Patent Application
20230416799
This invention relates to the process of production of sLox-1, more particularly to production of sLox-1 in the cultured blood clot, application of sLox-1 and the manner to produce sLox-1 in the cultured blood clot.
Lectin-like oxidized low-density lipoprotein receptor 1 (Lox-1) is a scavenger receptor that recognizes oxidized low-density lipoprotein (oxLDL) and activated platelets1,12. In healthy arteries, Lox-1 expression is scarcely detected, but a chronic diet rich in saturated fats and cholesterol induces Lox-1 expression on coronary artery endothelial cells10. Endothelial cells exposed to oxLDL upregulate adhesion receptors that capture platelets, and monocytes that can become Lox-1-expressing macrophages, scavenge oxLDL and convert into “foamy” fat-laden cells12,13.
Over time, these events contribute to stiffening of the arteries and the deposition of fatty plaques in the inner lining of the arteries. These structural changes contribute to hypertension which has a snowball effect of increasing vascular damage, ischemia, and cardiomyopathy. oxLDL also stimulates Lox-1-expressing cells to release pro-fibrotic mediators that drive tissue hypertrophy, fibrosis, atrial fibrillation14. Quivering of the atrium that occurs in diastolic or systolic dysfunction can lead to pooling of the blood and clot formation on the side-wall of the atrium. These clots can break free and lodge in the lung or brain tissue causing transient ischemia, strokes, and sudden death.
Although less-studied, cardiomyopathy can develop after severe trauma11. A subset of Warfighters recovering from battlefield injuries may therefore face an additional long-term struggle with chronic heart disease. An understanding of why some individuals develop cardiomyopathy while sparing others is lacking.
Lox-1 has an established role in cardiovascular disease1-10, and may have a role in trauma-induced cardiomyopathy11.
Shock trauma after resuscitation from massive hemorrhage stimulates intravascular protease activity (ADAM17) that sheds the glycocalyx from endothelial cells leading to vascular injury15. These same proteases were shown to cleave the Lox-1 extracellular domain from this type II transmembrane receptor, to release soluble Lox-1 (sLox-1) (
sLox-1 was proposed as a biomarker of early-stage acute coronary syndrome and myocardial infarction17,18, but the cells shedding the ectodomain have yet to be identified. In specific disease states (lung cancer, thrombotic influenza, thrombotic COVID), peripheral blood neutrophils can acquire Lox-1 expression19-21. In lung cancer patients, Lox-1 was identified as a biomarker of myeloid-derived suppressor cells (MDSCs) along with selected soluble mediators known to drive atrial hypertrophy and fibrosis (CXCL8, CXCL2, CCL2, IL1A, TNF, VEGFA)19,22. A subset of these mediators were found in blood samples of patients with obstructive sleep apnea, a condition characterized by intermittent hypoxia/reoxygenation (IHR)8,9,23.
These data suggest that Lox-1 and sLox-1 levels may be altered by hemorrhage, ischemia-reperfusion but in healthy human donors, endothelial cell expression of Lox-1 is scarce. The cells producing peripheral blood sLox-1 remain to be identified. It is currently unclear how to diagnose the risk or to treat trauma-induced cardiomyopathy. In patients with thrombocytopenia, is currently unclear how to diagnose low platelet counts caused by inadequate platelet production from low platelet counts caused by intravascular platelet activation (thrombosis).
Methods for producing sLox-1 are currently limited to complex systems involving cultured cells, mainly bacterial cells that cannot properly fold, dimerize or glycosylate sLox-1. Autologous sLox-1 with personalized glycosylations is not currently available for clinical use.
Further, research papers till the time of the present invention could not identify the cell source of sLox-1 and assumed that the endothelial cells were shedding sLox-1 into the bloodstream.
In an embodiment, present invention provides a method of generating, ex vivo production of soluble Lox-1 (sLox-1), comprising: introducing a sample containing a blood free from an anti-coagulant factor into a device; adding a coagulation enhancing material in the blood; incubating the device; forming a cultured blood clot in the device; and shedding of the sLox-1 outside the cultured blood clot, wherein the method is configured to shed sLox-1 more than an anti-coagulated blood.
