WO2020198063A1 - Administration of eicosapentaenoic acid and its derivatives to correct the susceptibility of apob-lipoproteins aggregation - Google Patents

Abstract

Methods, systems and kits for treating selected patients with eicosapentaenoic acid and its derivatives, especially icosapent ethyl, in order to correct the susceptibility of apoB-lipoproteins to aggregation in the presence of secretory sphingomyelinase and thereby achieve effective treatment.

Description

Methods and Systems for Administration of Eicosapentaenoic Acid and its Derivatives to Correct the Susceptibility of apoB-Lipoproteins to Aggregation in the Presence of

Mammalian Secretory Sphingomyelinase

Cross Reference to Related Applications

This application claims the benefit of US provisional application serial number 62/824,628 filed March 27, 2019. The entire disclosure of the foregoing provisional application is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The field of this invention is the exogenous administration of eicosapentaenoic acid (EPA) and its derivatives in order to correct the susceptibility of apoB-lipoproteins, including LDL, to aggregation in the presence of mammalian secretory

sphingomyelinase.

BACKGROUND OF THE INVENTION

Oral administration of a highly purified preparation of eicosapentaenoic acid (EPA; 20:5; n-3, where n-3 is also known as omega-3) ethyl ester (a/k/a“icosapent ethyl”) was recently reported to decrease cardiovascular events by 25% in a high-risk population on statins (REDUCE-IT trial). (Bhatt et al. 2019). The authors addressed the possibilities that various parameters could explain, account for, or predict this degree of benefit. The parameters included reductions in plasma levels of apoB, triglyceride, C- reactive protein, low-density lipoprotein (LDL) cholesterol, and bilayer membrane- stabilizing effects. (See also Kastelein and Stroes, 2019). Ultimately, the authors concluded that the mechanisms responsible for the benefit of icosapent ethyl were still not known. Significantly, the mechanism that underlies the present inventions was not even considered. in order to optimize the use of icosapentyl ethyl and similar drugs, it would be advantageous to select, for drug treatment, those patients who will benefit most from administration of the drug and those who will benefit least or not at all. EPA has side- effects. The authors of the REDUCE-IT report noted that a larger percentage of patients in the icosapent ethyl group than in the placebo group were hospitalized for atrial fibrillation or flutter and that serious bleeding events occurred in a higher percentage of the patients in the icosapent ethyl group than in the placebo group. As a result, an optimization of the ratio of benefit to harm is needed. It would therefore be highly advantageous to identify in advance of treatment those patients who would particularly benefit from icosapent ethyl and related drugs. Similarly, it would be valuable to monitor the effectiveness of icosapent ethyl or similar drugs in order to be able do decide whether to continue the therapy if it is effective or to change the dose or the drug if it is ineffective.

BRIEF SUMMARY OF THE INVENTIONS

The inventions are based on the concept that patients with the highest susceptibility to mammalian acid SMase-induced aggregation of cholesterol-rich apoB- containing atherogenic lipoproteins, such as LDL, will be highly responsive to icosapent ethyl, and similarly structured drugs, that protect against atherosclerotic cardiovascular events. Further, monitoring the susceptibility to mammalian acid SMase-induced aggregation of cholesterol-rich apoB-containing atherogenic lipoproteins, such as LDL, during treatment of a patient with icosapent ethyl, or similarly structured drugs, and looking for a decrease in said susceptibility, is useful in guiding therapy. The present inventions are methods, systems, and kits reflecting that concept. DETAILED DESCRIPTION

Terminology

“The invention” or“the inventions” refers to those of the applicant/inventor for this patent application.

An“apoB-lipoprotein aggregation susceptibility assessment system” is a system (or test, or assay) that determines“apoB-lipoprotein aggregation susceptibility” - meaning how prone a person’s apoB-lipoprotein particles (for example, the person’s LDL) are to aggregation in the presence of a mammalian acid sphingomyelinase in that system. Such systems are described in more detail below.

The term“derivative of EPA” as used herein refers to molecules with a close structural relationship to EPA regardless of whether they were actually derived from EPA. They include for example molecules part of whose structure includes, as a carboxylic acid component, a fatty acid component the same as that of EPA. They also include esterified forms of EPA (a/k/a esterified forms of the fatty acid component of EPA), esterified forms of EPA that are triacylglycerols (also known as triglycerides), ethyl esters of EPA, and ethyl esters of the fatty acid component of EPA. They further include EPA or EPA esters in a mixture with docosahexaenoic acid (DHA, 22:6, n-3). EPA and its derivatives purified from natural sources and/or chemically synthesized are considered to be part of the inventions. Esterified forms of EPA in phosphoglycerides are considered to be included in the term“esterified forms of EPA” - they can be absorbed after oral administration and have advantages for administration parenterally, such as by intravenous or subcutaneous injection. However, the cholesterol content of cholesterol incorporated into an ester with EPA (the cholesteryl ester of EPA) makes it undesirable as an orally or parenterally administered agent and so the cholesteryl ester of EPA is not considered to be a useful derivative for the inventions described herein.

The term“EPA or one of its derivatives” contemplates that a mixture thereof - or a mixture of derivatives is considered to be included. An example is Epanova^®, which is a mixture of EPA and the omega-3 fatty acid, DHA.

A human is considered herein to be an“animal. The terms“patient”,“person" and“subject” may be used interchangeably herein.

“ApoB” refers to apolipoprotein-B, a term that comprises both the full-length form, apoB-ioo, and the naturally truncated form, apoB48.

The prefix“apo-” refers to a protein component of a lipoprotein, e.g.,

apolipoproteins that can be isolated after the lipid of the lipoprotein has been removed.

An apoB-lipoprotein particle is one that contains, as one of its components, apoB.

“LDL” and“LDL particles” both refer to low-density lipoprotein particles.

“SMase” refers to sphingomylenase. As used herein it is a general abbreviation for all sphingomyelinases. The main sphingomyelinase in the arterial wall involved in atherosclerotic plaque development is the secretory SMase (“S-SMase”), which is a product of the mammalian acid sphingomyelinase gene. (Schissel et al. 1996, Schissel et al 1998).

