Patent Application
20140213465
The methods and compositions described herein enable rapid, sensitive and/or efficient in vitro diagnosis, biotechnology tools and drug discovery and development of plaque-associated diseases.
Atherosclerosis is a complex, progressive and chronic inflammatory cardiovascular disease caused by assembly and progression of atherosclerotic plaque in the arteries (Lippy P et al 2011). According to American Heart Association, approximately, 40 million people in US are believed to have atherosclerosis without noticeable clinical symptoms and only 6 million are symptomatic. A number of analytical methods and tools are used to diagnose both symptomatic and asymptomatic subjects of the atherosclerosis (Naghavi et al, 2003)
Most of these diagnostic procedures and techniques are expensive and invasive often involving administration of chemical and/or radioactive compounds to localize or visualize the atherosclerotic plaques (Greenland et al, 2007). In addition, the results obtained from these procedures are not sufficient enough to conclusively suggest therapeutic intervention to the suspected patients. Further, limited knowledge about the mechanism of atherosclerotic plaque formation make drug discovery and development challenging. Thus, there is a need for simple, determinative, cost-effective solutions for the diagnosis, drug discovery and development of plaque-associated diseases such as atherosclerosis.
Amyloidosis are a group of more than twenty plaque-associated diseases characterized by protein aggregation including Alzheimer's Disease (AD), Parkinson's Disease (PD), prion-mediated diseases, Huntington disease (HD), Multiple sclerosis (MS), type 2 diabetes and the like. AD is a common neurodegenerative disease associated with progressive dementia caused mostly due to the deposition of Amyloid-beta (Abeta) peptides (Yankner, 1996). Abnormal processing of the Abeta precursor protein is an early and causative event in the pathogenesis of AD (Selkoe D J, 2003). Abeta peptides released from amyloid precursor protein by the action of β- and γ-secretases undergo structural transformation from monomers to oligomers and finally into amyloid fibrils/plaques (Dobson C M, 2003).
The pathological consequences of such senile plaque accumulation are neuronal loss, cerebrovascular inflammation, reduction in the cerebrovascular space and cognitive decline (Bell R D et al, 2009).
AD is definitively diagnosed through examination of brain tissue, usually at autopsy (Khachaturian et al 1985; McKhann et al, 1984). Post-mortem slices of brain tissue of subjects with AD exhibit the presence of amyloid in the form of proteinaceous extracellular cores of the neuritic plaques that are characteristic of AD.
Research efforts to develop methods for diagnosing AD include (1) genetic testing, (2) immunoassay methods and (3) imaging techniques. The limitations of these methods are several-fold. Genetic analysis of a large number of AD families has demonstrated that AD is genetically heterogeneous (George-Hyslop et al, 1990). Also, the genetic tests reveal risk factors rather than disease markers for AD. The immunoassay methods diagnosing presence of amyloid related protein in cerebrospinal fluid (CSF) for diagnosing AD have not been proven to detect AD in all patients, particularly at early stages of the disease. Imaging methods face the challenge of getting the imaging agent such as antibodies or radio-labeled peptides across the blood brain barrier. Identifying asymptomatic AD individuals is a challenging task and multiple tests involving neurophysiological and neuropathological techniques are currently used to diagnose these subjects (Thies W et al 2012). Clinical diagnosis of AD is not always accurate since the criteria are relatively subjective and the disease needs to be differentiated from other dementing illnesses. According to new recommendation from the National Institute on Aging and the Alzheimer's disease association, identification of novel biomarker is imperative both for AD diagnosis and drug discovery (McKhann G M et al, 2011; Sperling R A et al, 2011). The diagnosis of asymptomatic subjects in will help to initiate early therapeutic intervention to reduce life threatening risk associated with the progression of plaque-associated diseases including atherosclerosis and amyloidoses.
Accordingly, there remains an unmet need for novel, cost effective AD diagnostic methods and effective drug discovery and development technologies.
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
In some embodiments, the present invention provides a method of detecting plaque particle formation in a subject, the method comprising: preparing at least one plaque aggregate or self-formed plaque particle in vitro wherein the plaque aggregate or self-formed plaque particle is linked to a detectable label; contacting a biological sample from the subject with the at least one plaque aggregate or self-formed plaque particle; and then employing a device to detect the detectable label. In some embodiments, the label is a fluorescent label or luminescent label or dye. In some embodiments, the device is a flow cytometer or other fluorescence detector or luminescent detector or colorimeter.
In some embodiments, the biological sample is a biological fluid. In other embodiments, the biological fluid is selected from the group consisting of: blood, plasma, serum, cerebral spinal fluid, urine and saliva. In some embodiments, the contacting of biological sample results in addition of components to the at least one plaque aggregate or self-formed plaque particle such that at least one plaque particle is formed. In some embodiments, the contacting results in addition of components to the at least one plaque aggregate or self-formed plaque particle such that at least one plaque particle is formed and the plaque particles formed resembles a plaque associated with atherosclerosis, Alzheimer's disease, Autism, Parkinson's disease, multiple sclerosis, osteoarthritis, Mad Cow Sponsiform, Type II diabetes, dementia, systemic amyloidosis, dialysis-related amyloidosis, lysozyme amyloidosis, insulin-related amyloidosis, and/or amyotrophic lateral sclerosis.
In some embodiments, the at least one plaque particle formed is compared to a plurality of self-formed plaque particles. In other embodiments, the subject is identified as having, or being at risk of having, a plaque-associated disease if the at least one plaque particle is substantially similar to a self-formed plaque particle among the plurality of self-formed plaque particles. In some embodiments, the plaque-associated disease is atherosclerosis or amyloidosis. In some embodiments the subject has, is at risk of having, or is suspected of having, atherosclerosis or an amyloidosis including Alzheimer's disease. In some embodiments, the at least one plaque aggregate or a plurality of plaque aggregates are used. In some embodiments the method further comprises diagnosing or stratifying subjects based on plaque particle formation, plaque particle sub-types, plaque particle images, plaque particle count, or plaque particle profile.