In an embodiment, the coagulating enhancing material comprises a lipopolysaccharide (LPS).
In an embodiment, the anti-coagulant factor comprises heparin, citrate, or ethylene diamine tetraacetic acid (EDTA).
In an embodiment, detecting one or more interleukins in the device.
In an embodiment, the one or more interleukins comprises IL-6, IL-8 and/or IL-18.
In an embodiment, addition of the inflammation enhancing material in the blood spikes shedding of sLox-1 into the device by about 20% to 60% more compared to a cultured blood clot free of the coagulation enhancing material.
In an embodiment, the method is configured to shed 0.5 ng to 50 ng of the sLox-1 per ml of the blood sample.
In an embodiment, the method is configured to produce an autologous sLox-1.
In an embodiment, the device is incubated at a temperature ranging from 20° C. to 40° C. for a time period ranging from 1 hour to 18 hours.
In an embodiment, the sLox-1 so sheds in the device is a personalized anti-coagulant or anti-thrombogenic agent.
In an embodiment, the device comprises a thrombus mimetic device.
In an embodiment, the method is configured to detect IL-8 and IL-6 concentrations in the cultured blood clot and the anti-coagulated blood.
In an embodiment, concentration of the sLox-1 in serum of the cultured clot compared to the anti-coagulated blood is from 0 pg/mL to about 50 ng/mL.
In an embodiment, concentration of the IL-8 in serum of the cultured clot compared to the anti-coagulated blood is from 50 pg/mL to 50 ng/ml.
In an embodiment, concentration of the IL-6 in serum of the cultured clot compared to the anti-coagulated blood is from 0 to 100 ng/mL.
In an embodiment, a device comprising a vacutainer tube and a means of heating to maintain temperature of the device at about 37° C., wherein the device is configured to screen agents that promote or inhibit one or more of the following: cell apoptosis, scramblase activity, flippase activity, ADAM17 activity, ADAM10 activity, alpha secretase activity, sLox-1 sheddase activity, tumor necrosis factor activation.
In an embodiment, the device is configured to screen a drug, antibody, nanoparticle, microbial-derived component, biomaterial, neutraceutical/dietary supplement as causing an increase or decrease in cultured clot serum sLox-1 relative to untreated cultured clot serum sLox-1, without inducing cell necrosis.
In an embodiment, the drug is a chemotherapeutic agent intended to induce apoptosis.
In an embodiment, the drug is configured to decrease ADAM17 activity and TNF activation.
In an embodiment, the drug is configured to suppress TNF expression or activity.
In an embodiment, the drug comprises a serine-threonine phosphatase inhibitor.
In an embodiment, the drug comprises beta glycerol phosphate.
The accompanying drawings, which are included to provide further understanding of the present invention disclosed in the present disclosure and are incorporated in and constitute a part of this specification, illustrate aspects of the present invention and together with the description serve to explain the principles of the present invention. In the drawings:
In
In
Numbers (1-4) on x-axis refers to: 1) cultured clot+LPS, 2) cultured clot, 3) cultured heparin blood, and 4) cultured heparin blood+LPS. Number of genes in each cluster: a) 378, b) 95, c) 3, d) 1, e) 18, f) 912, g) 2, h) 587, i) 3, j) 50, k) 10, l) 5, m) 11, n) 356, o) 1, p) 190, q) 2, r) 1, s) 69, t) 594, u) 11, v) 757, w) 549, x) 1, y) 2, z) 9, aa) 56, bb) 5, cc) 1, dd) 486, ee) 128, ff) 16, gg) 80, hh) 6, ii) 3.
