Cross Reference to Related Applications The present application claims the benefit of United States provisional application

61/136,306, filed on August 26, 2009, the contents of which is hereby incorporated by reference in its entirety.

Field of the Invention The present invention is directed to diagnostic methods based upon circulating levels of pentraxin-3. The methods may be used to determine the likelihood of a subject developing metabolic syndrome (MetS) and assessing whether patients with metabolic syndrome are responding to treatment. In addition, the methods may be used to determine whether certain individuals are an increased risk of experiencing a cardiovascular event such as a stroke or myocardial infarction.

Background of the Invention

Inflammation contributes importantly to the pathogenesis of atherosclerosis and its acute thrombotic complications, including acute myocardial infarction, unstable angina, and stroke (Libby, Nature 420:868-874 (2002); Libby, et al., Circulation 705:1135-1143 (2002); Paoletti, et al, Circulation 109:11120-11126 (2004)). Recent clinical and preclinical evidence suggests that inflammation may also promote metabolic syndrome (Sutherland, et al., Metabolic Syndrome and Related Disorders 2:82-104 (2004); Esposito, et al., Nutr. Metab. Cardiovasc. Dis. 14:228-232 (2004)), a disease characterized by a group of metabolic risk factors including: 1) abdominal obesity (excessive fat tissue in and around the abdomen); 2) atherogenic dyslipidemia (high triglycerides; low HDL cholesterol and high LDL cholesterol); 3) elevated blood pressure; 4) insulin resistance or glucose intolerance; 5) a prothrombotic state (e.g., high fibrinogen or plasminogen activator inhibitor-1 in the blood); and 6) a proinflammatory state (e.g., elevated CRP in the blood). Metabolic syndrome has become increasingly common in developed countries and is closely associated with risk of coronary heart disease (Malik, et al., Circulation 110:1245- 1250 (2004); Irabarren, et al., J. Am. Coll. Cardiol. 45:1800-1807 (2006)). Pentraxin-3 is a protein that sharply increases in the blood of patients suffering from an acute myocardial infarction and is commonly detectable in plaques present in advanced arteriosclerosis (Fazzini, et al., Arthritis Rheumatism, 44:2841-2850 (2001); Peri, et al., Circulation 102:636-641 (2000), Rolph, et al., Arterioscler. Thromb. Vase. Biol. 22:elO-el4 (2002)). It has also been associated with an increased risk of stroke and myocardial infarction (US 20060286617). An high sensitivity enzyme-linked immunosorbent assay (ELISA) was recently developed for this protein and used to aid in the diagnosis of vascular inflammation (WO 2007/055340; WO 2005/080981). This is of significance since mild vasculopathy may precede a myocardial infarction.

Summary of the Invention

The present invention is based upon research suggesting that PTX3 levels are related to metabolic syndrome in a complex way. The basic concepts that have been developed and that underlie the invention are as follows: 1) PTX3 levels in patients with metabolic syndrome are inversely related to the severity of the syndrome. Non-diabetic subjects show a stronger inverse association between PTX3 levels and the severity of metabolic syndrome compared to all subjects.

2) There is a subgroup of metabolic syndrome patients that have higher

PTX3 levels than other patients and are at increased risk for having a cardiovascular event, particularly a stroke or myocardial infarction.

3) Low PTX3 levels are an indication that an individual is at increased risk for developing metabolic syndrome, particularly in people that already have some of the characteristics of this syndrome.

4) Patients that are obese and that have elevated plasma levels of PTX3 have a greater chance of having a cardiovascular event than obese individuals with lower PTX3 levels. Obese patients with low PTX3 levels are at greater risk of developing other characteristics associated with metabolic syndrome. In its first aspect, the invention is directed to a method of determining whether a subject has metabolic syndrome or subclinical metabolic syndrome. The term "subclinical metabolic syndrome" refers to a subject that does not have metabolic syndrome using accepted clinical criteria but has at least one of the characteristic symptoms of metabolic syndrome and, in addition, an abnormal blood level of PTX3. Characteristic symptoms include: hyperglycemia, hypertension, hypertriglyceridemia, low HDL, high LDL, insulin resistance, glucose intolerance and abdominal obesity (e.g., waist of 40 inches or more), BMI at >30 kg/m ² , a prothrombotic state (e.g., high fibrinogen or plasminogen activator inhibitor-1 in the blood), and a proinflammatory state (e.g., elevated C reactive protein in the blood). Usually, a diagnosis of metabolic syndrome requires the presence of at least 3 of these symptoms. In all cases, the existence of a symptom is determined using accepted clinical criteria well known in the art.

