Numerous epidemiologic studies have evaluated several inflammatory markers, including C-reactive protein, various cytokines, adhesion molecules, and white blood cell (WBC) count for their clinical usefulness in predicting risk of cardiovascular disease. (Zhang, “Association between Myeloperoxidase levels and risk of coronary artery disease” JAMA, 286(17), 2001).

Carotid intima-media thickness (IMT)

IMT increase predicts the risk of cardiovascular evetns, with relatively stronger prognostic power for crebral as compared with coronary vascular events. Inc reased IMT is considered to represetn a manifestation of subclinical atherosclerosis and has been incuded in the lsit of organ damage conditions in European hypertension guidlines. The lack of invasiveness and reepatability makes IMT measurement an attractive biomarker, potentially useful as a therapeutic target in subjects at increased cardiovascular risk. (J American Colelge of Cardiology, 56(24) 2010)

Above average C-reactive prtoein with statin therapy predicts failure of IMT regression in those with optimal LDL cholesterol (<100 mg/dl) but not if LDL cholesterol is <70 mg/dL (ultra-low levels). (Kent, American J. of Cardiology, 92(15), 2003). 

Cholesterol

A lipid test (chloresterol) is often performed to evaluate risks for heart disease. Cholesterol is an important normal body constituent, used in the structure of cell membranes, syntehsis of bile acids and synthesis of steroid hormones. Since cholesterol is water insoluble, most serum cholesterol is carried by lipoprotines (chylomicrons, VLDLC, LDLC, and HDLC). Excess cholesterol in the blood has been correlated with cardiovascular events. LDL is soemtimes referred to as “bad” cholestero, because elevated levels of LDL correlate most directly with cardiovascular events sucha s coronary heart disease. HDL is sometimes referred to as “good” cholesterol since high levels of HDL are correlated with a reduced risk of cardiovascular events sucha s coronary heart disease. The term chloesterol means “total” chloesterol, i.e., VLDLC+LDLC+HDLC. (US 14/886626, published as US 2016/026146). 

Preferably, cholesterol levels are measrued after fasting. The cholesterol measurement is typically reported in milligrams per deciliter (mg/dL). Typically, the higher the total cholesterol, the more at risk a human subject is for a cardiovascular event. A value of total cholesterol of less than 200 mg/dL is a “desirable” level. Levels over 240 mg/dL may put one at almost twice the risk of a cardiovascular vent such as coronary heart disease. (US 14/886626, published as US 2016/026146).

Since cholesterol is water insoluble, most serum cholesterol is carried by lipoproteins (chylomicrons, VLDLC, LDLC and HDLC). The term cholesterol measn “total” cholesterol, i.e., VLDLC + LDLC + HDLC. 

C reactive protein: 

CRP even in the absence of hyperlipidemia, is associated with an increased risk of coronary events. In terms of clinical application, CRP seems to be a stronger predictor of cardiovascular events than LDL cholesterol, and it adds prognostic information at all levels of calculated Framingham Risk and at all levels of the metabolic syndrome, Ridkey, Circulation, 107, 2003, 363-369. The rates of coronary events increase significantly with increases in levels of CRP (Ridker, N Engl J Med, 344(26), 2001).

The usefulness of CRP as a clinical biomarker of as a predicttor of cardiovascular risk is important because because almost half of all myocardial infarctions and strokes occur in persons without elevated levels of low-density lipoprotein cholesteroal (Clin. Cardiol. 26pp. III-39-III-44 (2003).

Herrmann (Cellular and Molecular Biology 50(8) 895-901, 2004) discloses that homocysteine and high sensitivity C-reactive protein (hs-CRP) are were independently associated with the risk of myocardial infarction, stroke and cardiovascular death. 

A CRP level of ≥1 is considered an elevated level. CRP levels of <1m 1 ti 3m abd >3 ng.K cirresoibd ti kiw0 niderate abd gugg risk groups for future cardiovascular vents. (Ridker, Circulation, 2003, 107: 363-369).

Ridker (US7,030,152 and US7,964,614) diclsoes methods for characterizing an individual’s risk profile of developing a future CVD disorder as well as evaluating the likelihood that an individual will benefit from treatment with an agent such as a statin for reducing the risk of future CVD by obtaining a level of a marker of systemic inflammation such as CRP.