In some embodiments, at least one plaque aggregate or self-formed plaque particle comprises one or more of the following: protein, protein derivative, cholesterol, cholesterol derivative, lipid, lipid derivative, Abeta-42, Abeta derivatives, Synuclein, prion, Amylin, Tau, phospholipids, cholesterol crystals, Serum Amyloid A, Beta Microglobulin, lysozyme, insulin, or super dioxide dismutase, and calcium-phosphate (CP).
In some embodiments, the method further comprises screening the biological sample against a plurality of plaque aggregates or a pair of plaque aggregates labeled with different fluorophores for generating fluorescence resonance energy transfer (FRET) or a plurality of self-formed plaque particles or a pair of self-formed plaque particles labeled with different fluorophores for generating fluorescence resonance energy transfer (FRET).
In some embodiments, the method further comprises monitoring the subject by repeating steps of preparing plaque aggregates or self-formed plaque particles linked to a detectable label, contacting biological sample to plaque aggregates or self-formed plaque particles and using a device to detect a detectable label at different points over time. In some embodiments, the invention provides a method for detecting plaque particle formation in a subject, comprises: preparing at least one plaque aggregate or self-formed plaque particle; contacting a biological sample from the subject with the at least one plaque aggregate or self-formed plaque particle; then contacting the product with detectable label or an antibody-linked detectable label; and then employing a device to detect the detectable label.
In some embodiments, the invention provides a method of screening a test agent comprise: preparing the at least one plaque aggregate or self-formed plaque particle in vitro wherein the plaque aggregate or self-formed plaque particle is linked to a detectable label; contacting the at least one plaque aggregates or self-formed plaque particle linked to a detectable label with at least one test agents; and then employing a device to detect the detectable label. In some embodiments, the at least one of test agent comprises a small molecule or protein or antibody library of test agents. In some embodiments, the effect of at least one test agent is to accelerate the formation of plaque particles. In other embodiments, the effect of at least one test agent is to reduce or slow or disrupt plaque particle formation. In yet other embodiments, the method further comprises identifying the test agents that prevent or disrupt or reduce plaque particle formation. In other embodiments, the method further comprises testing the efficacy of the test agent or agents at disrupting plaque particles or reducing the formation of plaque particles or further comprising testing the safety of the test agent. In some embodiments, the test agent is a nanoparticle or is formulated with a nanoparticle. In these embodiments, the method further comprises monitoring the efficacy of the test agent in subjects.
In some embodiments, the invention provides a method of screening a test agent comprising: preparing at least one plaque aggregate or self-formed plaque particle in vitro wherein the at least one plaque aggregate or self-formed plaque particle is linked to a detectable label; culturing mammalian cells with the at least one plaque aggregate or self-formed plaque particle linked to a detectable label wherein the mammalian cells express morphologic changes, pathological symptoms, cell adhesion molecules, cytokines and or apoptosis, inflammation; contacting the mammalian cells at least one test agent; and then identifying test agents that prevent or lessen the formation of pathological symptoms or morphological changes in the cells.
In some embodiments, the invention provides a method of biomarker identification in a subject, the method comprising: preparing at least one plaque aggregate or self-formed plaque particle in vitro wherein the plaque aggregate or self-formed plaque particle is linked to a detectable label; contacting a biological sample from the subject with the at least one plaque aggregate or self-formed plaque particle; and then identification from of a protein or antibody or metabolite or substance in the biological sample that contributed to accelerated plaque particle formation using proteomics or mass spectrometry analysis or the like.
In some embodiments, the invention provides the methods disclosed above are used in the screening blood or blood products for plaque particle formation. In other embodiments, the blood or blood products is administered to a recipient subject following the screening or testing wherein the negative result is a finding of few or no new plaques following the contacting of said blood or blood product with the plaque aggregates.
A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
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As used herein, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Abeta-42 refers to Amyloid beta peptide 1-42 and derivatives; Abeta-28 refers to Amyloid beta peptide 1-28 and derivatives; Abeta-17 refers to Amyloid beta peptide 1-17 and derivatives; Ch1 refers to cholesterol; LS refers to phospholipid; CP refers to calcium phosphate.
Two reagents used in the plaque array method are plaque aggregates and self-formed plaque particles. Plaque aggregates are prepared from organic or inorganic molecules under conditions that cause the molecules to aggregate. Plaque aggregates may be used directly in the plaque array method. For example, they could be used to screen a biological sample to diagnose if the subject has a plaque-associated disease. The second reagent disclosed herein is self-formed plaque particles. Self-formed plaque particles are formed from plaque aggregates. This occurs over time without contacting the plaque aggregates with biological sample. Like the plaque aggregates, the self-formed plaque particles can be used to screen a biological sample in the plaque array method. To perform the plaque array method biological sample is added to the reagent—either plaque aggregates or self-formed plaque particles. The addition of biological sample to the reagent results in formation of plaque particles which are detected by fluorescence or luminescence or colorimetry. The resulting plaque particles are referred to as plaque particles or in vitro plaque particles.
The “plaque particles” and “in vitro plaque particles” disclosed herein refer to the same reaction product formed in the presence of added biological sample and the terms are used interchangeably. These terms are different from the term “self-formed plaque particles” which are formed in the absence of added biological sample. “Self-formed plaque particles” refers to one type of reagent used in the plaque array assay.
The plaque aggregates (including cholesterol plaque aggregates, phospholipid plaque aggregates, Abeta plaque aggregates, hybrid plaque aggregates and the like disclosed herein are water soluble. The self-formed plaque particles and the plaque particles disclosed herein are water insoluble. The aggregates of various Abeta peptides disclosed herein as Abeta aggregates generally referred to in the literature as oligomers. As disclosed herein, an array or a panel refer to a plurality of plaque aggregates or self-formed plaque particles.
