Concentration of Lipoprotein (a) Cholesterol

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority under 35 U.S.C. §111(a) as a continuation of U.S. Provisional Patent Application Serial No. 63/248,837, entitled “Quantification of Lp(a) vs. non-Lp(a) apoB concentration: development of a novel validated equation,” filed on September 27, 2021, which is hereby incorporated by reference in its entirety for all purposes.

BACKGROUND

[0002] The application generally relates to systems and methods to determine the concentration of cholesterol in lipoprotein (a).

[0003] Cholesterol travels through the blood on lipoproteins. Lipoproteins include the commonly known high-density lipoprotein cholesterol (HDL-C), which is referred to as good cholesterol, and low-density lipoprotein cholesterol (LDL-C), which is referred to as bad cholesterol.

[0004] Lipoproteins also include lipoprotein (a). Lipoprotein (a) consists of an LDL-like particle and apolipoprotein(a), which is covalently bound to apolipoprotein B. Lipoprotein (a) also includes a particle of LDL cholesterol. The cholesterol portion of lipoprotein (a) is referred to as Lp(a)-C. When patients have high levels of lipoprotein (a), the levels of lipoprotein (a) can result in the patient having high LDL cholesterol results. [0005] High lipoprotein (a) levels increase the likelihood that a person will have a heart attack or stroke. Lipoprotein (a) may also cause increased clotting, which may lead to rapidly formed blockages in blood vessels. Lipoprotein (a) promotes inflammation, which increases the likelihood that plaques will rupture. Lastly, high lipoprotein (a) may lead to the narrowing of the aortic valve because chronic inflammation leads to calcium build up on the valve, which may result in reduced blood flow if the valve is unable to open completely.

[0006] Studies indicate that people born with elevated lipoprotein (a) may have a two-fold to four-fold increased risk of heart attacks and other serious events compared to people with low lipoprotein (a) levels.

SUMMARY

[0007] In some embodiments a method to determine a concentration of cholesterol in lipoprotein (a) includes receiving a fluid sample from a subject. The method further includes measuring a lipoprotein (a) value in nmol/L from the fluid sample. The method further includes determining the concentration of lipoprotein (a) apolipoprotein B by multiplying the lipoprotein (a) value by 0.0513. The method further includes determining the concentration of cholesterol in lipoprotein (a) by multiplying the concentration of lipoprotein (a) apolipoprotein B by 1.497.

[0008] In some embodiments, the method further includes determining a concentration of non-lipoprotein low density lipoprotein (LDL) cholesterol by subtracting the concentration of cholesterol in lipoprotein (a) from a concentration of LDL cholesterol. In some embodiments, the method further includes determining whether the concentration of cholesterol in lipoprotein (a) is above a predetermined threshold and responsive to the concentration of cholesterol in lipoprotein (a) exceeding the predetermined threshold, administering or prescribing a therapy to treat a cardiovascular condition. In some embodiments, the therapy is a proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor. In some embodiments, the therapy is Repatha. In some embodiments, the administering reduces LDL levels. In some embodiments, the method includes determining a risk of coronary heart disease based on determining the concentration of cholesterol in lipoprotein (a). In some embodiments, the method includes determining a risk of an ischemic stroke event based on determining the concentration of cholesterol in lipoprotein (a).

[0009] A device determines a concentration of cholesterol in lipoprotein (a) includes a processor and a memory coupled to the processor, with instructions stored thereon that, when executed by the processor, cause the processor to perform operations. The operations include receiving a fluid sample from a subject, measuring a lipoprotein (a) value in nmol/L from the fluid sample, determining the concentration of lipoprotein (a) apolipoprotein B by multiplying the lipoprotein (a) value by 0.0513, and determining the concentration of cholesterol in lipoprotein (a) by multiplying the concentration of lipoprotein (a) apolipoprotein B by 1.497.

[0010] In some embodiments, the operations further include determining a concentration of non-lipoprotein low density lipoprotein (LDL) cholesterol by subtracting the concentration of cholesterol in lipoprotein (a) from a concentration of LDL cholesterol. In some embodiments, the operations further include determining whether the concentration of cholesterol in lipoprotein (a) is above a predetermined threshold and responsive to the concentration of cholesterol in lipoprotein (a) exceeding the predetermined threshold, administering or prescribing a therapy to treat a cardiovascular condition. In some embodiments, the therapy is a proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor. In some embodiments, the therapy is Repatha.