The first step in the method involves obtaining a test sample of blood, plasma or serum from a subject that has not been diagnosed as having metabolic syndrome but who, nevertheless, has one or more of the characteristics described above and determining the concentration of pentraxin-3 (PTX3) present. This may be accomplished using any method known in the art but the use of an ELISA (see WO 2005/080981) is preferred. The results obtained from the test sample are compared to results obtained using one or more control samples. If the concentration of PTX3 in the test sample is lower than the concentration in the control sample, this is an indication that the subject has subclinical metabolic syndrome and that treatments aimed at preventing further progression, e.g., a diet and exercise program, should be initiated. Control samples may be blood, plasma or serum obtained from the total population of metabolic syndrome patients or an average range based upon PTX3 levels among all metabolic syndrome patients. It will be understood that once a reference range is known, it may be compared to results of test samples rather than concurrently run controls. An abnormally low concentration would preferably be 20-40% lower than the reference range. Subjects with a history of cardiovascular disease, a cardiovascular event (e.g., a stroke or myocardial infarction), or diabetes would be of particular interest for testing.

In another aspect, the invention is directed to a method of determining whether an obese test subject that has, or has not, been diagnosed as having metabolic syndrome is at increased risk of having a coronary event relative to other obese subjects. The term "increased risk" as used herein means that, statistically, a person having a given characteristic (e.g., higher than normal PTX3 levels) has a greater chance of experiencing a particular outcome (e.g., a myocardial infarction) than other people in a particular population (e.g., obese people without elevated PTX3 levels). At a minimum, 10% more people in an "at risk" group should experience the outcome, with 20%, 40% or more being preferred. An obese subject is defined as someone with a BMI of 30 kg/m ² or more. In order to assess risk, a test sample of blood, plasma or serum is obtained from the obese test subject and the concentration of PTX3 present is determined, e.g. using an ELISA. Results are compared with those of one or more control samples and a conclusion is reached that the test subject is at increased risk of having a cardiovascular event if the concentration of PTX3 in the test sample is higher than the concentration in the control samples. In this case, the control samples may either be the general population of obese patients or, preferably, obese patients known not to be at increased risk of a coronary event and the PTX3 concentration of the control samples can be determined in advance The term "cardiovascular event" or "coronary event" refers to a stroke or myocardial infarction. As a general rule, in order to be suggestive of increased risk, the concentration of PTX3 in the test sample should be at least 20-40% higher than in control samples or, for example, 4.0 ng/mL or higher.

In addition, the invention is directed to a method of determining whether metabolic syndrome is progressing or regressing in patients. This is accomplished by: obtaining a test sample of blood, plasma or serum from the patient, determining the concentration of PTX3 present and comparing the results with those from one or more reference samples obtained from the same patient at an earlier time. Metabolic syndrome that is progressing will be indicated by a concentration of PTX3 in the test sample that is lower than the concentration in the reference samples and metabolic syndrome that is regressing will be indicated by a concentration of PTX3 in the test sample that is higher than the concentration in said one or more reference samples. This test will be useful, for example, in determining if a therapeutic regimen is working. Preferably, in order to make a determination whether metabolic syndrome is progressing or regressing, the concentration of PTX3 in the test and reference samples should differ by at least 20%. In general, it is expected that the concentration of PTX3 in the test sample will be from 2 ng/ml to 3.5 ng/ml or lower. The invention also includes a method of determining whether a test patient diagnosed as having metabolic syndrome is at increased risk of having a coronary event relative to other patients with metabolic syndrome. As in the assays discussed above, a test sample is first obtained and assayed to determine the concentration of pentraxin. A comparison is made with the concentration of PTX3 in control samples derived from metabolic syndrome patients in general or, preferably, metabolic syndrome patients known not to be at increased risk for a cardiovascular event. If desired, the PTX3 concentration of the control samples can be determined in advance. The test subject is considered to be at increased risk if the concentration of PTX3 in the test sample is higher than the concentration in the control sample. Preferred patients for testing will be those with a history of cardiovascular disease and the preferred test method is by ELISA. To be of diagnostic value, the concentration of PTX3 in the test sample should preferably be at least 20%, and, more preferably, at least 40%, higher than the control samples. By way of example, patients with a concentration of 4.0 ng/ml might be considered to be at increased risk.