For simplicity and clarity of illustration, the drawing figures illustrate the general manner of construction, and descriptions and details of well-known features and techniques may be omitted to avoid unnecessarily obscuring the present disclosure. Additionally, elements in the drawing figures are not necessarily drawn to scale. For example, the dimensions of some of the elements in the figures may be exaggerated relative to other elements to help improve understanding of embodiments of the present disclosure. The same reference numerals in different figures denote the same elements.
The terms “first,” “second,” “third,” “fourth,” and the like in the description and in the claims, if any, are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances such that the embodiments described herein are, for example, capable of operation in sequences other than those illustrated or otherwise described herein. Furthermore, the terms “include,” and “have,” and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, device, or apparatus that comprises a list of elements is not necessarily limited to those elements, but may include other elements not expressly listed or inherent to such process, method, system, article, device, or apparatus.
The terms “left,” “right,” “front,” “back,” “top,” “bottom,” “over,” “under,” and the like in the description and in the claims, if any, are used for descriptive purposes and not necessarily for describing permanent relative positions. It is to be understood that the terms so used are interchangeable under appropriate circumstances such that the embodiments of the apparatus, methods, and/or articles of manufacture described herein are, for example, capable of operation in other orientations than those illustrated or otherwise described herein.
No element, act, or instruction used herein should be construed as critical or essential unless explicitly described as such. Also, as used herein, the articles “a” and “an” are intended to include items, and may be used interchangeably with “one or more.” Furthermore, as used herein, the term “set” is intended to include items (e.g., related items, unrelated items, a combination of related items, and unrelated items, etc.), and may be used interchangeably with “one or more.” Where only one item is intended, the term “one” or similar language is used. Also, as used herein, the terms “has,” “have,” “having,” or the like are intended to be open-ended terms. Further, the phrase “based on” is intended to mean “based, at least in part, on” unless explicitly stated otherwise.
The present invention may be embodied in other specific forms without departing from its spirit or characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.
As defined herein, “approximately” can, in some embodiments, mean within plus or minus ten percent of the stated value. In other embodiments, “approximately” can mean within plus or minus five percent of the stated value. In further embodiments, “approximately” can mean within plus or minus three percent of the stated value. In yet other embodiments, “approximately” can mean within plus or minus one percent of the stated value.
Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, health monitoring described herein are those well-known and commonly used in the art.
The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. The nomenclatures used in connection with, and the procedures and techniques of embodiments herein, and other related fields described herein are those well-known and commonly used in the art.
For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
The percentage given any should be construed based on the weight %, unless indicated otherwise.
The following terms and phrases, unless otherwise indicated, shall be understood to have the following meanings.
As used herein, the terms “subject”, “patient” and “subject in need thereof” may be used interchangeably and refer to a subject in need of administration of the pharmaceutical composition of the invention, or in need of pre-operative, post-operative, or periodic blood sampling to document health status. The term “subject” denotes a mammal, such as a rodent, a feline, a canine, an equine, a goat, a pig, a transgenic pig, a bovine, and a primate.
As used herein, the term “ex vivo” refers to the process by which cells are removed from a living organism and grown outside the organism (eg, a test tube). As used herein, the term “in vitro” refers to the process by which cells known to grow only in vitro (eg, various cell lines, etc.) are cultured.
Ex vivo production of sLox-1, according to this aspect of the present invention, provides white blood cells (WBC) with conditions for cell growth ex vivo, culturing WBC ex vivo with or without cytokine, thereby allowing production of sLox-1. In another aspect of the present invention, Ex vivo provides neutrophils with conditions for cell growth, culturing neutrophils ex vivo with or without cytokine, thereby allowing production of sLox-1.
As used herein, “culturing” provides the chemical and physical conditions (eg, temperature, gas) and growth factors required for the maintenance of blood clot. In an embodiment, culturing provides the chemical and physical conditions for maintenance of neutrophils.
As used herein, “cultured blood clot” or “cultured clot” refers to a process when the blood is allowed to clot, for example: a small volume of blood is collected in a sterile container and allowed to clot within an effective duration time to form a clot. In an embodiment, the clot could be collected and put into in a suitable broth to collect serum.