As used herein,“human recombinant sphingomyelinase (hrSMase)” and“human recombinant acid sphingomyelinase” (also abbreviated as“hrSMase”) refer to the protein product of the human acid sphingomyelinase gene made in an artificial expression system. (Wasserstein et al. 2018).

As used herein,“mammalian acid sphingomyelinase (mASMase)” refers to the protein product of a mammalian acid sphingomyelinase gene, whether made in vivo, by mammalian cells in vitro from their own acid sphingomyelinase gene, or made through the use of an artificial expression system. Examples useful in the current inventions are discussed below. Thus, S-SMase and hrSMase are examples of mASMases.

“VLDL” refers to very-low-density lipoprotein particles.

“IDL” refers to intermediate-density lipoprotein particles.

“Lp(a)” refers to lipoprotein(a), a form of LDL that includes the apolipoprotein(a).

“C-TRL” refers collectively to cholesterol- and triglyceride-rich apoB-containing lipoproteins, a group that comprises, in particular, cholesterol- and triglyceride-rich apoB-containing remnant lipoproteins, chylomicron remnants, IDL, triglyceride-rich VLDL remnants, and small VLDL particularly when under 70 nm in diameter.

“TRL” refers to triglyceride-rich lipoproteins, a group that comprises triglyceride- rich apoB-containing remnant lipoproteins.

“b-VLDL” (i.e., beta-VLDL), refers specifically to a type of cholesterol-rich remnant lipoprotein particle seen in type III dyslipoproteinemia and in apoE knock-out mice.

“sVLDL,”“small VLDL”, and“sVLDL particles” refers to small very-low-density lipoprotein particles.

The term“atherogenic lipoprotein particle” as used herein refers to atherogenic apolipoprotein particles that comprise apolipoprotein B. This group comprises all apoB- lipoproteins under about 70 nm in diameter when in plasma, before entering the arterial wall and aggregating there. (Boren J and Williams KJ., 2016).

“TG-rich apoB-lipoproteins” as used herein refers to atherogenic TG-rich apoB- lipoproteins.

“Plurality” means more than one.

ApoB-lipoprotein aggregation susceptibility assessment systems

As noted above, such a system (or test, or assay) is one that determines how prone a person’s apoB-lipoprotein particles (for example, the person’s LDL) are to aggregate in the presence of a mammalian acid sphingomyelinase in that system. The time allowed for aggregation to occur in the system, the initial starting concentration of such particles, what constitutes a result reflecting aggregation, and other details of how the system are used, are preferably kept consistent among persons tested in order to be able to use, with a patient at different times and also between patients, data previously acquired with the system. As an example, aggregation of purified LDL ex vivo in the presence of human recombinant acid SMase (hrSMase), a type of mammalian acid sphingomyelinase, can be assessed by dynamic light scattering, to assess average aggregate size, at regular time intervals, such as every hour, or else in real time, and typical measures that have discriminatory power between individuals include the inflection points in the curves of aggregate size vs. incubation time, as well as LDL aggregate size at the 2-h time point (which correlates tightly and significantly with the inflection point). (Ruuth et al. 2018) In addition to the average size of the aggregates, the size distribution of aggregates, including what percentage of the initial apoB-lipoproteins participate in aggregation (versus those remaining as monomeric particles), are also informative measures of the extent of aggregation. As other examples, LDL aggregation and/or retention in arterial segments ex vivo or in vivo can be used.

Mammalian acid sphingomyelinase (mASMase)

As noted above, that term refers to the protein product of a mammalian acid sphingomyelinase gene, whether made in vivo, by mammalian cells in vitro from their own acid sphingomyelinase gene, or through the use of an artificial expression system. Thus, S-SMase and hrSMase are examples of mASMases.

The apoB-lipoprotein aggregation susceptibility assessment systems used to implement the present inventions utilize an mASMase. These enzymes depend on the divalent zinc cation (Zn²⁺). (Schissel et al. 1996; Schissel SL, Keesler GA, et al 1998) Thus, the current invention can be implemented using assessment systems that comprise a Zn²⁺-dependent SMase, preferably either a Zn²⁺-dependent vertebrate SMase or a Zn²⁺-dependent mammalian SMase, and most preferably a hrSMase.

It is understood that SMase from Bacillus cereus (bcSMase) fails to provide discriminatory power between individuals in terms of susceptibility of their LDL to aggregate (Ruuth et al. 2018) and is unsuitable for use in the current invention. Methods of the invention

The invention in one general aspect is a method of treatment with a drug, said drug being EPA or one of its derivatives. This method comprises (1) a patient selection step based at least in part on the patient’s apoB-lipoprotein aggregation susceptibility level and (2) a subsequent treatment step with the drug for a patient whose apoB- lipoprotein aggregation susceptibility level is considered to be predictive of a positive response to the drug.

The criteria used by the person or organization that makes the selection in step (1) may include criteria in addition to apoB-lipoprotein aggregation susceptibility level. Preferably step (1) is done according to documented selection criteria in the

organization where that step is carried out.

What constitutes an apoB-lipoprotein aggregation susceptibility level that is considered to be predictive of a positive response to the drug is determined as described below. Preferably that level is determined by studies with a plurality of patients prior to utilization of the method to select a particular patient for treatment.