Finally, PTX3 levels may be diagnostically useful with respect to conditions that are closely related to metabolic syndrome such as cardiovascular calcification. It has been found that inflammation promotes arterial and valvular calcification (Aikawa, et al., Circulation 775:377-86 (2007); Aikawa, et al., Circulation 776:2841-50 (2007)). In the same way that abnormally low levels of PTX3 may be used diagnostically to detect metabolic syndrome, assays of PTX3 on blood, plasma or serum may be used to detect subclinical arterial or aortic valve calcification.

Brief Description of the Drawings

Figure 1 : Figure 1 shows the amino acid sequence of human PTX3 (SEQ ID NO:1).

Detailed Description of the Invention

The present invention is based upon the discovery of that circulating levels of PTX3 may be used diagnostically in evaluating metabolic syndrome and the risk of certain patients having a cardiovascular event. However the relationships are complex. For example, it has been found that levels of PTX3 are inversely related to the severity of metabolic syndrome but, paradoxically, directly related to the likelihood of a patient with metabolic syndrome having a coronary event. Thus, in a patient receiving treatment for metabolic syndrome, an increase in PTX3 levels is an indication that the treatment is working, i.e., promoting a regression of the syndrome. However, if the levels reach a point where they are substantially higher than those in metabolic syndrome patients in general, the patient is at increased risk of having a stroke or myocardial infarction. Thus, the goal of therapy must be to raise PTX3 levels to that seen in low risk individuals but not substantially beyond.

Low levels of PTX3 in subjects that have no characteristics associated with metabolic syndrome or some characteristics but an insufficient number to conclude that this condition is present, is an indication that the individual is likely to develop other characteristics associated with the condition. In instances where one or more characteristics associated with metabolic syndrome are already present, the tendency to develop additional characteristics is considered especially strong and these individuals have been termed as having "subclinical metabolic syndrome." Higher levels of PTX3 do not indicate that characteristics associated with metabolic syndrome are likely to develop but, based on results seen with metabolic syndrome patients, these patients are likely to be at greater risk for a coronary event. High levels of PTX3 in obese patients are a particularly strong indicator that coronary problems may develop.

Breaking things down more specifically, individuals with low PTX3 levels relative to the average are likely to develop insulin resistance, glucose intolerance, hypertension, hypertriglyceridemia, abdominal obesity, a BMI of 30 kg/m ² or more, prothrombotic tendencies (e.g., high fibrinogen or plasminogen activator inhibitor-1 in the blood), and proinflammatory tendencies (e.g., elevated C reactive protein in the blood). If a patient already has been diagnosed as having metabolic syndrome, especially low levels, e.g., less than 3 ng/ml, suggests that the condition is, or is likely to become, particularly severe.

The complete amino acid and nucleotide sequence of human PTX3 are known (see Figure 1 for the amino acid sequence) and can be used to develop assays for determining the concentration of PTX3 in blood, plasma or serum. For example, based upon the amino acid sequence, antibodies may be developed that are specific for PTX3 and used in immunoassays or ELISA assays. Guidance concerning assays that can be used may be found in Innoue, et al., {Arterioscler. Thromb. Vase. Biol. 27:161-167 (2007)); US 20060286617; and WO 2005080981 and it appears that there is at least one assay that is commercially available (R&D Systems, Minneapolis MN).

Diagnostic criteria for metabolic syndrome and tests that can be used in determining whether this condition is present are well known in the art of medicine. Guidelines have been established, for example, by the American Association of Clinical Endocrinologists (see Bloomgarden, Diabetes Care 26:933-939 (2003)) and the American Heart Association/National Heart Blood and Lung Institute (see Beilby, Circulation 109:433-43% (2004)) and may e applied to the present invention.

Treatments for metabolic syndrome are well known in the art and include changes in diet and exercise as well as the administration of drugs to treat high blood pressure, and reduce cholesterol levels (Wagh, Exp. Rev. Cardiovasc. Ther. 2:213-228 (2004); Esposito, et al., Metab. Syn. ReI. Disorders 5:291-295 (2007); Mallare, et al, Diabetes Spec. 18:220- 1228 (2005)). The methods discussed herein may be used to determine the extent to which these therapeutic approaches appear to be stopping or reversing the progression of metabolic syndrome. In patients, that are found to have subclinical metabolic syndrome, to be at risk for developing the condition or to be at increased risk of having a coronary event, treatments may be introduced by physicians to prevent disease progression.