In an embodiment, “cultured clot” is different from “fresh blood clot.” The fresh blood clot is formed by incubation at room temperature for at least 45 minutes and up to as long as standard laboratory tests for patient serum allow.
The advantage of clot culture is that serum can be collected and investigated for serological tests. The cultured blood clot could be either treated or untreated.
As used herein, term “treated cultured blood clot” or “treated cultured clot” refers to when the formation of the blood clot is positively influenced by addition of a drug, inflammatory factor, enhancing factor, coagulant factor, antibody, nanomaterial, nucleic acid, enzyme, pathogen-derived factor etc.
As used herein term, “untreated cultured blood clot” refers to when the formation of the blood clot happens due to its natural process with only the addition of inert anionic surfaces (e.g., glass or silicates) to initiate coagulation.
As used herein, “cultured clot serum” refers to serum that is collected from a cultured clot. In an embodiment, the cultured clot is centrifuged to collect the serum supernatant free of any cells. The supernatant free of any cell works as a cultured clot serum.
As used herein, the expression “effective duration” of culturing blood clot refers to time required for production of sLox-1. The duration of culturing blood clot suitable for use in some embodiments of the present invention typically ranges from zero to 10 mins to 20 mins, about 0.5 hours to 4 hours to about 5 weeks; varying from 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 10 hours, 15 hours, 20 hours, 24 hours, 2 days, 3 days, 4 days, 5 days or more.
As used herein, the expression “anti-coagulant factor” refers to an agent or class of agents that prevents coagulation or clotting of blood. For example but not limited to agents which function by chelating calcium as known in the art. These anticoagulants function by combining with, precipitating and effectively removing calcium ions normally present in the blood. They therefore reduce the concentration of calcium ion in the blood below normal physiological levels. Generally, the anticoagulants which fall within this definition include the citrate anticoagulants, for example, acid citrate dextrose (ACD), citrate phosphate dextrose (CPD), and trisodium citrate (TSC). Ethylenediaminetetraacetic acid (EDTA) may also be used. CPD is the most preferred anticoagulant of the available citrates, as described in U.S. Pat. No. 4,359,463A, which is incorporated herein by reference in its entirety. In an embodiment, heparin is an anticoagulant. In an embodiment, sLox-1 is an anti-coagulant.
As used herein, the term “coagulant” or like refers to a substance capable of causing whole blood or a blood component (plasma, platelets) to form a clot.
In an embodiment, the present invention may preferably use calcium ions to reverse the action attic citrate anticoagulant present in plasma feedstock, as described in US20040120942A1, which is incorporated herein by reference in its entirety.
As used herein, the term “blood” used herein means whole blood including hemocytes (erythrocytes, leucocytes, platelets) and plasma (serum) that is a liquid component, and liquid containing at least one of these (for example, blood collected by apheresis). The term “blood” refers to “fresh blood” that is not coagulated. In some embodiments, fresh blood is also interchangeably referred as an anticoagulated blood.
As used herein, the term “serum” means a pale-yellow liquid obtained by allowing collected blood to stand, resulting in reduction of the fluidity, followed by separation from the red coagulated block (clot). As used herein, the term “autologous blood” refers to a patient's own blood. As used herein, the term “homologous blood” refers to that obtained from a blood donor other than the individual for whom the coagulant is prepared.
As used herein, the term, “coagulation” is also known as clotting, is the process by which blood changes from a liquid to a gel, forming a blood clot. The mechanism of coagulation involves activation, adhesion and aggregation of platelets, as well as deposition and maturation of fibrin.