The phrase“predictive of a positive response to the drug” minimally means that the probability of a positive response exceeds 10 percent. Rephrased, it means that minimally (1) there is more than a 10 percent probability that the drug will lower the risk of a major cardiovascular event or (2) more than a 10 percent probability that it will result in the patient living longer than would be expected without drug treatment or (3) more than a 10 percent relative reduction in the probability of a major cardiovascular event. Any of those criterion (1) or (2) or (3) can be used.“Major cardiovascular events” that would qualify are those listed below for the REDUCE-IT trial, the JELIS study, the GREACE study, the Jupiter trial, or the American College of Cardiology website. An individual or organization may require a higher benchmark than that the probability of a positive response exceeding 10 percent. It may for example, require that it exceed 15 percent, 20 percent or other percentage greater than 20. Such a choice would be still be an example of the above-noted method of the invention. The risk of a major cardiovascular event in the absence of intensive drug treatment or the expected additional life span in the absence of intensive drug treatment can be determined as described below or based on data in the medical literature. The REDUCE-IT trial and the JELIS study provide examples of such data. Other examples include the GREACE study (Athyros VG et al. 2003), the JUPITER trial (Ridker PM et al. 2008), and those at the American College of Cardiology (ACC) ASCVD Risk

Estimator Plus. The ACC examples can be accessed at their web address: http://tools.acc.orq/ASCVD-Risk-Estimator-Plus/#!/calculate/estimate/ ,

(accessed 22 March 2019)) under“Resources”, then“Clinician Resources”, then“App terms and concepts” and then their definition of ASCVD events, namely:“atherosclerotic cardiovascular disease, defined as coronary death or nonfatal myocardial infarction, or fatal or nonfatal stroke".

Any other cardiovascular event of interest can, if desired by an investigator, also be considered to be a major cardiovascular event for purpose of the present inventions. For example, clinically significant peripheral vascular disease, such as claudication, amputation, or peripheral ischemia, is also included as a major cardiovascular event.

In a second general aspect of the invention, the above-noted method is preceded by a step in which the apoB-lipoprotein aggregation susceptibility levels of a plurality of patients are tested using an apoB-lipoprotein aggregation susceptibility assessment system, and the relationship between apoB-lipoprotein aggregation susceptibility level and the therapeutic success of EPA or one of its derivatives is determined. The accuracy of determining that relationship will increase with the number of persons tested. The accuracy also will increase to the extent the homogeneity (age, gender, etc.) of the persons being considered increases. This step will provide standard benchmarks for aggregation susceptibility levels that indicate high vs intermediate vs low likelihood of therapeutic benefit from EPA administration.

As an illustration of aggregation susceptibility benchmarks that can be used to select patients for EPA therapy, figure 1 in Ruuth et al. 2018 shows aggregation susceptibilities ex vivo of LDL obtained from healthy subjects but in the worst quartile for aggregation (most highly susceptible to aggregation), as well as LDL obtained from coronary heart disease patients who subsequently survived or subsequently died.

In the REDUCE-IT trial, the primary end point was a composite of cardiovascular death, nonfatal myocardial infarction, nonfatal stroke, coronary revascularization, or unstable angina, each of which qualifies as a major cardiovascular event for purposes of the present inventions.

In the earlier JELIS trial, also testing EPA ethyl ester in atherosclerotic cardiovascular disease (ASCVD) events, the primary endpoint was any major coronary event, including sudden cardiac death, fatal and non-fatal myocardial infarction, and other non-fatal events including unstable angina pectoris, angioplasty, stenting, or coronary artery bypass grafting. (Yokoyama et al. 2007) Each of these qualifies as a major cardiovascular event for purposes of the present inventions.

In the GREACE study (Athyros VG et al. 2003) primary endpoints were all-cause mortality, coronary mortality, coronary morbidity (non-fatal myocardial infarction, revascularization, unstable angina, congestive heart failure), and stroke. Each of these qualifies as a major cardiovascular event for purposes of the present inventions.

In the JUPITER study (Ridker PM et al. 2008) primary endpoints were nonfatal myocardial infarction, nonfatal stroke, arterial revascularization, hospitalization for unstable angina, or confirmed death from cardiovascular causes. Each of these qualifies as a major cardiovascular event for purposes of the present inventions.

At the aforementioned American College of Cardiology (ACC) web site, as noted above, their definition of ASCVD events includes atherosclerotic cardiovascular disease, defined as coronary death or nonfatal myocardial infarction, or fatal or nonfatal stroke. Each of these qualifies as a major cardiovascular event for purposes of the present inventions.

For purposes of the present invention, improvements in any of the foregoing can be used as indicators of therapeutic success. It is also understood that icosapent ethyl administration causes side-effects, such as an increase in the percentage of patients hospitalized for atrial fibrillation or flutter, as well as possibly serious bleeding events.(Bhatt DL et al. 2019). As a result, there is a need to maximize benefit over harm from EPA, and the present inventions are directed to that need.

In the REDUCE-IT trial (Bhatt et al. 2019), patients received 2 g of icosapent ethyl twice daily (total daily dose, 4 g).

A related aspect of the invention is a method of modifying the drug dose in a human or other animal during the course of its treatment based on their apoB- lipoprotein aggregation susceptibility levels during the course of treatment said method comprising the steps of:

1) administering a dose (“reference dose”) of the drug to a human or other animals so as to change the susceptibility, in said human or other animal, of

atherogenic particles (LDLs or other apoB-containing particles) to aggregation induced by mASMase, preferably by hrSMase;

2) assessing, for said human or other animal, a result obtained using an apoB- lipoprotein aggregation susceptibility assessment system before and then after said dose, said result selected from the group consisting of less aggregation ex vivo, a change in atherogenic lipoprotein particle composition indicating less susceptibility to aggregation, less retention in an arterial wall in vivo or ex vivo, and a decreased in an assessment of an adverse response in an artery to aggregated LDL or other apoB- lipoproteins (such as macrophage accumulation, activation, or M1 polarization, and/or expression of a protease, protease activity, tissue factor, or atherogenic cytokine); and

3) if the reference dose leads to little or no decrease in said susceptibility, then administering the next drug dose such that said next dose is larger than the reference dose and/or the time interval between said reference dose and said next dose is shorter than the time interval between the reference dose and the dose preceding said reference dose. In a subset of the foregoing method, the method is not applied to a human with dyslipidemia. In another subset of the foregoing method, after reaching a maximal dose of EPA or EPA derivative but with little or no decrease in said apoB- lipoprotein aggregation susceptibility, there is either a discontinuation of the EPA or EPA derivative or a change to a different EPA or EPA derivative, or a change to a different class of ASCVD therapy.