Examples

A prospective, nested, case-control study within the Cholesterol and Recurrent Events (CARE) trial was conducted. CARE was a randomized, placebo-controlled trial of pravastatin among 4159 patients who suffered acute myocardial infarction 3 to 20 months before enrollment. The institutional review board at each participating clinical center approved the study, subjects gave informed consent, and all procedures followed institutional guidelines. Baseline total cholesterol was <240 mg/dL, LDL cholesterol 115 to 174 mg/dL, and triglycerides <350 mg/dL. The median follow-up duration was 5 years. During the trial, 486 patients experienced the primary end point of coronary death, or myocardial infarction. Sufficient plasma for analysis was available from both screening visits for 413 of these cases. Control subjects were randomly selected from among patients who did not have a primary end point, matched to the cases on decade of age (e.g., 40 to 49, 50 to 59 years) and sex. Among the 419 matches, 53 patients were excluded who had coronary bypass surgery, coronary angioplasty, or stroke after randomization during the follow-up. Clinical backgrounds of the patients were shown in Table 1.

Table 1

A fasting blood sample was taken from each patient on each of 2 screening visits, > 1 week apart, and sent by overnight delivery in cooled containers to the core laboratory in St. Louis, Missouri. EDTA served as an anticoagulant and preservative. Plasma was separated in a refrigerated centrifuge, and 1-mL aliquots were placed in polypropylene vials and stored continuously at -80 ⁰ C until analysis. PTX3 was measured by the high-sensitivity ELISA system (Perseus Proteomics Inc, Tokyo). Plasma total cholesterol, HDL and LDL cholesterol, triglycerides, fasting blood glucose, and apolipoproteins CIII and E were measured as previously described. Plasma insulin levels were measured by a commercially available ELISA kit (Alpco Diagnostics, USA). All personnel at the laboratories were blinded to the case-control status, which was maintained at the Data Coordinating Center, University of Texas School of Public Health, Houston.

We defined metabolic syndrome using the modified 2005 NCEP-ATP III definition as 3 or more of the following: waist circumference > 40 inches in men, >35 inches in women; blood pressure > 130/85 mmHg; triglycerides > 150 mg/dL; HDL cholesterol ≤ 40 mg/dL in men, 50 mg/dL in women; and glucose > 100 mg/dL. We categorized body mass index (BMI) according the WHO criteria: BMK25 kg/m ² = healthy weight, BMI 25-29.9 kg/m ² = overweight and BMI>30 kg/m ² = obese.

For analysis comparing cases and controls, continuous variables are shown as means (standard deviation) or medians (inter quartile range) and the unpaired t test or the Wilcoxon unpaired rank sum test was used to compare cases and controls. Proportions were compared using the χ ² test. We used Spearman correlations to assess the relation between continuous variables. To predict mean concentration of PTX3 within categories of metabolic and cardiovascular risk factors, we used multivariate linear regression to calculate least-squares mean and standard errors, adjusting for potential confounders. Additionally, we used multivariate linear regression to calculate least-squares mean and standard errors to estimate components of the MetS within quartiles of PTX3. Insulin and HOMA-IR were not normally distributed, thus these variables were log-transformed in the linear regression model and geometric means were estimated. For these analyses, we used only the healthy control subjects, as they represent the underlying population from which the cases arose and would not be subject to bias due to underlying disease pathology. We used unconditional logistic regression to calculate the odds ratio and 95% confidence interval (CI) of coronary heart disease (CHD). Participants were categorized into quartiles based on the distribution of PTX3 levels in the control participants. Models were adjusted for various coronary risk factors. Tests for linear trend for both the linear regression and logistic regression models were conducted by assigning the median value for each quintile of intake and treating this new variable as continuous. A probability value of 0.05 (2-sided) was considered significant.

Key Findings We observed modest but significant inverse correlations between PTX3 and BMI, waist circumference, triglycerides, apo CIII, apo E and tPA and modest positive correlations between PTX3 and HDL and age. Plasma PTX3 levels were not significantly correlated with total and LDL cholesterol levels, plasma glucose levels, or systolic and diastolic blood pressure.