In some embodiments disclosed herein the plaque array method uses building block materials that are normally present in atherosclerotic and amyloid plaques, including lipids, proteins, cholesterol, calcium, amyloid peptides, endothelial cells, bacteria, and minerals. These components exist inside the in vivo plaques as soluble aggregates and mature plaque/crystalline forms and contribute to origin and progression of the plaque-associated diseases (
Some embodiments disclosed herein relate to atherosclerosis. In these embodiments, the plaque aggregates or self-formed plaque particles used to screen biofluids or biological samples effect on plaque particle formation may comprise one or more of the following: protein, protein derivative, cholesterol, cholesterol derivatives—(cholestatrienol(=cholesta-5,7,9(11)-triene-3β-ol), 22-NBD-cholesterol(=22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3-ol),α-epoxycholesterol-Cholestan-5α,6α epoxy-3beta diol; 35-hydroxy cholesterol; 7-keto cholesterol, cholesterol monohydrate etc. (Examples shown in
Some embodiments disclosed herein relate to amyloidosis. In these embodiments, the plaque aggregates or self-formed plaque particles used to screen biofluids or biological samples effect on plaque particle formation may comprise one or more of the following: Abeta peptides and derivatives
Amyloid (1-42); N27A-DAEFRHDSGYEVHHQKLVFFAEDVGSAKGAIIGLMVGGVVIA etc), Synuclein, prion, Amylin, Tau, phospholipids, cholesterol crystals, Serum Amyloid A, Beta Microglobulin, lysozyme, insulin, or super dioxide dismutase. In some embodiments, the method comprises a mixture of fluorescently-labeled calcium-phosphate (CP), lipids and cholesterol.
In some embodiments, plaque aggregates or self-formed plaque particles comprise at least one component known to persons of ordinary skill in the art to be present in in vivo formed plaques in subjects with symptomatic and asymptomatic amyloidosis. In these embodiments, the component may be linked to a detectable label. In other embodiments, plaque aggregates or self-formed plaque particles comprise Abeta-42 peptides. In yet other embodiments, plaque aggregates or self-formed plaque particles comprise Abeta-28 peptides.
In some embodiments, plaque aggregates or self-formed plaque particles comprise at least one component known to persons of ordinary skill in the art to be present in in vivo formed plaques in subjects with atherosclerosis. In these embodiments, the component of may be linked to a detectable label. In other embodiments, plaque aggregates or self-formed plaque particles comprise cholesterol or its derivatives. In yet other embodiments, plaque aggregates or self-formed plaque particles comprise phospholipid or its derivatives. In some embodiments, the plaque aggregates comprise a single component while in other embodiments they are hybrid aggregates and comprise more than one component. In some embodiments, the self-formed plaque particles comprise a single component while in other embodiments they are hybrid self-formed plaque particles and comprise more than one component.
In some embodiments, the plaque aggregates and self-formed plaque particles are prepared in phosphate buffered saline (PBS) or phosphate buffers. A person of ordinary skill in the art would recognize that any suitable aqueous solution may be used instead. In some embodiments, the plaque aggregates or self-formed plaque particles are prepared using organic solvents such as alcohol. In some embodiments, the reactions forming plaque aggregates and self-formed plaque particles are performed at 37° C. In other embodiments, the reaction is performed at a temperature and a time which are appropriate for progression of a reaction. In some embodiments the reactions using the plaque aggregates and self-formed plaque particles in diagnostic or drug discovery or development or other context are performed at 37° C. In other embodiments, the reaction is performed at a temperature and a time which are appropriate for progress of a reaction.
The formation of Abeta plaque aggregates is determined by the concentration of the peptides in the buffer, incubation time at 37° C. and presence of metal ions such as copper, iron, aluminium and zinc. We found that incubation of Abeta peptides at 37° C. up to 6 hrs is sufficient to prepare plaque aggregates. Incubation at 37° C. for 24 to 48 hrs produces self-formed Abeta plaque particles. Similarly, cholesterol or phospholipid plaque aggregates are prepared (0 hr) in Phosphate buffered saline (PBS) and used for plaque array assay. Incubation at 37° C. for 12 to 48 hrs leads to the formation of self-formed cholesterol or phospholipid plaque particles. The formation of plaque particles from plaque aggregates in the absence and presence of biofluids is determined by the concentration of the plaque aggregates and incubation time at 37° C. The resulting self-formed plaque particles or plaque particles formed in the serum are insoluble in water, phosphate buffers, Tris-HCL buffers and the like.
In one aspect, the invention is embodied in a method for formation of plaque particles from the plaque aggregates or from self-formed plaque particles. In some embodiments disclosed herein, the formation of plaque particles is accelerated in the presence of biological samples including biofluids. In other embodiments, the formation of plaque particles is accelerated by artificial growth medium, chemical medium and the like.
Any biological sample may be tested according to the disclosed methods. Such a sample may be cells, tissue, blood, urine, semen, or a fraction thereof (e.g., plasma, serum, urine supernatant, urine cell pellet or prostate cells), which may be obtained from a patient or other source of biological material, e.g., autopsy sample or forensic material. Prior to contacting the plaque aggregates or self-formed plaque particles, the sample may be processed to isolate or enrich the sample for the desired molecules using a variety of standard laboratory practices may be used for this purpose, such as, e.g., centrifugation, immunocapture, cell lysis. Biofluid is one category of biological sample. As disclosed herein, the term biofluid is a fluid biological sample and is used interchangeably with the term biological fluid. While the biofluid used in the Examples disclosed herein is serum from human subjects, in some embodiments the biofluid may comprise plasma or saliva. In other embodiments the biofluid may comprise urine, or cerebrospinal fluid. In yet other embodiments the biofluid may comprise blood.
Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples include cerebrospinal fluid, sputum, bronchial washing, bronchial aspirates, urine, lymph fluids, and various external secretions of the respiratory, intestinal and genitourinary tracts, tears, saliva, milk, biological fluids such as cell culture supernatants, tissue, cell, and the like.
In some embodiments, the self-formed plaque particles comprise a linked detectable label which is be detected using fluorescence, luminescence, colorimetry and the like. In another embodiment, the invention comprises an array method for in vitro formation of atherosclerotic or amyloid plaque particles and indirect way of detection of plaque particles or plaque particle sub-types using fluorescent dye or proteins or fluorescently or luminescently-labeled antibodies, the method comprising first converting organic or inorganic molecules into plaque aggregates (0 hr) or self-formed plaque particles (24 hrs), wherein the organic or inorganic molecules is chosen from the group consisting of unlabeled cholesterol or its derivatives or lipid or its derivatives or Abeta-42 or its derivatives, contacting the plaque aggregates or self-formed plaque particles with biological sample so the plaque aggregates interact with the proteins, lipids or carbohydrates and other molecules present in the biological sample leading to in vitro formation of plaque particles. The detection of the resulting plaque particles or plaque particles sub-types employs fluorescently or luminescently-labeled antibodies that bind to components of the plaque particles. This is an indirect method of detecting plaque particles and sub-types that will help to identify the molecules that are attached to the plaque particles and using those molecules as biomarkers for predicting atherosclerosis and AD.