In the foregoing method, the time interval between doses can vary depending on the treatment protocol. Possible time intervals include those in of the order of half a day (the REDUCE-IT trial) up to preferably not more than 30 days, preferably not more than 7 days.

In particular aspects of the methods, it is intended to reduce the mASMase- induced aggregation of atherogenic lipoprotein particles selected from the group consisting of LDL, remnant lipoproteins, cholesterol- and triglyceride-rich remnant lipoproteins (together, referred to C-TRLs), very low-density lipoprotein (VLDL), small VLDL (sVLDL), cholesterol-rich remnant lipoproteins, b-VLDL, VLDL remnants, chylomicron remnants, postprandial remnants, intermediate-density lipoprotein (IDL), lipoprotein(a) [Lp(a>], and triglyceride-rich remnant lipoproteins (TRL).

In further particular embodiments of the methods of the invention, methods are ones in which the EPA or its derivative is administered in order to achieve a positive therapeutic result in which there is inhibition of an atherosclerosis-related phenomena selected from the group consisting of: formation of crystals of unesterified cholesterol, abnormal cholesterol-enrichment of cell membranes, denaturation of apoB, the development of other harmful materials derived from apoB-lipoproteins aggregated in the presence of sphingomyelinase, inflammasome activation (particularly the NLRP3 inflammasome), activation of proatherogenic T-cells, release of harmful cytokines (such as IL1 b and IL6), plaque progression and destabilization, and release of C-reactive protein (“CRP”).

In vivo apoB-lipoprotein aggregation susceptibility assessment systems include, but are not limited to, assays of apoB-lipoprotein accumulation within the arterial wall, apoB-lipoprotein aggregation within the arterial wall, cholesterol crystal formation within the arterial wall, inflammasome activation, inflammasome activation within the arterial wall, T cell activation, T cell activation within the arterial wall, release of harmful cytokines such as active IL1 R and IL6, release of IL2, and levels of the marker CRP. An example of an assay for apoB-lipoprotein accumulation within the arterial wall is administration of labeled lipoproteins then assessment of the accumulation of their label within the arterial wall.

An example of an assay for apoB-lipoprotein aggregation within the arterial wall is administration of doubly labeled lipoproteins such that their aggregation either quenches or enhances the label.

An example of an assay for cholesterol crystal formation within the arterial wall is administration of labeled lipoproteins such that cholesterol nucleation enhances the signal (as in Guarino et al. 2004). An example of an assay for inflammasome activation is the release of related cytokines and downstream products, such as IL1 b, IL6, and CRP.

An example of an assay for inflammasome activation within the arterial wall is inflammasome-specific imaging.

An example of an assay for T-cell activation in vitro is the release of T-cell- specific cytokines, such as IL2. (Ruuth M et al. 2018). An example of an assay for macrophage activation in vitro is the release of MMP7. (Ruuth M et al. 2018).

An example of an assay for T-cell activation within the arterial wall is T-cell- specific imaging.

An example of an assay for release of harmful cytokines (such as IL1 b, IL6 and IL2) is a quantitative assay of their concentrations in plasma or serum or other body fluids.

An example of an assay for levels of CRP is well-known in the art and

commercially available from routine clinical chemistry laboratories.

Other drugs

Preferably the methods and systems described herein for EPA or one of its derivatives are those where“derivatives” refers to those derivatives selected from the group consisting of the fatty acid of EPA itself as a carboxylic acid, esterified forms of EPA, esterified forms of the fatty acid of EPA, esterified forms of EPA involving triacylglycerols (also known as triglycerides), ethyl esters of the EPA, ethyl esters of the fatty acid of EPA, an n-3 fatty acid preparation of EPA, an omega-3-carboxylic acid of EPA, and a mixture of EPA and the omega-3 fatty acid, DHA - for example, Epanova. However, the cholesterol content of the cholesteryl ester of EPA makes it undesirable as a drug for the present inventions.

Treatment systems reflecting the invention

A treatment system is a system that has available all the equipment and reagents needed for measuring apoB-lipoprotein aggregation susceptibility levels and

administering the drug. Different parts of the system may be at different locations. A preferred system is one where apoB-lipoprotein aggregation susceptibility levels and administering drugs are done by or under the control of a single entity. Examples of such entities include, but are not necessarily limited to, hospitals, medical systems comprising a plurality of hospitals, health care providers, physicians, nurses, nurse practitioners, physician assistants, and medical practices that comprise a plurality of physicians.

Kits of the invention

A kit provides (1) one or more reagents useful for measuring an apoB-lipoprotein aggregation susceptibility level, and (2) a drug to be administered. Preferably it also contains printed notice as to the purpose of the kit. Most preferably it contains instructions on how the kit is to be used. All components of the kit are preferably in single container. However, they may be provided in two or more containers such that at least one container, or an enclosed notice, indicates its contents can be used in conjunction with those of the other container (or other containers) to help achieve the kit’s desired goal of assessing a person’s apoB-lipoprotein aggregation susceptibility level and, if they have appropriate levels, administering the drug of interest.

An example of such a kit is one comprising:

(1) EPA or one of its derivatives;

(2) Materials for implementation of an apoB-lipoprotein aggregation

susceptibility assessment system for assessing the susceptibility of a person’s apoB-lipoproteins to aggregation induced by a mammalian acid SMase, preferably hrSMase; and

(3) printed notice indicating that the kit can be used to decrease the susceptibility of atherogenic lipoprotein particles to aggregation in a human (or other animal).

In particular aspects of the kit, it is intended to reduce the mASMase-induced aggregation of atherogenic lipoprotein particles selected from the group consisting of LDL, remnant lipoproteins, cholesterol- and triglyceride-rich remnant lipoproteins (together, referred to C-TRLs), very low-density lipoprotein (VLDL), small VLDL

(sVLDL), cholesterol-rich remnant lipoproteins, b-VLDL, VLDL remnants, chylomicron remnants, postprandial remnants, intermediate-density lipoprotein (IDL), lipoprotein(a) [Lp(a)], and triglyceride-rich remnant lipoproteins (TRL).