[0011] In some embodiments, a method to treat a subject in need thereof includes: receiving a fluid sample from the subject, measuring a lipoprotein (a) value in nmol/L from the fluid sample, determining a concentration of lipoprotein (a) apolipoprotein B by multiplying the lipoprotein (a) value by 0.0513, determining the concentration of cholesterol in lipoprotein (a) by multiplying the concentration of lipoprotein (a) apolipoprotein B by 1.497, and responsive to the concentration of cholesterol in lipoprotein (a) exceeding a predetermined threshold, administering or prescribing a therapy to treat a cardiovascular condition.

[0012] In some embodiments, the fluid sample is selected from the group consisting essentially of: blood plasma or serum. In some embodiments, the administering reduces LDL levels. In some embodiments, the administering reduces lipoprotein (a) levels. In some embodiments, the therapy includes statins. In some embodiments, the therapy is a proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor. In some embodiments, the therapy is Repatha.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The disclosure is illustrated by way of example, and not by way of limitation in the figures of the accompanying drawings in which like reference numerals are used to refer to similar elements.

[0014] Figure 1 illustrates a block diagram of an example network environment to determine the concentration of cholesterol in lipoprotein (a), according to some embodiments described herein.

[0015] Figure 2 is a graph that illustrates a comparison of a non-lipoprotein (a) cholesterol concentration calculated from a commonly-used equation and a non-lipoprotein (a) cholesterol concentration calculated from a molar concentration equation, according to some embodiments described herein. [0016] Figure 3 is a graph that illustrates a comparison of a non-lipoprotein (a) cholesterol concentration calculated from a commonly-used equation and non-lipoprotein (a) cholesterol concentration calculated from a molar concentration equation based on sex, according to some embodiments described herein.

[0017] Figure 4 is a graph that illustrates a comparison of a non-lipoprotein (a) cholesterol concentration calculated from a commonly-used equation and non-lipoprotein (a) cholesterol concentration calculated from a molar concentration equation based on ethnicity, according to some embodiments described herein.

[0018] Figure 5 is a flowchart of an example method to determine a concentration of cholesterol in lipoprotein (a), according to some embodiments.

DETAILED DESCRIPTION

[0019] Lipoprotein (a) consists of a low-density lipoprotein (LDL) particle with a molecule of apolipoprotein B linked by a disulfide bond to a glycoprotein termed apolipoprotein (a). High lipoprotein (a) levels have been associated with an increased risk for coronary heart disease and ischemic stroke events among adults independent of a history of cardiovascular disease. In some embodiments, the risk of coronary heart disease or the risk of an ischemic stroke event may be determined based on the level of lipoprotein (a). For example, patients with lipoprotein (a) levels greater than 50 milligram/deciliter (mg/dL) may be considered at higher stroke recurrence risk than those with lipoprotein (a) levels less than 50 mg/dL. In some examples, a normal value is considered below 30 mg/dL.

[0020] A patient may be prescribed a therapy or a doctor may administer a therapy to treat a cardiovascular condition if the patient has an apolipoprotein (a) level that exceeds a predetermined threshold. For example, a high apolipoprotein (a) may be considered greater than 50 mg/dL. Other values may apply, for example, depending on a patients age, ethnicity, family history, etc.

[0021] In some embodiments, the therapy may include a proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor or statins. For example, the PCSK9 inhibitor may include the generic drug evolocumab or the brand-name drug Repatha. In another example, the statin may include the brand-name drug Lipitor.

[0022] In some embodiments, the administering may include reducing LDL levels. In some embodiments, the administering may include reducing lipoprotein (a) levels. For example, a doctor may administer a drug, such as a PCSK9 inhibitor or statins to a patient.

[0023] Since the measurement of LDL-cholesterol also comprises the cholesterol bound to lipoprotein (a), studies evaluating the associations of lipoprotein (a) and atherosclerotic cardiovascular risk have estimated the cholesterol content in lipoprotein (a) from the measurement of its mass. Specifically, the literature typically estimates that 30-45% of lipoprotein (a) is composed of cholesterol. The estimation of lipoprotein (a) cholesterol is used to calculate lipoprotein (a)-corrected LDL cholesterol by subtracting lipoprotein (a) cholesterol from LDL cholesterol using different equations or occasionally “direct” LDL-cholesterol measurements. Because the concentration of lipoprotein (a) cholesterol is incorrectly estimated, in some instances calculating the non-lipoprotein (a) cholesterol results in a negative value.