Compared with control subjects, the cases had higher BMI and waist circumference. Case subjects were more likely to be current smokers, have diabetes and hypertension, and take cardiovascular medications than did the controls. The cases had significantly higher total cholesterol, LDL cholesterol, triglycerides, and plasma glucose, and lower levels of HDL cholesterol than the controls. Case subjects also had higher mean plasma PTX3 levels than the controls, although the difference was not statistically significant. However, PTX3 was not significantly associated with higher risk of CHD (nonfatal MI and fatal coronary heart disease), even after adjustment for other coronary risk factors.

We further explored the relation between PTX3 and various components of the metabolic syndrome. Among 779 patients, plasma PTX3 levels were inversely associated with various parameters associated with MetS, including body mass index (P trend < 0.001), triglycerides (P trend = 0.004), insulin levels (P trend = 0.002), insulin resistance (HOMA-IR, P trend = 0.002) and positively associated with HDL cholesterol (P trend = 0.002) after adjustment for age. Subjects with metabolic syndrome also had significantly lower PTX3 levels (mean=3.80 ng/niL; S. E. =0.11) than those without metabolic syndrome (4.20 ng/niL; S.E.=0.09) (P = 0.01), after adjustment for age, gender, smoking, apoCIII, apoE, and tPA. We also explored the association of PTX3 with BMI and diabetes. Adjusted mean PTX3 concentration was lower among the obese subjects (mean=3.77 ng/mL; S.E.=0.13) than among the non-obese subjects (mean=4.15 ng/mL; SE=O.08) (P = 0.01). On the other hand, adjusted mean PTX3 concentration was higher among patients with diabetes (mean=4.53 ng/mL; S.E.=0.11) than among controls without diabetes (mean=3.96 ng/mL; SE=O.07) (P = 0.002). To clarify the association of PTX3 on obesity independent of diabetes mellitus, we stratified the control subjects by diabetes status. Compared to subjects with a healthy weight (BMI < 25), both overweight subjects (25 ≤ BMI < 30) and obese subjects (BMI > 30) had significantly lower mean levels of plasma PTX3 in non-diabetic subjects (N=655) after adjustment age, gender, smoking, HDL cholesterol, triglyceride, history of hypertension, apoCIII, apoE, and tPA (P = 0.06, P = 0.001, respectively). In diabetic subjects (N= 124) had no significant difference in mean plasma PTX3 levels in different categories of BMI. As a result, the correlation of PTX3 with obesity is stronger in non-diabetric subjects than those in diabetic subjects.

We further explored the association between PTX3, metabolic syndrome and diabetes mellitus. The total number of components of MetS was inversely associated with mean plasma PTX3 levels in all subjects (N=779), even after adjustment for age, gender, smoking, apoCIII, apoE, and tPA (P trend = 0.006). After exclusion of diabetic subjects, non-diabetic subjects (N=655) showed a stronger inverse association between mean plasma PTX3 levels and the total number of metabolic syndrome components (P trend = 0.004).

Due to the strong correlations between PTX3 and components of the metabolic syndrome, we explored whether PTX3 was a predictor of coronary events among subjects with the metabolic syndrome. Among subjects with the MetS, cases had significantly higher plasma PTX3 levels (mean = 4.01 ng/mL; SE=O.16) than the controls (mean = 3.44 ng/mL;

SE = 0.14), after adjustment for age, gender, smoking, apoCIII, apoE, and tPA (P = 0.01).

There was no significant difference in PTX3 concentration between cases and controls in subjects without the MetS (p=0.39).

Although PTX3 is an inflammatory mediator and obesity is an inflammatory disease, obese subjects had significantly lower plasma PTX3 levels than non-obese subjects. Therefore, we investigated whether PTX3 could serve as a significant predictor of coronary event in obese subjects. Whereas no significant difference in plasma PTX3 levels between the cases and controls appeared in non-obese subjects (P = 0.27), PTX3 was significantly higher among the obese case subjects than the obese controls (P = 0.01). In multivariate models, there was a suggestion that PTX3 was a stronger predictor of risk among obese subjects. Compared to subjects in the lowest quartile of PTX3, obese subjects in the highest quartile had an odds ratio of 1.79 (95%CI: 0.63-5.13), while the odds ratio for the same comparison among the non-obese subjects was 1.09 (0.65-1.84).

All references cited herein are fully incorporated by reference. Having now fully described the invention, it will be understood by those of skill in the art that the invention may be practiced within a wide and equivalent range of conditions, parameters and the like, without affecting the spirit or scope of the invention or any embodiment thereof.