The printed notice may be on sheet of paper, a label, or a package. The printed notice requirement of the kit of the invention is satisfied if the kit comprises a printed notice of where the user can go (for example to a website) to find out that the kit can be used to decrease the aggregation susceptibility of atherogenic lipoprotein particles in a human (or other animal with a closed circulatory system).

In particular aspects, the kit of the invention is combined with an apoB-lipoprotein aggregation susceptibility assessment system for measuring extent of aggregation of atherogenic lipoprotein particles and/or their retention in an artery, arteries, or arterial segment of a human or other animal. Such apoB-lipoprotein aggregation susceptibility assessment systems are discussed above in relation to the methods of the invention, specifically as to systems that can be used to determine whether the EPA dose should be modified.

More particular variations and details of the invention are described herein in the text (specification) of the application. The claims below (in the section, What is Claimed) also describe aspects of the invention. It is clear to a person of ordinary skill that various aspects of the invention can reasonably be combined to create additional inventive methods, systems and kits.

ApoB-lipoprotein aggregation susceptibility systems (a/k/a Susceptibility Assessment systems)

For assessment of susceptibility of apoB-lipoproteins to aggregation, such a susceptibility assessment system, is an assay in vitro and/or a system for assessment of those particles in vivo, and/or by an assay of the level of aggregation indicators in the human or other animal of interest. The assay in vitro (or ex vivo) for assessment of susceptibility to aggregation is preferably selected from the group consisting of (1) measurement of the extent or rate of aggregation of LDL that has been isolated from plasma and then incubated ex vivo with a mASMase (i.e., the susceptibility of said LDL to aggregation induced by mASMase), (2) the aggregation of apoB-lipoproteins isolated from plasma and then incubated ex vivo with a mASMase, (3) the aggregation of apoB-containing lipoproteins still in plasma isolated from the human or other animal or with other plasma

components, (4) the aggregation of apoB-containing lipoproteins in the presence of plasma components to which one adds a mASMase, (5) incubations of LDL or other atherogenic lipoproteins with arterial segments ex vivo, and (6) a system for the determination of the composition of an apoB-lipoprotein.

A system for the determination of the composition of an apoB-lipoprotein would be any system that can determine the relative concentrations of the components of an apoB-lipoprotein. Susceptibility can be inferred from such a determination. Examples regarding lipid composition of said apoB-lipoprotein are provided in Ruuth M et al. 2018; further, the incorporation of EPA or EPA esters into an apoB-lipoprotein is envisioned here. Protein composition of said apoB-lipoprotein is also envisioned, such as the content of apoC-lll (See Schissel SL, Jiang X-c, et al. 1998;273:2738-2746. doi:

10.1074/jbc.273.5.2738)

The arterial-wall enzyme used in the assay in vitro for assessment of

susceptibility to aggregation is preferably selected from the group consisting of a product of a mammalian acid SMase gene, a human secretory SMase (S-SMase), a human recombinant SMase from the acid SMase gene (preferred), a mammalian SMase used at an acidic pH, a Zn²⁺-dependent SMase, and a lysosomal acid SMase.

It is understood that assays of LDL aggregability or composition (or of other atherogenic apoB-lipoproteins) can be automated, such as on a clinical autoanalyzer, automated mass spectrometry, nephelometry, ELISA and ELISA-like assays,

turbidometric analyses, rate-zonal centrifugation, and/or dynamic light scattering (DLS).

The susceptibility system for assessment of the retention of the atherogenic particles in the arteries in vivo is preferably selected from the group consisting of an assay of apoB-lipoprotein aggregation and/or retention within the arterial wall in vivo, an imaging method of retained and/or aggregated lipoproteins within the arterial wall, an assay of lipoprotein aggregation and/or retention in a healthy arterial segment, an assay of lipoprotein aggregation and/or retention in a healthy but atherosclerosis-prone arterial segment (Schwenke DC and Carew TE, 1989), an assay of lipoprotein aggregation and/or retention in a diseased arterial segment, an imaging method (such as cardiac catheterization, intravascular ultrasound [IVUS], an MRI, an MRI with contrast, a CT scan, a scan with contrast, an imaging method with a contrast agent wherein said contrast agent comprises a nanoparticle, and a nuclear medicine study), a method that involves injection of said apoB-lipoprotein into an animal, a method that involves labeling of said apoB-lipoprotein followed by its injection into an animal (said animal comprising a human and a non-human animal), and a method that involves

assessments of endogenous apoB-lipoproteins in vivo. Arterial retention of artificial nanoparticles is also contemplated (For example see Cormode et al. 2009). EXAMPLES

Example 1

ApoB-lipoprotein aggregation Susceptibility System measurements

This example is designed to show a preferred option for an in-vitro assay measuring susceptibility of LDL to aggregation. The assay is based on that done with mouse LDL (see Williams 2018; see also Ruuth 2018) but can also be based on that done with human LDL.

Blood is taken from a human into tubes with an anticoagulant, and plasma is prepared by low-speed centrifugation. The density of the plasma samples is raised to a density of 1.063 g/ml, typically by addition of KBr or NaBr, and then ultracentrifuged, a process that floats up VLDL and LDL. The supernatant is subjected to size-exclusion chromatography through a Superose 6 column to separate VLDL from LDL, the latter of which is smaller and therefore elutes later. To ensure purity of the LDL, some of the LDL samples are passed a second time over the size-exclusion column. An alternative is to subject the plasma to sequential ultracentrifugation to isolate the LDL fraction as d = 1.019 to 1.063 g/mL.