In some embodiments, the in vitro formed plaque particles resemble a plaque associated with, atherosclerosis, Alzheimer's disease, Autism, Parkinson's disease, multiple sclerosis, osteoarthritis, Mad Cow Sponsiform, Type II diabetes, dementia, systemic amyloidosis, dialysis-related amyloidosis, lysozyme amyloidosis, insulin-related amyloidosis, and/or amyotrophic lateral sclerosis.
In certain embodiments, the present invention comprises a method of diagnosing, categorizing, evaluating, quantitating, or predicting atherosclerotic disease or amyloid diseases including Alzheimer's disease in a test subject. Such method may comprise: (a) contacting the blood, plasma, serum, urine, saliva, cerebral spinal fluid of a test subject with a plurality of one or more luminescence or fluorescently-labeled plaque aggregates or self-formed plaque particles; and employing a device to detect the detectable label and identify molecules within the blood, plasma or serum that accelerate plaque particle formation of said one or more plaque aggregates or self-formed plaque particles
The present invention may comprise a method of diagnosing, detecting, analyzing, evaluating or administrating a therapeutically effective dose of a drug, biological compounds, proteins, antibodies or chemical compound to a subject, wherein said drug, biological compound, or chemical compound has been previously screened for its ability to bind, penetrate, disassemble, disrupt, or prevent atherosclerotic plaque or amyloid plaque.
In another embodiment, the invention comprises an array method for identification of molecules present in the biofluids that contribute to assembly of in vitro plaque particles. The cholesterol or phospholipids or amyloid peptide plaque aggregates interact with the proteins, lipids or carbohydrates and other molecules present in the human serum or plasma leading to in vitro formation of plaque particles. Mass spectrometry and proteomics analysis may help to identify the molecules that are attached to the plaque particles and involved in the plaque assembly process. These molecules may be used as biomarkers for detecting the course of in vivo atherosclerosis and amyloid disease development and progression.
In some embodiments disclosed herein the plaque array technology permits the discovery of both novel mechanisms and molecules that catalyze the accelerated plaque particle assembly when treated with the biological samples including biofluids. In some embodiments the plaque array enables the evaluation of the pathogenicity of plaques of varying compositions.
The present invention also embodies a plaque array kit to aid in the diagnosis, prediction, prognosis, or detection of a plaque-associated disease such as AD and atherosclerosis. The method may also comprise obtaining or generating a designation of the risk potential of AD or atherosclerosis against constituent of the panel of plaque particles. In some embodiments, the kit comprises one or more molecules for preparing plaque aggregates, or self-formed plaque particles or plaque particles as described herein. In other embodiments, the kit includes composition of one or more plaque aggregates or self-formed plaque particles or plaque particles with a carrier, e.g. salt, buffer, booster and the like. In other embodiments, the kit further includes reagents of plaque array assay and detection of plaque particles by flow cytometer or luminescence detector.
The present invention also includes plaque array kits that can be used for diagnosis, drug discovery and drug development of plaque-associated diseases. In some embodiments instructions teaching the use of the kit according to the various methods and approaches described herein are provided. Such kits may also include information, such as scientific literature references, package insert materials, clinical trial results, and/or summaries of these and the like, which indicate or establish the activities and/or advantages of the agent. Such information may be based on the results of various studies, for example, biochemical plaque assays, studies using experimental animals involving in vivo models and studies based on human clinical trials. Kits described herein can be provided, marketed and/or promoted to health providers, including physicians, nurses, pharmacists, formulary officials, and the like.
In another embodiment, the invention involves the use of the plaque array method disclosed herein to screen agents for the inhibition or stimulation of the in vitro formation of the plaque particles. As such, agents including but not limited to chemical compounds, small molecule compounds, therapeutic drugs, biological molecules, oligomers, ligands, proteins, antibodies or other components, capable of binding the plaque aggregates or self-formed plaque particles or plaque particles in the presence or absence of biofluids, preventing their assembly, disassembling these aggregates or self-formed plaque particles or plaque particles once already formed, or reducing their pathogenic properties, are tested for their potential as therapeutic leads for diagnosing, preventing, treating, and/or curing amyloid plaque diseases. Since the methods or processes disclosed herein are capable of isolating the steps of in vitro plaque particle formation, anti-plaque agents targeting different stages of plaque development are also capable of being identified. The term “anti-plaque agents” and “anti-plaque therapeutics are used interchangeably herein and refer to compounds or drugs which are effective in a) dissolving, inhibiting or disrupting the architecture, or structure of a plaque aggregates or self-formed plaque particles or plaque particles described herein, and/or b) inhibiting, preventing, or alleviating the detrimental effects that the plaque may have on other cells, tissues or organs of humans.
In certain embodiments, the present invention comprises a method for screening drug, chemical or biological compounds including proteins and antibodies controlling formation of in vitro plaque particles when plaque aggregates or self-formed plaque particles are treated with serum, defined or synthetic medium or plasma samples. This is done by culturing mammalian cells with the at least one plaque aggregates or self-formed plaque particles described herein causing the cells to express morphologic changes, cell death, inflammation, DNA damage, aging or age related degenerative process; and then using the system to screen drug, chemical or biological compounds that prevent or lessen the formation of plaque particle induced pathological symptoms, cell death or morphological changes in the cells. In certain embodiments, the plaque particles resemble an atherosclerotic plaque or amyloid plaque.
In some embodiments, the present invention comprises a method of profiling or categorizing or evaluating efficacy, testing safety of the drug or drug formulations, nanomaterials. In some embodiments, one or more of the plaque aggregates or self-formed plaque particles described herein are used to evaluate drug efficacy, drug safety, pathogenicity in atherosclerotic or amyloid disorders animal models. The animal may be a mouse, a rat, a pig, a horse, a non human primate, a guinea pig, a hamster, a chicken, a frog, a dog, a sheep, a cow, or a human.