As used herein, the term, “coagulation enhancing factor or coagulation enhancing material” or like are agents that stimulate the contact pathway of coagulation (membranes containing phosphatidylserine that activate Factor XII) or like agents that respond in a cascade to form fibrin strands, which strengthen the platelet plug. The platelet plug is also known as the hemostatic plug or platelet thrombus, is an aggregation of platelets formed during early stages of hemostasis in response to one or more injuries to blood vessel walls. After platelets are recruited and begin to accumulate around the breakage, their “sticky” nature allows them to adhere to each other. This forms a platelet plug, which prevents more blood from leaving the body as well as any outside contaminants from getting in. The plug provides a temporary blockage of the break in the vasculature. As such, platelet plug formation occurs after vasoconstriction of the blood vessels but before the creation of the fibrin mesh clot, which is the more permanent solution to the injury. The result of the platelet plug formation is the coagulation of blood. For example, but not limited to Tissue factor (TF), a transmembrane glycoprotein in combination of binding factor FVII/FVIIa, lipopolysaccharide (LPS), Cancer procoagulant, etc.
As used herein, the term, “inflammation enhancing material” or like refers to the material that either leads or increases the inflammation reaction in mammals known to a person skilled in the art. For example, but not limited to Bacterial pathogens such as Lipopolysaccharide, Vasoactive amines, eicosanoids, peptidoglycan, to viral particles and etc. The term “enhancing material” refers to any material that increases the level of sLox-1 in cultured blood clot. For example, but not limited to phorbol esters such as phorbol myristate acetate (PMA), etc.
As used herein, the term, “viability” of the cells refers to cells not undergoing necrosis or late apoptosis. According to some embodiments, the term “viable cells” refers to cells having an intact plasma membrane. Assays for determining cell viability are known in the art, such as using alamarblue metabolic dye which may be detected in the serum by fluorimetry, or propidium iodide (PI) or calcein AM staining which may be detected by epifluorescence microscopy of the clot or flow cytometry of single cells. Accordingly, according to some embodiments, viable cells are cells which metabolize calcein AM and do not show propidium iodide intake. Necrosis can be further identified, by using light, fluorescence or electron microscopy techniques, or via uptake of the dye trypan blue.
As used herein, the term, “necrosis” of the cells is another mode of cell death as known in the art. A “necrotizing cell disease” includes trauma, ischemia, stroke, myocardial infarction, carbon lethal toxin-induced septic shock, sepsis, LPS-induced cell death and HIV-induced T cell death leading to immunodeficiency, it refers to acute diseases not limited to the above. The term “necrotic cell disease” also refers to chronic neurodegenerative diseases (eg Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, Alzheimer's disease, infectious encephalopathy, dementia such as HIV-related dementia). But is not limited.
As used herein, the term, “activating agents” or like is used to provide a surface for reaction. Preferably, the activating agent provides a negatively charged catalytic surface that simulates the contact pathway of coagulation, or by exposure of blood to collagen type I. The intrinsic clotting cascade pathway is initiated by a process called contact activation, a surface and charge dependent phenomenon centered on the activation of Factor XII. Factor XII is highly susceptible to proteolysis because it is bound to surfaces via a charge interaction. The Factor XII precursor has areas of net positive charge that can interact with surfaces with a net negative charge. This charge binding induces conformational changes that enhance the molecule's ability to undergo activation by plasma kallikrein and Factor HK. “Activating agent” may also include thrombin.
Materials commonly used for contact activation include borosilicate glass (i.e., hematology glass), silicates, diatomaceous earth, ceramics, ellagic acid and kaolin. Ion exchange resins may also be suitable. Ion exchange resins can provide three separate process' functions in one single material: anionic activation of plasma proteins, a source of calcium to neutralize citrate, and molecular exclusion absorbance to remove water and low molecular weight fluids, thus, concentrating the high molecular weight constituents of the final serum. It has been demonstrated that a borosilicate glass with an anionic surface charge has these preferred features and can be used as an effective activating agent, as described in US20040120942A1 which is incorporated herein by reference it its entirety.
As used herein, the term, “interleukins (IL)” refers to a group of cytokines with complex immunomodulatory functions, including cell proliferation, maturation, migration and adhesion. In an embodiment of the invention, interleukins include human interleukin. ILs known to a person skilled in the art is covered according to an embodiment of the invention.