The purified LDL sample is extensively dialyzed against a buffer that removes KBr (or NaBr), adjusts the pH to 5.5 (the optimum for mASMase), and supplies zinc (Zn²⁺), a required cofactor for mASMase. This LDL is then brought to a“standard concentration”, typically 0.2 mg of protein per mL, and then incubated with mASMase (preferably human recombinant sphingomyelinase [hrSMase] at a standard

concentration, typically 75 pg/ml, that nearly completely hydrolyzes the sphingomyelin content of LDL within the first hour of incubation at pH 5.5 at 37°C), preferably for times up to 6-25 hours (because aggregation takes additional time after enzymatic digestion of LDL sphingomyelin). A standard LDL concentration means that it is the same LDL concentration from one run of the assay to the next - and from one sample of LDL to the next. For standard LDL concentration and standard hrSMase concentration, see Supplementary methods in Ruuth M et al. 2018.

The assay mixture (LDL in buffer, plus hrSMase) can be incubated in the wells of microtiter plates that can be read with a plate reader. For an example of an incubation of LDL in microtiter plates at pH 5.5 at 37°C with hrSMase, see (Sneck, Nguyen, et al 2012). Other high-throughput adaptations, such as use of a clinical autoanalyzer, are also contemplated. The mean diameter of the LDL aggregates is determined by dynamic light scattering (DLS) at different time points during the incubation (Ruuth et al. 2018). Aggregation of each LDL sample at each time point is quantified as the average size of the aggregates (mean diameters) as determined by dynamic light scattering. Values between LDL samples are compared at each time point, for example, using Student’s two-tailed unpaired t-test, preferably at the 2-h time point.

As noted, in addition to average size of the aggregates, the size distribution of aggregates, including what percentage of the initial apoB-lipoproteins participate in aggregation (versus the percentage remaining as monomeric particles), is also informative. Size distributions of aggregates of apoB-lipoproteins can be obtained by several methods, such as dynamic light scattering and size-exclusion chromatography.

Reproduction of the Claims of the provisional application

The claims of the present application differ from those of the provisional application Serial Number 62/824,628 to the extent that the phrases“any of the foregoing claims” or“any of the foregoing methods” have been replaced by reference to a single claim number. That is done because retention of those phrases might lead to “extra claim” fees if and when application is filed in certain countries. In order to retain, in this PCT application, the information disclosed in the claims of the provisional application, those claims are reproduced here immediately below with two main exceptions: Instead of using the numbering system in the provisional application (e.g., Claim 1 to Claim 9) the claims are referred to here as, for example, Method 1 to Method 9. Additionally, Claim 15 of the provisional application has been split in the“WHAT IS CLAIMED” section of this PCT application into three claims, Claims 15, 16 and 17, and that is reflected by including equivalent language in this section,“Reproduction of the Claims of the Provisional Application”.

Method 1 : A method comprising the steps of:

(1) testing apoB-lipoprotein particles of a patient as to their susceptibility to aggregation in the presence of a mASMase in order to determine whether or not the patient will be expected to benefit (or even particularly benefit) from

administration of a drug, said drug being EPA or one of its derivatives, such that the more susceptible the patient’s apoB-lipoprotein particles are to mASMase- induced aggregation, the greater the likelihood that the patient will benefit from administration of the drug and the less susceptible their LDL is to mASMase- induced aggregation, the lower the likelihood that they will benefit from such administration; and

(2) if the patient is determined by the test to be one that is expected to benefit (or particularly benefit) from the drug, then administering the drug to the patient; wherein said apoB-lipoprotein particles are LDLs or other apoB-containing particles.

Method 2: The method of Claim 1 wherein the apoB-lipoprotein particles are selected from the group consisting of LDL, IDL, VLDL, chylomicron remnant lipoproteins, Lp(a), a cholesterol- and triglyceride-rich apoB-containing lipoprotein, b-VLDL, and an apoB-containing lipoprotein at or below 70 nm in diameter.

Method 3: A treatment method comprising the steps of (1) testing the apoB- lipoprotein-containing particles of a patient at a plurality of times during the course of his or her treatment with a drug as to those particles’ susceptibility to

aggregation in the presence of a mASMase, said drug being EPA or one of its derivatives and if there is little (not more than 2 percent) or no decrease in said susceptibility over a treatment time interval then (2) either terminating treatment with the drug or increasing in dose or dose frequency of the drug, wherein said particles are LDLs or other apoB-containing particles.

Method 4: The method of Method 3 wherein as to the possibilities selected from the group consisting of little decrease in susceptibility and no decrease in susceptibility, no decrease in susceptibility is observed in Step (1).

Method 5: A method wherein (1) a plurality of patients are tested for their susceptibility to mASMase-induced LDL aggregation and (2) are also treated with a drug, said drug being EPA or one of its derivatives.

Method 6: The method of Method 5 further comprising the step (3) wherein based on the test results, the relationship between that susceptibility and the therapeutic success of the drug is determined.

Method 7: The method of any of the foregoing Methods, wherein the apoB- containing lipoprotein is selected from the group consisting of LDL, IDL, VLDL, chylomicron remnant lipoproteins, Lp(a), a cholesterol- and triglyceride-rich apoB- containing lipoprotein, b-VLDL, and an apoB-containing lipoprotein at or below 70 nm in diameter.

Method 8: The method of any of the foregoing Methods wherein the mASMase is selected from the group consisting of a human recombinant sphingomyelinase (hrSMase), a secretory sphingomyelinase, a product of a mammalian acid sphingomyelinase gene, a lysosomal sphingomyelinase, an arterial-wall acid sphingomyelinase, and a Zn²⁺-dependent sphingomyelinase.

Method 9: The method of any of the above Methods wherein the time between step (1) and the start of step (2) is preferably not more than 30 days, more preferably not more than seven days.

System 1 : A treatment system that has available all the equipment and reagents needed for measuring susceptibility levels and administering a drug as set forth in any of the above claims, wherein said drug is EPA or one of its derivatives.

Kit 1 :A kit for decreasing the susceptibility of atherogenic lipoprotein particles to aggregation in a human (or other animal), said kit comprising:

(1) A drug, said drug being EPA or one of its derivatives;

(2) Materials for implementation of an assessment system for assessing the susceptibility of a person’s apoB-lipoproteins to mASMase-induced lipoprotein particle aggregation; and

(3) printed notice indicating that the kit can be used to decrease the

susceptibility of atherogenic lipoprotein particles to aggregation in a human.