The Example illustrated in
The results of flow cytometry displayed herein are typically presented as one dimensional histogram on a logarithmic scale or two-dimensional displays (dot plot) with logarithmic axes that can extend over a four- to five-decade range.
Cholesterol, lipids and calcium crystals are major components present in the atherosclerotic plaques. Specifically, vulnerable atherosclerotic plaques (type-IV, Va) contain significant accumulated cholesterol, lipids and calcified crystals. To develop the plaque array method, first fluorescently-labeled plaque aggregates (0 hr) comprising cholesterol, phospholipids, Abeta peptide and/or calcium-phosphate were prepared. Then, self-formed plaque particles (24 hrs) were synthesized from plaque aggregates by incubating for 24 hrs. The self-formed plaque particles can be detected using a flow cytometer.
The preparation of plaque aggregates from individual molecules was reported earlier (Madasamy, 2009, USPTO Application #: 20090104121). Briefly, 1 mg of fluorescently-labeled cholesterol or cholesterol derivatives (Ex/Em=495 nm/507 nm) was solubilized in 1 mL of 100% alcohol. From this stock solution, 100 μL was taken and mixed in 900 μL of PBS. The transfer of esterified cholesterol molecules from organic medium (alcohol) to PBS buffer caused transformation of individual molecules into cholesterol plaque aggregates (0 hr). The samples were centrifuged for 5 min. at 5000 rpm to remove precipitates, if any, and the supernatant containing soluble aggregates were used for plaque array assay.
Next, 1-10 μg of the fluorescently-labeled cholesterol plaque aggregate was incubated at 37° C. for 24 hrs to analyze self-formation of plaque particles in the absence of any added biofluids. Both plaque aggregates (0 hr) and self-formed plaque particles (24 hrs) were analyzed using flow cytometer.
Next, to prepare fluorescently-labeled phospholipids (LS) plaque aggregates, 1 mg of fluorescently-labeled-phospholipids or its derivatives (Ex/Em=495 nm/507 nm) was solubilized in 1 mL of 100% alcohol. From this stock solution, 100 μL was taken and mixed in 900 μL of PBS. The samples were centrifuged for 5 min. at 5000 rpm to remove precipitate, if any, and the supernatant containing plaque aggregates were used for plaque array assay. The transfer of esterified phospholipids molecules from organic medium (alcohol) to PBS buffer caused transformation of these molecules into phospholipid plaque aggregates. It is notable, when the cholesterol or lipid molecules transform-into soluble or insoluble aggregates they acquire new conformations (Stapronos et al 2003; McCourt et al 1997) thus the resulting plaque aggregates are structurally different from their soluble forms. Next, 1-10 μg of the fluorescently-labeled phospholipid plaque aggregates were incubated at 37° C. for 24 hr to allow synthesis of self-formed plaque particles in the absence of any added biofluids. For detection of both plaque aggregates (0 hr) and self-formed plaque particles (24 hrs) a flow cytometer was used.
Together, these results indicate that the 0 hr Ch1, LS and Abeta-42 plaque aggregates are soluble so they were not efficiently detected by flow cytometry. However, when they are incubated at 37° C. for 24 or more hrs they interact and become insoluble plaque particles that can be detected by flow cytometer (
Different combinations of fluorescently-labeled plaque aggregates or self-formed plaque particles are prepared to mimic various stages of atherosclerosis and used for screening human serum and plasma samples. For each assay using the fluorescently-labeled plaque aggregates, the plasma or serum samples obtained from atherosclerotic subjects and normal healthy subjects are first centrifuged at 5,000 rpm for 5 min. and the supernatants are transferred to new centrifuge tubes. Next, the supernatants are diluted in PBS to make 50% of the serum and plasma samples and used to treat plaque aggregates or self-formed plaque particles to examine if in vitro plaque particle synthesis occurs. Each assay is performed in a 200 μL reaction (100 μL of 50% plasma or serum) and 100 μL (10 μg) of the Ch1-plaque aggregates and the mixtures are incubated at 37° C. for 1 hr. After the incubation, 300 μL sheath fluid is added to the mixture and the samples are used for acquisition (1-2000 events/particles for 1 min) in flow cytometer.
A significantly higher number of plaque particles were produced in the incubations with serum samples of the atherosclerosis subjects compared to the controls. We conclude that the presence of serum from subjects with atherosclerosis accelerates the synthesis of plaque particles from plaque aggregates.
A time course study was performed to monitor the formation of plaque particles in the presence and absence of serum from subjects with atherosclerosis. After incubation of fluorescently-labeled cholesterol plaque aggregates (0 hr) with serum, samples were collected at various time points and used for acquisition (1-2000 events/particles for 1 min) in the flow cytometry.
The dot plots reveal a significantly higher number of plaque particles formed in the incubations with serum of subjects with atherosclerosis subjects compared to normal subjects. In addition, the time course shows that the number of plaque particles formed in a sample increased over time from 0 to 24 hrs and analyzed at different time points.
These results suggest that serum samples of the subjects with known history of atherosclerosis related cardiovascular diseases contain factors that ‘catalyze’ in vitro formation of plaque particles from plaque aggregates. In the controls, a small number of plaque particles were detected suggesting the serum samples of the normal subjects may either lack factors that contribute to the plaque particle formation or contain factors that inhibit the in vitro plaque particle formation. It is notable that the human serum or plasma is a complex biological fluid containing approximately 289 proteins and 107 variants of circulating immunoglobulins at a given point in time (Molina H et al, 2005). The accelerated formation of plaque particles may be due to binding of plaque aggregates with a number of molecules including proteins, antibodies, lipids, carbohydrates, metals and metabolites that are present in the serum of atherosclerotic subjects. It would seem likely that substances involved in the formation or assembly of in vitro plaque particles might be specific and correlate with in vivo atherosclerotic plaque development.