[0024] Despite the lack of knowledge of the cholesterol content in individual lipoprotein (a) particles, the amount of lipoprotein (a) cholesterol as reported in the literature varies from 30% to 45% of the total lipoprotein (a) mass without any sound data to support these estimates. Therefore, previous attempts to determine the amount of lipoprotein (a) cholesterol involves multiple assumptions that compound the error, thus resulting in implausible estimates, particularly in individuals with low LDL-cholesterol and high lipoprotein (a) values.

[0025] In some embodiments, the concentration of lipoprotein (a) cholesterol is accurately calculated using the Rosenson-Marcovina equation (Equation 3 below). The Rosenson- Marcovina equation advantageously replaces the older techniques that assumed that the concentration of lipoprotein (a) cholesterol was 30%-45% of the total mass of lipoprotein (a).

[0026] In some embodiments, equation 1 assumes that there is one mole of apolipoprotein A and one mole of apolipoprotein B in lipoprotein (a) particles. Because lipoprotein (a) particles may vary in size, measuring the total mass of apolipoprotein (a) in the blood in milligrams per deciliter (mg/dL) is not as accurate as measuring lipoprotein (a) in nanomoles per liter (nmol/L) because nmol/L reflects the number of particles in a patient’s blood instead of the mass.

[0027] Equation 1 assumes that by measuring the lipoprotein (a) in nanomoles per liter (nmol/L), the concentration of apolipoprotein B in milligrams per deciliter (mg/dL) is calculated using the following equation:

[0028] Lp(a) nmol/L x 0.0513 = Lp(a) apoB mg/dL (Eq. 1)

[0029] where Lp(a) represents lipoprotein a and Lp(a) apoB represents the concentration of apolipoprotein B in lipoprotein (a).

[0030] Although different molecular weights for apolipoprotein B have been reported, the molecular weight of 513,000 kilodalton (kDa) chosen in equation 1 is obtained by amino acid analysis.

[0031] The following equation reflects the overall average ratio of LDL cholesterol to apolipoprotein B : [0032] LDL-C/apoB = 1.497 (Eq. 2)

[0033] where LDL-C is the amount of cholesterol in LDL and apoB is the amount of apolipoprotein B in LDL-C.

[0034] The following equation determines the concentration of cholesterol in lipoprotein (a) particles based on the amount of apolipoprotein B:

[0035] Lp(a)-C mg/dL = Lp(a) apoB mg/dL x 1.497 (Eq. 3)

[0036] where Lp(a)-C is the concentration of cholesterol in lipoprotein (a) and Lp(a) apoB is the concentration of apolipoprotein B in lipoprotein (a).

[0037] As a result of the above equations, the concentration of cholesterol in lipoprotein (a) can be calculated based on using the cholesterol to apolipoprotein B ratio when the lipoprotein (a) values are measured in nmol/L.

[0038] Network Environment 100

[0039] Figure 1 illustrates a block diagram of an example environment 100 to determine a concentration of cholesterol in lipoprotein (a). In some embodiments, the environment 100 includes a cholesterol device 101, a user device 115a, a user device 115n, and a network 105. Users 125a, 125n may be associated with respective user devices 115a, 115n. In some embodiments, the environment 100 may include servers or devices not shown in Figure 1. In Figure 1 and the remaining figures, a letter after a reference number, e.g., “115a,” represents a reference to the element having that particular reference number. A reference number in the text without a following letter, e.g., “115,” represents a general reference to embodiments of the element bearing that reference number.

[0040] The cholesterol device 101 may include a processor, a memory, and network communication hardware. In some embodiments, the cholesterol device 101 is a hardware server. The cholesterol device 101 is communicatively coupled to the network 105 via signal line 102. Signal line 102 may be a wired connection, such as Ethernet, coaxial cable, fiber-optic cable, etc., or a wireless connection, such as Wi-Fi®, Bluetooth®, or other wireless technology. In some embodiments, the cholesterol device 101 sends and receives data to and from one or more of the user devices 115a, 115n via the network 105. The cholesterol device 101 may include a cholesterol application 103a and a database 199.