The Methods, Systems, or Kits of any of the foregoing claims, in which the drug is EPA or a derivative of EPA, and the derivative is selected from the group consisting of the fatty acid component of EPA as a carboxylic acid, esterified forms of EPA, esterified forms of the fatty acid component of EPA, esterified forms of EPA that are triacylglycerols (also known as triglycerides), ethyl esters of EPA, ethyl esters of the fatty acid component of EPA, an n-3 fatty acid preparation of EPA, an omega-3-carboxylic acid of EPA, and a mixture of EPA and the omega-3 fatty acid DHA, for example Epanova, provided that the derivative selected is not the cholesteryl ester of EPA.

A Method, System, or Kit of any of the foregoing claims, in which the drug is icosapent ethyl.

REFERENCES

Bhatt DL, Steg PG, Miller M, Brinton EA, Jacobson TA, Ketchum SB, Doyle RT, Juliano RA, Jiao L, Granowitz C, Tardif J-C, and Ballantyne CM, for the

REDUCE-IT Investigators. Cardiovascular risk reduction with icosapent ethyl for hypertriglyceridemia. N Engl J Med. 2019; 380:11-12. doi:

10.1056/NEJMoa1812792.

Kastelein JJP and Stroes ESG. FISHing for the miracle of icosapentaenoic acid. N Engl J Med 2019; 380:89-90. doi: 10.1056/NEJMe1814004.

Ruuth M et al. Susceptibility of low-density lipoprotein particles to aggregate depends on particle lipidome, is modifiable, and associates with future

cardiovascular deaths. Eur Heart J. 2018;39:2562-2573. doi:

10.1093/eurheartj/ehy319.

Schissel SL, Schuchman EH, Williams KJ and Tabas I. Zn²⁺-stimulated sphingomyelinase is secreted by many cell types and is a product of the acid sphingomyelinase gene. J Biol Chem. 1996;271 :18431-18436.

Schissel SL, Keesler GA, Schuchman EH, Williams KJ and Tabas I. The cellular trafficking and zinc dependence of secretory and lysosomal sphingomyelinase, two products of the acid sphingomyelinase gene. J Biol Chem.1998;273: 18250- 18259.

Schissel SL, Jiang X-c, Tweedie-Hardman J, Jeong T-s, Camejo EH, Najib J, Rapp JH, Williams KJ and Tabas I. Secretory sphingomyelinase, a product of the acid sphingomyelinase gene, can hydrolyze atherogenic lipoproteins at neutral pH: implications for atherosclerotic lesion development. J Biol Chem.

1998;273:2738-2746. doi: 10.1074/jbc.273.5.2738

Wasserstein MP, Diaz GA, Lachmann RH, Jouvin M-H, Nandy I, Ji AJ and Puga AC. Olipudase alfa for treatment of acid sphingomyelinase deficiency (ASMD): safety and efficacy in adults treated for 30 months. J Inherit Metab Dis.

2018;41 :829-838. doi: 10.1007/s 10545-017-0123-6.

Boren J and Williams KJ. The central role of arterial retention of cholesterol-rich apoB-containing lipoproteins in the pathogenesis of atherosclerosis: a triumph of simplicity. Curr Opin Lipidol. 2016;27:473-83. doi:

10.1097/MOL.0000000000000330.

Yokoyama M, Origasa H, Matsuzaki M, Matsuzawa Y, Saito Y, Ishikawa Y, Oikawa S, Sasaki J, Hishida H, Itakura H, Kita T, Kitabatake A, Nakaya N,

Sakata T, Shimada K, and Shirato K, for the Japan EPA lipid intervention study (JELIS) Investigators. Effects of eicosapentaenoic acid on major coronary events in hypercholesterolaemic patients (JELIS): a randomised open-label, blinded endpoint analysis. Lancet. 2007;369:1090-8. doi: 10.1016/S0140- 6736(07)60527-3.

Guarino AJ, Tulenko TN and Wrenn SP. Cholesterol crystal nucleation from enzymatrically modified low-density lipoproteins: combined effect of

sphingomyelinase and cholesterol esterase. Biochemistry. 2004: 43:1685-93. Doi: 10. 1021bi035747r.

Schwenke DC and Carew TE. Initiation of atherosclerotic lesions in cholesterol- fed rabbits. II. Selective retention of LDL vs. selective increases in LDL permeability in susceptible sites of arteries. Arteriosclerosis. 1989;9:908-918. Cormode DP, Frias JC, Ma Y, Chen W, Skajaa T, Briley-Saebo K, Barazza A, Williams KJ, Mulder WJ, Fayad ZA and Fisher EA. FIDL as a contrast agent for medical imaging. Clin Lipidol. 2009;4:493-500. doi: 10.2217/clp.09.38.

Williams KJ. Methods and Kits for Reducing the Susceptibility of Lipoprotein Particles to Atherogenic Aggreggation Induced by Arterial Wall Enzymes.

International Patent Application Number PCT/US2017/049351 , International Publication Number WO 2018/045015 A1 , Publication Date March 8, 2018.

Sneck M, Nguyen SD, Pihlajamaa T, Yohannes G, Riekkola M-L, Milne R, Kovanen PT, Oorni K. Conformational changes of apoB-100 in SMase-modified LDL mediate formation of large aggregates at acidic pH. J Lipid Res.

1012;53(9): 1832-9.

Athyros VG, Papageorgiou AA, Symeonidis AN, Didangelos TP, Pehlivanidis AN, Bouloukos VI, and Mikhailidis DP, for the GREACE Study Collaborative Group. Early benefit from structured care with atorvastatin in patients with coronary heart disease and diabetes mellitus. A subgroup analysis of the GREek Atorvastatin and Coronary Heart Disease Evaluation (GREACE) Study. Angiology.

2003;54:679-90. doi: 10.1177/000331970305400607).