Next, fluorescently labeled cholesterol self-formed plaque particles prepared by 24 hrs incubation of cholesterol plaque aggregates (0 hr) at 37° C. were used for incubation with serum samples. For the control experiment, fluorescently-labeled cholesterol plaque particles incubated in PBS in the absence of serum. Each in vitro plaque particle formation assay is performed in a 200 μL reaction (100 μL of 50% plasma or serum and 100 μL (10 μg) of the fluorescently-labeled cholesterol self-formed plaque particles and the mixtures are incubated at 37° C. for 1 hr. After the incubation, 300 μL sheath fluid is added to the mixture and the samples were analyzed in the flow cytometer.
Next, to further examine whether accelerated plaque particle synthesis observed with the Ch1-plaque aggregates is unique mechanism or common to other lipid plaque aggregates, fluorescently-labeled LS-plaque aggregates were prepared and used for screening the diluted human serum and plasma samples. Experiments were performed with serum samples collected from subjects identified for atherosclerosis indications and normal healthy subjects. For control experiment, fluorescently-labeled plaque aggregates were incubated in PBS and not treated with the serum. Each in vitro plaque particle formation assay is performed in a 200 μL reaction (100 μL of 50% plasma or serum and 100 μL (10 μg) of the LS-plaque aggregates and the mixtures are incubated at 37° C. for 1 hr. After the incubation, 300 μL sheath fluid is added to the mixture and the samples were analyzed by flow cytometry.
As shown in
Dot plot analysis reveals that, as observed earlier with the cholesterol plaque aggregates, phospholipid plaque aggregates were not detectable by flow cytometry (
The in vivo atherosclerotic plaques contain immunoglobulin isoforms IgG, IgM and IgA that are co-localized with lipids and cholesterol deposits. However, their role in the manifestations of the atherosclerosis is not completely understood (Hannson G et al, 1984). We have previously identified antibodies in the serum samples of atherosclerotic subjects that bind to self-formed plaque aggregates (Madasamy S, 2009, USPTO Application #: 20090104121). The results described in Examples 3 to 5 demonstrate that serum of subjects with atherosclerosis contain factors that contribute to enhanced in vitro plaque particle formation.
In order to delineate the role of immunoglobulins for in vitro plaque particle formation, IgG-depleted serum of subjects with atherosclerosis was examined using cholesterol plaque aggregates. The IgG depleted serum was prepared by incubating (1 hr at RT) diluted serum in micro-titre plate pre-coated with protein A and protein A/G and pre-blocked with blocking buffer. After incubation, the serum supernatant was screened for its ability to accelerate in vitro plaque particle formation from plaque aggregates. Each assay was performed in a 200 μL reaction (100 μL of diluted serum final concentration is 25% and 100 μL of the aggregates) and the mixtures are incubated at 37° C. for 1 hr for in vitro plaque particle formation. Sheath fluid (300 μL) was added to the mixture and samples were analyzed by flow cytometry for detection and counting of plaque particles.
Abnormal metabolism of cholesterol is implicated in the development of vascular dementia in Alzheimer's disease (Umeda T, et al, 2012). Increasing evidence shows a strong correlation between impaired metabolism of cholesterol and Abeta peptides and that together they play a role in the development of vascular dementia (Pac-Soo C, et al, 2011). Although, the serum samples were collected from subjects with known history of atherosclerosis, we decided to examine whether these subjects had any amyloid plaque related disorders. In order to probe this, as performed earlier, the serum samples were screened using Abeta-42 aggregates and analysis of the resulting dot plot data showed ˜20% of the atherosclerosis subjects were positive for Abeta-42 plaque particles compared to the controls (Table 2). Together, these results strongly suggest the plaque array method could be successfully used to identify and stratify atherosclerosis and AD subjects using different types of plaque aggregates or self-formed plaque particles.
Using atherosclerotic mouse models, mice carrying ApoE-gene mutation and normal C57BL/6 mice with same age group were fed with atherogenic diet from 8 weeks to 20 weeks. Serum samples collected at different time points were used for detection of disease progression based on in vitro plaque particle formation from fluorescently-labeled cholesterol plaque aggregates.
Taken together, the plaque array method using fluorescently-labeled Ch1-plaque and LS-plaque aggregates or self-formed plaque particles in combination with serum samples or other biofluids provides a novel serological diagnosis test that helps to measure and profile serum samples from known and unknown subjects. In general, serologic assays are useful both for evaluating the high risk individuals and also complementing risk analysis using data obtained from other diagnosis methods for ultimate decision-making. It is possible the titers of serum molecules that promote plaque particle formation have unique patterns of rise and fall during the longer window period of the atherosclerotic plaque developments. Finally, applying this plaque array method significantly helps rapidly diagnose asymptomatic individuals and take appropriate preventive measures at the early stage of disease development.
The goal was to examine whether serum from subjects with AD accelerates the synthesis of plaque particles from Abeta-42 aggregates. To prepare fluorescently-labeled Abeta-42 aggregates, 1 mg of fluorescently-labeled Abeta-42 peptide was suspended in 1 mL of PBS and the sample was incubated at 37° C. for 6 hrs. The samples were centrifuged for 5 min. at 5000 rpm to remove precipitate, if any, and the supernatant containing aggregates were used for plaque array assay. Similarly, to prepare unlabeled Abeta-42 aggregates, 1 mg of Abeta-42 peptide was suspended in 1 mL of PBS and the sample was incubated at 37 for 6 hrs. For Abeta-28 aggregates preparation, 1 mg of Abeta-28 was suspended in 1 mL of PBS and the sample was incubated at 37° C. for 6 hrs. The Abeta aggregates prepared by incubation for 6 hrs were not detectable by flow cytometer whereas the self-formed Abeta particles prepared by incubations at 37° C. for 36-48 hrs were detected by flow cytometer. This suggests prolonged incubation of aggregates leads to self-formed plaque particles in the absence of biofluids (
As shown in
Next, the serum samples of subjects with AD were screened against three different plaque aggregates to determine the profiles of plaque particle formation among patients. The serum of subjects with AD was tested against Abeta-42 Abeta-28 and cholesterol plaque aggregates for in vitro plaque particle formation according to the protocol described in Example 8 above. As shown in
These results suggest each patient serum forms different levels of amyloid and atherosclerotic plaque particle formation which in turn might determine the development and progression of multiple plaque related disorders. The profiles of in vitro plaque particle formation can be applied to distinguish patients affected with Abeta related dementia or vascular dementia caused by abnormal metabolism of Abeta peptide and cholesterol, respectively. These results are in good agreement with our preceding observations with atherosclerotic plaque particle formation and indicate serum samples of subjects with AD contain molecules that catalyze in vitro plaque particle formation.
