Negative correlations were observed with PIIINP (r=-0.3461, p=0.0036), CITP (r=-0.3050, p=0.0121), MMP2 (r=-0.4194, p=0.0005), IL-6 (r=-

0.3848, p=0.0011), IL-8 (r=-0.3457, p=0.0048), age (r=-0.3686, p=0.0228), left atrial volume (r=-0.3507, p=0.0039), e'(r=-0.4067, p=0.0008) and left ventricular posterior wall thickness (r=-0.5138, p<0.0001).

SAP detects disease progression

Thirty patients with worsening LVDD were identified and matched with 30 non- progressors. SAP concentration was found to be significantly lower (p = 0.0143) at the second time point (1 year apart) in patients with progressive LVDD (Fig 4). Levels did not change significantly in the non-progressor group over time (p=0.4942). There was no significant difference between progressors and non-progressors at time point 2. Discussion

The main findings of this study are the following: (1) chronic administration of SAP is associated with a reduction of LVH in SHRs; (2) perivascular fibrosis is attenuated in the myocardium of SHRs on chronic SAP therapy; (3) serum MCP-1 and CXCL-1 levels are decreased in SHRs treated with SAP; and (4) serum levels of SAP are lower in human subjects with more extensive cardiac dysfunction and in those with progressive DD.

The parameters necessary to generate a LVM measurement using Devereux's formula include IVSd, LVIDd and LVPWd [28]. LVM is one of the most accurate measures of LVH in vivo and this is made more sensitive when indexed to body surface area in humans or tibial length in rats [29]. In essential hypertension, a reduction in

LVM during treatment is a favorable prognostic marker that predicts a lesser risk for subsequent cardiovascular morbid events [30, 3 1 ]. Such an association is independent of baseline LV mass, baseline clinic and ambulatory blood pressure, and degree of blood pressure reduction [30]. A trend in reduction in IVSd and a significant reduction in LVPWd culminated in a very significant (p<0.01 ) overall reduction in LVMi in the SAP treated SHRs; an outcome that is likely to reduce cardiovascular events.

Perivascular collagen to vessel lumen area is a well described method for measuring the early fibrotic response to increased afterload [25-27]. The finding that perivascular collagen was lower but interstitial col lagen similar in the SHR-SAP and WKY-V groups compared to the SHR-V group probably reflects the early stages of disease in these young adult rats. We did not reproduce the findings made by Hermida et al. that 22 week old WK.Y rats exhibit a 45% lower CVF than SHRs [32].

Nevertheless the reduction in PVI in the SAP treated animals confirms that the anti- fibrotic properties of this protein, prev iously demonstrated in acute models of organ fibrosis, also applies to this chronic treatment model. Reduction in the burden of myocardial fibrosis decreases the incidence of cardiovascular events such as arrhythmia, heart failure and death.

CXCL-1 is a small cytokine produced by macrophages, neutrophils and epithelial ceils, and is involved in neutrophil chemoattractant activity. It plays a role in angiogenesis, tumorigenesis, wound healing and inflammation. MCP- 1 , known as CCL- 2, is also a small cytokine produced by monocytes, macrophages and dendritic cells which recruits monocytes, memory T-cells, and dendritic cel ls to sites of tissue injury, infect ion and inflammation. Pre-clinical st udies suggest that the development of fibrosis and scarring is attenuated by blocking or reducing the activ ity of these cytokines [38, 39]. The significant reduction in these cytokines found in the SAP treated animals may represent lower numbers of recruited in flammatory cells and indeed M2 macrophages, and may in part explain the reduction in aberrant collagen deposition or PVI. These findings may relate to the mechanism by which SAP prevents monocyte differentiation into M2 macrophages via an interaction with Fcl ^' Rs as described by Murray [21]. The finding that MCP-1 levels were higher in the WKY-V rats compared to the SHR-V animals may relate to a difference in strain.

The results of the biomarker studies are in keeping with the initial observation made based on our proteomic discovery analysis. Castano et al. hypothesised that consumption of SAP through recruitment to the site of injury may explain why plasma concentrations fall in patients with inflammatory kidney diseases [ 17]. This theory which likens the behaviour of SAP to complement could also explain our biomarker findings. HFPEF patients have significantly lower SAP levels than AH patients and therefore SAP measurement may provide additional information as a tool when diagnosis is uncertain. However, SAP is not cardiac specific. SAP levels also become depleted in asymptomatic hypertensive patients with echocardiographic evidence of progressive LVDD. A more realistic clinical role for SAP relates to how it tracks progression of LVDD and may serve as a biomarker reflecting the left ventricular remodelling responsible for worsening LVDD.

In conclusion, this is the first study to observe the effects of chronic SAP therapy on a model of solid organ fibrosis. It is also the first study to investigate the effects of SAP on a pre-clinical model of HHD; and to examine the role of SAP as a biomarker across a spectrum of LV remodelling in patients with hypertension. The fact that administration of SAP induces anti-inflammatory responses in our study indicates that a functional deficiency of SAP exists in the hearts of hypertensive rats. The theory suggesting SAP consumption through recruitment to the site of injury may also explain why serum concentrations fall in patients with progressive LVDD and HFPEF. Our findings support a role for SAP as a treatment for HHD and as a biomarker in progression of LV remodelling.

In an attempt to identify molecules associated with the progression of cardiac fibrosis and hypertrophy we have recently completed a proteomic analysis of coronary sinus serum from HHD patients. We have shown that expression of serum amyloid P- component is reduced in hypertensive patients that are at risk of developing myocardial fibrosis (remodelling) and HFPEF and propose that this may contribute to the progression of disease. The words "comprises/comprising" and the words "having/including" when used herein with reference to the present invention are used to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.

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