[0041] The cholesterol application 103a may include code and routines operable to receive a fluid sample from a subject, measure a lipoprotein (a) value in nmol/L from the fluid sample, determine a concentration of lipoprotein (a) apolipoprotein B by multiplying the lipoprotein (a) value by 0.0513, and determine the concentration of cholesterol in lipoprotein (a) by multiplying the concentration of lipoprotein (a) apolipoprotein B by 1.497.

[0042] In some embodiments, the media application 103a may be implemented using hardware including a central processing unit (CPU), a field-programmable gate array (FPGA), an application-specific integrated circuit (ASIC), any other type of processor, or a combination thereof. In some embodiments, the media application 103a may be implemented using a combination of hardware and software.

[0043] The database 199 may store medical information about patients. For example, the database 199 may store electronic health records for patients that include information about their cholesterol, such as levels of HDL, LDL, dates measured, etc.

[0044] The user device 115 may be a computing device that includes a memory and a hardware processor. For example, the user device 115 may include a desktop computer, a mobile device, a tablet computer, a mobile telephone, a wearable device, or another electronic device capable of accessing a network 105.

[0045] In the illustrated implementation, user device 115a is coupled to the network 105 via signal line 108 and user device 115n is coupled to the network 105 via signal line 110. The media application 103 may be stored as media application 103b on the user device 115a or media application 103c on the user device 115n. Signal lines 108 and 110 may be wired connections, such as Ethernet, coaxial cable, fiber-optic cable, etc., or wireless connections, such as Wi-Fi®, Bluetooth®, or other wireless technology. User devices 115a, 115n are accessed by users 125a, 125n, respectively. The user devices 115a, 115n in Figure 1 are used by way of example. While Figure 1 illustrates two user devices, 115a and 115n, the disclosure applies to a system architecture having one or more user devices 115.

[0046] In some embodiments, the user device 115a includes a cholesterol application 103b that is operable to receive information about a patient’s cholesterol and display the results on a display. For example, the user 125a may be a doctor that views the results of a cholesterol measurement on the user device 115a.

[0047] Study of Different Methods for Calculating Lipoprotein (a)

[0048] The data for this study was gathered from 239 participants enrolled in a placebo- controlled trial of evolocumab in patients at high cardiovascular risk with well-controlled type 2 diabetes. Evolocumap is a human monoclonal antibody that is classified as a proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor. PCSK9 inhibitors reduce the levels of LDL circulating in the blood. The brand-name for evolocumab is Repatha, which comes as a 140 mg/mL prefilled syringe or autoinjector or as a 420 mg/3.5 mL infusor that is placed on the body.

[0049] The data was used to compare a non-lipoprotein (a) cholesterol concentration calculated from a commonly-used equation and a non-lipoprotein (a) cholesterol concentration calculated based on the Rosenson-Marcovina equation.

[0050] LDL cholesterol was directly measured by beta quantification when the LDL cholesterol was less than 40 mg/dL or triglycerides were greater than or equal to 400 mg/dL. When the LDL cholesterol was greater than or equal to 40 mg/dL, the Sampson equation was used. The Sampson equation is as follows:

[0051] LDL-C = TC/0.948-HDL-C/0.971-(TG/8.56+TGx non-HDL-C/2140 TG ² /16100)- 9.44 (Eq. 4)

[0052] where TC is total cholesterol, HDL-C is high-density lipoprotein cholesterol, and TG is triglycerides.

[0053] The study included determining the following statistics: mean, standard deviation, median, QI, Q3, and minimum and maximum values for the measured parameters. All summaries were performed for all records without considering study visits and treatment groups. Records with non-detectable lipoprotein (a) levels, which was defined as <2.8 nmol/L, were excluded from the analyses.

[0054] Figure 2 is a graph 200 that illustrates a comparison of a non-lipoprotein (a) cholesterol concentration calculated from a commonly-used equation and a non-lipoprotein (a) cholesterol concentration calculated from a molar concentration equation. The x-axis represents the LDL-cholesterol levels and the y-axis represents the Lp(a) corrected LDL-C. The mean ±95% confidence level is illustrated within the bar graph.