The role of immune system in the development of AD and associated neuroinflammation is well established. Autoantibodies against Abeta peptides have been identified in AD serum samples (Maetzler W, et al, 2011: Hou H, et al, 2012). The results described in the Example 9 demonstrate that serum samples of AD subjects contain factors that contribute to enhanced in vitro plaque particles formation. In order to delineate the role of immunoglobulins in in vitro plaque particle formation, IgG-depleted serum samples of Abeta positive subjects were examined using Abeta-42 aggregates. For antibody response assay, IgG-depleted serum was prepared by incubating diluted serum in microtitre plates pre-coated with protein A and protein A/G and pre-blocked with blocking buffer (1 hr at RT). After incubation, the serum supernatant was incubated with Abeta-42 plaque aggregates and in vitro plaque particle formation was determined. Each assay was performed in a 200 μL reaction and the mixtures were incubated at 37° C. for 1 hr. Sheath fluid (300 μL) was added to the mixture and samples were analyzed by flow cytometry for detection and counting of plaque particles.
Flow cytometric analysis of unlabeled Abeta-42 plaque aggregates incubated for 1 hr with: (left) serum from subject with AD; (middle) IgG-depleted serum i.e. serum from subject with AD that was pretreated with protein A/G and (right) IgG-depleted serum i.e. serum from subject with AD that was pretreated with protein A. The plaque particles were detected using Thioflavin S dye. It was observed that IgG-depleted serum samples showed a significant reduction in the number of plaque particle formation compared with the respective controls that are not treated with protein A/G for IgG-depletion (
AD mice model carrying APPSWE/PS-1 mutant genes and C57BL/6 normal mice with same age group were fed with normal diet from 8 weeks to 20 weeks. Serum samples collected at different time points were used for detection of plaque progression using plaque array method. Abeta-42 plaque aggregates were used to examine in vitro plaque particle formation using serum samples from AD model mice and normal mice. The results indicate progressive increase in the number of Abeta-42 plaque particle formation for AD mice model serum samples that are collected from week 8 to week 20. Compared to the AD mice model, a small number of plaque particle formation was observed for control or normal mice (
Fluorescence Resonance Energy Transfer (FRET) is a versatile biochemical method widely used to study the interaction between bimolecular (Selvin P R 2000). In order to further examine the self-assembly process of plaque aggregates and their transformation in to self-formed plaque particles in the absence of biofluids, the following experiments were carried out. The aforementioned examples demonstrated that fluorescently-labeled plaque aggregates interact or self-assemble with each other leading to the formation of self-formed plaque particles albeit slowly (
For the FRET assay, we used the donor Abeta-42 aggregate labeled with a fluorophore (Ext/Emi=485/520) and the acceptor Abeta-42 plaque aggregates labeled with another fluorophore (Ext/Emi=540/570). When the fluorophore of the donor Abeta-42 aggregates is excited directly at 485 nm, a portion of that energy is acquired by the acceptor Abeta-42 followed by emission at the 570 nm wavelength of the donor fluorophore. Significantly, such energy transfer can only occur when the two dyes are in close spatial proximity (typically less than 100 Å) so the extent of signal will depend on the extent of interaction between the donors and acceptors.
As shown in
In the control FRET experiment, the Abeta-42 plaque aggregates labeled with two different fluorophores when incubated in PBS samples will self-assemble into plaque particles over time leading to the generation of some signal in an end point FRET assay. In the presence of serum of subjects with AD, the rate of formation Abeta-42 plaque particles from Abeta-42 plaque aggregates is significantly accelerated so an enhanced level signal is detected in an end point FRET assay (
As shown in the
For developing a combination FRET assay to screen both AD and atherosclerosis serum samples using one pair of plaque aggregates, the donor Abeta-42 plaque aggregates was labeled with a fluorophore (Ext/Emi=485/520) and the acceptor cholesterol plaque aggregates was labeled with another fluorophore (Ext/Emi=540/570). In this case, when the fluorophore of the donor Abeta-42 aggregates is excited directly at 485 nm, a portion of that excitation energy is acquired by the acceptor cholesterol followed by emission at the 570 nm wavelength of the donor dye. In the control FRET assay, the Abeta-42 plaque and cholesterol aggregates labeled with fluorophores with two different fluorescent excitation/emissions when incubated in PBS or normal serum samples interact with each other leading to the generation of less plaque particles and less FRET signal. In the presence of AD or atherosclerosis serum samples, the interaction between the abeta-42 plaque aggregates is accelerated to form higher number of plaque particles which in turn lead to generation of enhanced the FRET signal. Accordingly, screening of serum samples using the combination FRET assay will help to identify asymptomatic subjects of atherosclerosis and AD.
Next, to further understand the mechanism of plaque particles assembly enhanced by serum samples, the images of the in vitro formed Abeta-42 plaque particles were analyzed. Accordingly, to capture the images of individual Abeta-42 plaque particles, serum samples of the AD subjects and the Abeta-42 plaque aggregates (6 hrs) are incubated at 37° C. for 1 hr. After incubation with diluted serum, 10 μL, of Thioflavin S (Ex/Em=430 nm/550 nm) fluorescent dye (10 μg) was added and the sample was incubated for an additional 30 min. at 37° C. Following incubation, images of Abeta-42 plaque particles were acquired using a Imaging flow cytometer (Amnis Corporation, Seattle, Wash., USA). Analysis of images of plaque particles showed at least are three different sizes of Abeta-42 plaque particles (
To further understand the mechanism of plaque particles assembly in the serum samples the images of the in vitro plaque particles were analyzed. Accordingly, to capture the images of individual cholesterol plaque particles, serum samples of atherosclerotic subjects and fluorescently-labeled cholesterol plaque aggregates are incubated at 37° C. for 1 hr. Acquisition of the resulting samples by Imaging flow cytometer and dot plot analysis showed that there are three major species of cholesterol plaque particles produced as a result of incubation with serum samples (
Next, to further confirm the results (Examples 14 and 15) indicating sub-types of cholesterol and phospholipid plaque particles the following experiments were performed. As aforementioned in Examples 4 to 8, fluorescently-labeled cholesterol and phospholipid plaque aggregates were prepared and used for incubation in the diluted human atherosclerotic serum. In the aforementioned examples (Examples 4 to 8), the samples containing in vitro formed cholesterol and phospholipid plaque particles were acquired using flow cytometer for detection of up to 0 to 2000 events/particles per minute. In the present assay, in order to identify rare/all species of plaque particles, more sample volume was acquired in the flow cytometer to capture greater than 10,000 events/plaque particles per minute.