Ridker PM, Danielson E, Fonseca FA, Genest J, Gotto AM, Jr., Kastelein JJP, Koenig W, Libby P, Lorenzatti AJ, MacFadyen JG, Nordestgaard BG, Shepherd J, Willerson JT, and Glynn RJ, for the JUPITER Study Group. Rosuvastatin to prevent vascular events in men and women with elevated C-reactive protein. N Engl J Med. 2008;359:2195-207. doi: 10.1056/NEJMoa0807646).

Claims

WHAT IS CLAIMED:

1. A method comprising the steps of:

(1) testing apoB-lipoprotein particles of a patient as to their susceptibility to aggregation in the presence of a mASMase in order to determine whether or not the patient will be expected to benefit (or even particularly benefit) from

administration of a drug, said drug being EPA or one of its derivatives, such that the more susceptible the patient’s apoB-lipoprotein particles are to mASMase- induced aggregation, the greater the likelihood that the patient will benefit from administration of the drug and the less susceptible their LDL is to mASMase- induced aggregation, the lower the likelihood that they will benefit from such administration; and

(2) if the patient is determined by the test to be one that is expected to benefit (or particularly benefit) from the drug, then administering the drug to the patient; wherein said apoB-lipoprotein particles are LDLs or other apoB-containing particles.

2. The method of Claim 1 wherein the apoB-lipoprotein particles are selected from the group consisting of LDL, IDL, VLDL, chylomicron remnant lipoproteins, Lp(a), a cholesterol- and triglyceride-rich apoB-containing lipoprotein, b-VLDL, and an apoB-containing lipoprotein at or below 70 nm in diameter.

3. A treatment method comprising the steps of (1) testing the apoB-lipoprotein- containing particles of a patient at a plurality of times during the course of his or her treatment with a drug as to those particles’ susceptibility to aggregation in the presence of a mASMase, said drug being EPA or one of its derivatives and if there is little (not more than 2 percent) or no decrease in said susceptibility over a treatment time interval then (2) either terminating treatment with the drug or increasing in dose or dose frequency of the drug, wherein said particles are LDLs or other apoB-containing particles.

4. The method of Claim 3 wherein as to the possibilities selected from the group consisting of little decrease in susceptibility and no decrease in susceptibility, no decrease in susceptibility is observed in Step (1).

5. A method wherein (1) a plurality of patients are tested for their susceptibility to mASMase-induced LDL aggregation and (2) are also treated with a drug, said drug being EPA or one of its derivatives.

6. A method of Claim 5 further comprising the step (3) wherein based on the test results, the relationship between that susceptibility and the therapeutic success of the drug is determined.

7. A method of Claim 1 , wherein the apoB-containing lipoprotein is selected from the group consisting of LDL, IDL, VLDL, chylomicron remnant lipoproteins, Lp(a), a cholesterol- and triglyceride-rich apoB-containing lipoprotein, b-VLDL, and an apoB-containing lipoprotein at or below 70 nm in diameter.

8. A method of Claim 1 wherein the mASMase is selected from the group consisting of a human recombinant sphingomyelinase (hrSMase), a secretory

sphingomyelinase, a product of a mammalian acid sphingomyelinase gene, a lysosomal sphingomyelinase, an arterial-wall acid sphingomyelinase, and a Zn²⁺- dependent sphingomyelinase.

9. A method of Claim 1 wherein the time between step (1) and the start of step (2) is preferably not more than 30 days, more preferably not more than seven days.

10. A treatment system that has available all the equipment and reagents needed for measuring susceptibility levels and administering a drug as set forth in any of the above claims, wherein said drug is EPA or one of its derivatives.

11.A kit for decreasing the susceptibility of atherogenic lipoprotein particles to

aggregation in a human (or other animal), said kit comprising:

(4) A drug, said drug being EPA or one of its derivatives;

(5) Materials for implementation of an assessment system for assessing the susceptibility of a person’s apoB-lipoproteins to mASMase-induced lipoprotein particle aggregation; and

(6) printed notice indicating that the kit can be used to decrease the

susceptibility of atherogenic lipoprotein particles to aggregation in a human.

12. A method of Claim 1 in which the drug is EPA or a derivative of EPA, and the

derivative is selected from the group consisting of the fatty acid component of EPA as a carboxylic acid, esterified forms of EPA, esterified forms of the fatty acid component of EPA, esterified forms of EPA that are triacylglycerols (also known as triglycerides), ethyl esters of EPA, ethyl esters of the fatty acid component of EPA, an n-3 fatty acid preparation of EPA, an omega-3-carboxylic acid of EPA, and a mixture of EPA and the omega-3 fatty acid DHA, for example Epanova, provided that the derivative selected is not the cholesteryl ester of EPA.

13. A system of Claim 10 in which the drug is EPA or a derivative of EPA, and the derivative is selected from the group consisting of the fatty acid component of EPA as a carboxylic acid, esterified forms of EPA, esterified forms of the fatty acid component of EPA, esterified forms of EPA that are triacylglycerols (also known as triglycerides), ethyl esters of EPA, ethyl esters of the fatty acid component of EPA, an n-3 fatty acid preparation of EPA, an omega-3-carboxylic acid of EPA, and a mixture of EPA and the omega-3 fatty acid DHA, for example Epanova, provided that the derivative selected is not the cholesteryl ester of EPA.

14. A kit of Claim 11 in which the drug is EPA or a derivative of EPA, and the derivative is selected from the group consisting of the fatty acid component of EPA as a carboxylic acid, esterified forms of EPA, esterified forms of the fatty acid

component of EPA, esterified forms of EPA that are triacylglycerols (also known as triglycerides), ethyl esters of EPA, ethyl esters of the fatty acid component of EPA, an n-3 fatty acid preparation of EPA, an omega-3-carboxylic acid of EPA, and a mixture of EPA and the omega-3 fatty acid DHA, for example Epanova, provided that the derivative selected is not the cholesteryl ester of EPA.

15. A method of Claim 1 , in which the drug is icosapent ethyl.

16. A system of Claim 10. in which the drug is icosapent ethyl.

17. A kit of Claim 11 in which the drug is icosapent ethyl.