[0055] In this cohort, LDL cholesterol levels ranged from 48.0 to 220.1 mg/dL in the placebo group and 12.2 to 346.7 mg/dL in the evolocumab group. Lipoprotein (a) concentrations ranged from 2.9 to 884.2 nmol/L overall. In general, variation in lipoprotein (a)-corrected LDL cholesterol increases when the LDL cholesterol level increases and is consistent in both the common and molar equations. The p-values of a homogeneity test in non-lipoprotein (a) cholesterol between the two methods are less than 0.05 for all LDL cholesterol deciles except the LDL cholesterol less than 10% and greater than or equal to 90% groups.

[0056] Figure 3 is a graph 300 that illustrates a comparison of a non-lipoprotein (a) cholesterol concentration calculated from a commonly-used equation and non-lipoprotein (a) cholesterol concentration calculated from a molar concentration equation based on sex. Figure 4 is a graph 400 that illustrates a comparison of a non-lipoprotein (a) cholesterol concentration calculated from a commonly-used equation and non-lipoprotein (a) cholesterol concentration calculated from a molar concentration equation based on ethnicity.

[0057] Variation in lipoprotein (a)-corrected LDL cholesterol level increases when the LDL cholesterol increases. This was found consistently in each sex and race group as illustrated in Figure 3 and Figure 4, except when the race is categorized as other due to a small sample size and blacks versus whites. While there were not consistently larger non-lipoprotein (a) cholesterol determinations in women as compared to men, the variations of non-lipoprotein (a) cholesterol determinations are in general larger than males in most LDL cholesterol deciles and are consistent using both methods. Using the common equation, 4.6% of the measurements with LDL cholesterol levels equal to or less than 12.2 mg/dL and 2.3% with LDL cholesterol levels between greater than 12.2 and less than 42 mg/dL had negative values when calculating the concentrations of lipoprotein (a) cholesterol.

[0058] In clinical practice and clinical trials of LDL lowering therapy, including PCSK9 inhibitors, the determine of lipoprotein (a) cholesterol was performed by assuming lipoprotein (a) cholesterol as 30% of the total lipoprotein (a) mass may result in negative values for the corrected LDL cholesterol. Thus, this determine introduces errors that preclude complete data analysis and accurate interpretation of lipoprotein (a) versus non-lipoprotein LDL cholesterol used in clinical trials of lipid therapies that also lower lipoprotein (a) including PCSK9 inhibitors, or selective therapies for lipoprotein (a) lowering. In addition, these negative numbers provide misleading data for clinicians, clinical investigators, and patients/ study participants.

[0059] In summary, quantification of lipoprotein (a) and non-lipoprotein (a) particle concentrations using the Rosenson-Marcovina equation provides a more reliable determination of the relative atherogenic contribution of lipoprotein (a) and non-lipoprotein (a) cholesterol than that obtained by the commonly used flawed determine of lipoprotein (a) cholesterol.

[0060] Example Method 500

[0061] Figure 5 is a flowchart of an example method 500 to determine a concentration of cholesterol in lipoprotein (a), according to some embodiments. In some embodiments, the steps are performed in whole or in part by a cholesterol application 103 that is stored on the cholesterol device 101 or the user device 115 in Figure 1.

[0062] The method may start at block 502. At block 502, a fluid sample is received from a subject. The fluid sample may be blood plasma, serum, or urine. Block 502 may be followed by block 504.

[0063] At block 504 a lipoprotein (a) value in nmol/L is measured from the fluid sample. Block 504 may be followed by block 506.

[0064] At block 506, a concentration of lipoprotein (a) apolipoprotein B is determined by multiplying the lipoprotein (a) value by 0.0513. Block 506 may be followed by block 508. [0065] At block 508, the concentration of cholesterol in lipoprotein (a) is determined by multiplying the concentration of lipoprotein (a) apolipoprotein B by 1.497. Block 508 may be followed by block 510.

[0066] At block 510, responsive to the concentration of cholesterol in lipoprotein (a) exceeding a predetermined threshold, a therapy is administered or prescribed to treat a cardiovascular disease. For example, a patient is prescribed a PCSK9 inhibitor, such as Repatha, or a statin, such as Lipitor. In another example, a doctor administers a PCSK9 inhibitor or a statin.