As shown in
Similarly, in order to identify rare/all species of phospholipid plaque particles, more sample volume was acquired in the flow cytometer to capture greater than 10,000 events/plaque particles per minute. As shown in
The data collected from multiple plaque array analysis such as detection of accelerated plaque particles formation, counting the plaque particle numbers, role of antibodies in plaque particles formation, the phenotype analysis of plaque particles using imaging and identification of plaque particle sub-types together help to develop “Plaque Fingerprinting” (PF). The application of “Plaque Fingerprints” includes clinical diagnosis, developing companion diagnosis for a particular drug and develops personalized medicine for subjects associated with plaque related disorders including AD and atherosclerosis.
The preceding examples strongly suggest that based on accelerated plaque particles formation both asymptomatic and symptomatic individuals of amyloid diseases including Alzheimer's disease and atherosclerosis disease could be rapidly diagnosed using this non invasive plaque array method. It is anticipated that the profile of accelerated plaque particles formation would vary from normal individual to suspected individuals and comparing their profiles would help to diagnose patients and predict severity of the AD and atherosclerotic plaque development non-invasively. Finally, detecting the asymptomatic individuals using the plaque array method would help to identify the suspected asymptomatic individuals early and treat them with appropriate therapy.
The preceding examples clearly proved that serum samples of atherosclerotic and Alzheimer's disease subjects contain molecules that contribute to the accelerated formation of the plaque particles. Accordingly, both the mechanisms of in vitro plaque particles formation in the biofluids and the factors contributing to this process are potential targets for drug discovery. Any drug or drug like molecules that would be identified for disrupting or accelerating or inhibiting these processes would be a novel therapeutic candidate for treatment of atherosclerosis and other amyloid plaques related diseases.
The first objective of the drug discovery efforts is to identify molecules that bind to the plaque aggregates so that the initiation of the plaque particles assembly in the biofluids could be altered. To achieve this goal, screening is carried out using phage display cDNA library of human endothelial cells. For binding of plaque aggregates to phage libraries, fluorescently-labeled cholesterol plaque aggregates (10 μg) or Abeta-42 plaque aggregates (10 μg) are mixed with either cDNA library (pfu 1×106) or phage display peptide library (pfu 1×106). After 30 min. incubation at 37° C., the samples are detected by flow cytometry.
As shown in
A similar approach could be employed to successfully identify small molecules after screening the chemical libraries and positive hits could be characterized and identified by mass spectrometry. The above described results clearly indicate that the plaque array is a powerful drug discovery platform for accelerated drug discovery. Also, the platform enables one to screen molecule libraries to identify novel anti-atherosclerotic and anti-amyloid compounds that would effectively disrupt multiple interactions contributing to in vitro plaque particle formation. Because of its unique and innovative nature, the plaque array based drug discovery platform makes it possible to rapidly identify novel therapeutic modalities to prevent or cure atherosclerosis, Alzheimer's and other plaques related diseases.
In order to determine the interplay between the plaque components and cells, the fluorescently-labeled plaque aggregates or self-formed plaque particles were incubated with both endothelial cells To achieve this, human coronary artery endothelial cells (HCAECs) are grown to confluence in 75 cm2 culture flasks containing 20 mL of endothelial cell growth medium (ECGM). After removing the medium, the cells are scraped off the plate and washed once with ice cold PBS buffer. After centrifugation and cell count, the HCAECs (500,000) were incubated with fluorescently-labeled Ch1 plaque aggregates (5 μg) and LS plaque aggregates (5 μg). After 30 min. incubation at room temperature the plaque aggregate treated cells were detected by flow cytometry.
Next, to probe the consequences of binding of plaque aggregates with the endothelial cells the plaque infected cells were analyzed for apoptosis. First, the hybrid plaque aggregates such as calcium phosphate (CP)-Ch1-LS and Ch1-LS are prepared as reported earlier (Madasamy S, 2009, USPTO Application #: 20090104121) and incubated with the HCAECs. For the control experiment, the calcium containing hybrid plaque aggregates CP-Ch1-LS were incubated with Flu 3 fluorescent dye that specifically binds to calcium moiety of the plaque aggregates. Second, the plaque aggregate treated cells are grown for 12 hrs. After removing the medium, the cells are scraped off the plate and washed once with ice cold PBS buffer. After centrifugation and cell count, the HCAECs (500,000) are assayed for apoptosis using fluorescently-labeled Annexin V (FITC) and propidium iodide (PI) fluorescent DNA binding dye. After 20 min. incubation at room temperature the cells are sorted in the flow cytometer. The dot plot analysis showed that the control cells that were not treated with plaque aggregates showed no significant apoptosis (
This plaque aggregates infected cell culture system using HCAECs or PBMCs enables assembly of different plaque sub types to mimic atherosclerotic plaques sub types such as pre-atheroma (type 1 to III), containing high lipids content and atheroma type (type IV and Va) containing CP, Ch1, lipids and fibrin clots. Identification of drug compounds that prevent or inhibit the pathogenic effect of plaque aggregates or plaque particles on HCAECs or PBMCS could be successful therapeutic candidates for treating atherosclerosis and other plaques related diseases. In addition, the plaque based HCAEC and PBMC cell culture model system could be used to test efficacy and safety of the anti-atherosclerotic or anti-amyloid drugs.
| Number | Date | Country | |
|---|---|---|---|
| 61566580 | Dec 2011 | US |