Ridker (US11244716) disclose that CRP levels can also be used to determine whether or not statin treament should be continued or changed.

High-Density Lipoprotein (HDLs) (“good” cholesterol)

High-density lipoprotein (HDL): HDL cholesterol concentration are inversely associated with occurrence of acrdiovascular events. Although measurement of HDL cholesterol concentration is useful as part of initial cardiovascular risk assessment, HDL cholesterol concentrations are not predictive of reisdual vascular risk among patients treated with statins who attain very low concentration of LDL (Ridker, Lancel 2010, 376, 333-339).

Low-density lipoprotein (LDL): (“Bad” cholesterol) 

LDLC levels are predictors of risk of cardiovascular event. Typically, the igher the LDLC, the mosre at risk . Levels of LDLC over 160 mg/dL may put a subject at higher risks as compared to levels less than 160 mg/dL. Levels of LDLC over 130 mg/dL with one or more risk factors for a future cardiovascular event may put a subject at higher risks of a cardiovascular event as compared to someone with a level less than 130 mg/dL. A level of LDLC less than 100 mg/dL is desirable in one with a prior cariovascualr event and is on therapy to reduce to risk of a future cardiovascualr vent. A elvel of LDLC less than 70 mg/dL is even more desirable. (US 14/886626, published as US 2016/026146).

An optimal low-density lipoprotein (LDL) cholesterol is less than 100 mg/dl for patients at high cardiovascular risk (Kent, “usefulness of lowering low-density lipoprotein cholesterol to <70 mg/dl and usefulness of C-reactive Protein in Pateint selection” American J. of Cardiology, 92(15), 2003). 

However, almost half of all myocardial infarctions and strokes occur in persons without elevated levels of LDL, showing that other factors than hyperlipidemia contribut to the development of atherosclerosis (Ridker, Clin. Cardiol. 26(III) 2003. 

CRP and LDLC:  

Kent, (“usefulness of lowering low-density lipoprotein cholesterol to <70 mg/dl and usefulness of C-reactive Protein in Pateint selection” American J. of Cardiology, 92(15), 2003) discloses that the likehood of carotid intima-media thickness (CIMT) was related to on therapy CRP value. Among patients with an LDL cholesteraol between 70 and 100 mg/dl, those with a CRP below the median value (0.17 mg/dl) had a more than threefold greater likelihood of CIMT regression than those with a CRP above the median. In contrast the likelihood of CIMT regression if LDL cholesterol was ultra low (<70 mg/dl) or above optimal (>100 mg/dl) was unrelated to CRP levels. 

Ridker, US 2007-0292960 discloses a method of obaining the level of CRP and LCLC from a patient in the course of treatment with a stain  and based on the level of CRP compraed to a control, continuing therapy with the stain when the level of CRP is below the control and changing therapy when the CRP level is above the control, when the level of LDLC is below 70 mg/dL or is above 100 mg/DL.

Because CRP and LDL chloresterol measurements tend to identify different high risk groups, screening for both markers provides a better prognostic information than screening for either alone for cardiovascular events. (Ridker “comparison of C-reactive protein and low-density lipoprotein cholesterol levels in the prediction of first cardiovascular events” N Engl J Med 2002: 347: 1557-1565).

Myeloperoxidase (MPO): 

MPO is an enzyme secreted from activated neutrophils, monocytes and certain tissue macrophages which may also be involved in the development of coronary artery disease (CAD). MPO synthesis occurs during myeloid differentiation in bone marrow and is completed within granulocytes prior to their entry into the circulation. The enzyme is stored within primary granules of neutrophils and monocytes and is not released until leukocyte activation and degranulation. MPO forms free radicals and diffusible oxidants with antimimicrobial activity. However, MPO also promotes oxidative damage of host tissues at sites of inflammation, including atherosclerotic lesions. (Zhang, “Association between Myeloperoxidase levels and risk of coronary artery disease” JAMA, 286(17), 2001). Stimulated phagocytes secrete this enzyme at inflammatory sites, where it generates a powerful reactive oxygen species, hypochlorous acid (HOCl), at physiological chloride concentrations (Sugiyama (American J. Pathology, 158(3), 2001). Hypochlorous acid/hypochlorite (HOCL/OCl-) is formed from H2O2 and chloride ions by MPO. HOCL reacts with a wide range of biological substrates and is a powerful oxidant. The oxidative modification of low density lipoprotein (LDL) is thought to be an important step in the formation of an atherosclerotic lesion (Malle, Eur. J. Biochm. 2, 4495-4503 (2000).  

Daugherty J Clin Invest, 94, 1994, 437-444 also identified MPO in human vasclular lesions and proposed that MPO may contribute to atherogenesis by catalyzing oxidative reactions in the vasclular wall.

Terletskaya (Vrach Delo, 1989, 3: 13-14 (in Russian)) note that during the necrosis of myocardial muscle in MI pateints, MPO apparently gets secreted into the intercellular medium and then to the blood, which causes the reduction of the MPO activity of nuetrophilic granuloctyes. Hazen (US11/753799) discloses a method for administering a therapeutic agent targeted to cardiovascular disease to a patient based on elevated levels of MPO by determining levels of MPO in blood or serum or plasma. eloperoxidase (MPO) is a naturall-occurring heme protein associated with some types of white loo cells. It functions as an oxidant converting inert substrates to reactive oxygen species toxic to pathogens, to aid in phagocytosis. Atherosclerosis (the major cause of coronary artery disease) was known to be a chronic inflammatory disorder and high blood levels of other metabolites had been correlated to CVC. While MPO had been found to be present at elevated levels in atherosclerotic lesions, it had not been shown that MPO was present at elevated levels in blood samples form pateints with atherosclerotic CVC. The patents dislcosed several methods of measuring a patient’s blood MPO level. 

Cleveland Clinic held US Patent Nos: 7,223,552, 9,575,065, 9,581,597 which were held invalid under 35 USC 101 as directed to an ineligible natural law. (Cclevaland Clinic v. True Health Diagnostics LLC, 859 F.3d 1352 (Fed. Cir. 2017) and Cleveland Clinic v. True Health Diagnostics, US Fed. Cir. 2019). At the time of the invention, cardiovascular disease (CVD) was understood to be multifactorial and physicians were developing predictive algorithms based on genetic, environmental and lifestyle factors. However, these factors alone did not fully predict an individual’s risk of developing CVD, in particular, a large number of cardiovascular disorders occured in individuals with apparently low to moderate risk profiles. The Court held that the patents starts and ends with observation of naturally occurring phenomena as in Ariosa Diagnostics v. Sequenom, 788 F.3d 1371 (Fed. Cir. 2015) and becasue the patents did not purport to have invented any of the biological technqiues used to detect MPO or the statistical methods used to compare a patient’s MPO levels to the control goup, the claims recited no further inventive concept sufficient to transform the nature of the claims into a patent-eligible application of the natural law. (the claim in Ariosa’s US Patent 6,258,540 was held ineligible because it was directed to the disovery that paternally inherited cffDNA exists in maternal blood plasma and the amplificaiton and detection techniques were concededly known in the art). 

Oxidation Products

Oxidation may play a role in the initiation and progression of atherosclerosis. Although it is not known how lipoprotein oxidation is initated, diverse oxidants produced by endothelial and inflammatory cells may oxidze lipoproteins. Nitric oxide (NO) and super-oxide radical (O2) are rleeased by these cells and can react with each other to form the strong oxidant and nitrating species peroxynitrite (ONOO-) which is capable of oxidizing lipoproteins. The peroxynitrite reaction with proteins yield nitrotyrosine athat is a stable product formed by the addition of a nitro group to the ortho position of tyrosine. Nitrotyrosine has been determeined in many pathologies and is a useful marker for peroxynitrite detection in human atherosclerosis (Moriel, Biochemical & Biophysical Research Communicatios 232, 332-335 (1997). 

Nitroytyrosine: Hazen (US11/313/012) teaches detecting protein bound nitrotyrosine by an anti-nitrotyrosine antibody on a serum or plasma sample, comparing the levels to a control and adminsitering a lipid lowering agent such as a statin if the level is greater than the control.