As used herein, the term, “coagulopathy” (also called a bleeding disorder) is a condition in which the blood's ability to coagulate (form clots) is enhanced or impaired. This condition can cause a tendency toward microthrombus formation or platelet depletion followed by prolonged or excessive bleeding (bleeding diathesis), which may occur spontaneously or following an injury or medical and dental procedures. The condition could result from multiple pathological, inheritable, trauma-induced or transfusion induced conditions, causing hypercoagulable or hypocoagulable states that may endanger life. Coagulopathy also occurs following acute trauma and hemorrhage in patients. In an embodiment, coagulopathy refers to platelet level dropped below 80,000 per microliter.
Trauma-induced coagulopathy is often the underlying cause of uncontrolled internal bleeding and, according to some accounts, leads to up to a fivefold increase in patient mortality. In order to normalize blood coagulation condition, a hemostasis therapy is essential that includes transfusion of whole blood or tissue factor concentrates. An early detection of the coagulopathy in patients and monitoring of coagulation metrics during the hemostasis therapy to guide therapeutic endpoints is important.
As used herein, the term, “cardiomyopathy” refers to diseases of the heart muscle. These diseases have many causes, signs and symptoms, and treatments. In cardiomyopathy, the heart muscle becomes enlarged, thick, or rigid. In rare cases, the muscle tissue in the heart is replaced with scar tissue. As cardiomyopathy worsens, the heart becomes weaker. It is less able to pump blood through the body and maintain a normal electrical rhythm. This can lead to heart failure or irregular heartbeats called arrhythmias. In turn, heart failure can cause fluid to build up in the lungs, ankles, feet, legs, or abdomen. The weakening of the heart also can cause other complications, such as heart valve problems.
The main types of cardiomyopathies are dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, and arrhythmogenic right ventricular dysplasia. Other types of cardiomyopathies sometimes are referred as “unclassified cardiomyopathy.” Cardiomyopathy can be acquired or inherited, with hypertrophic cardiomyopathy and arrhythmogenic right ventricular dysplasia substantially being inherited disorders. In some subjects, inherited cardiomyopathies are not evident until the occurrence of a catastrophic event (e.g., heart attack). Cardiomyopathy can be induced by other diseases or conditions, or by various toxins or drugs. For example, dilated cardiomyopathy can result from coronary heart disease, heart attack, high blood pressure, diabetes, thyroid disease, viral hepatitis, and HIV; infections, especially viral infections that inflame the heart muscle can result in cardiomyopathy; alcohol, especially in conjunction with a poor diet, etc.
As used herein, the term, “LOX-1” or “Lox-1” as used herein is a type II transmembrane cell surface receptor, lectin-like oxidized low density lipoprotein receptor 1, first identified in endothelial cells as one of the main receptors for oxidized-LDL (ox-LDL). Besides ox-LDL, this receptor has been shown to bind many different ligands including other modified lipoproteins, advanced glycosylation end products, aged red blood cells, apoptotic cells, bacteria and activated platelets. Interestingly LOX-1 has been involved in many different pathological conditions including atherogenesis, myocardial ischemia, hypertension, vascular diseases, stroke, and thrombosis11.
Expression of LOX-1 can be induced by a wide array of stimuli including pro-inflamatory factor (TNF-α, IL-1 or IFN-γ), angiotensin II, endothelin-1, modified lipoproteins and free radicals12. Engagement of LOX-1 can lead to induction of oxidative stress, apoptosis, endothelial dysfunction, fibrosis and inflammation through the activation of the NF-κB pathway. LOX-1 has also been described to play a role in tumorigenesis. Indeed, LOX-1 up-regulation has been observed during cellular transformation into cancer cell and can have a pro-oncogenic effect by activating the NF-κB pathway, by increasing DNA damage through increase ROS production and by promoting angiogenesis and cell dissemination. The nucleic, acid sequence for the gene encoding LOX-1 (gene name OLR1) can be found in databases such as NCBI, i.e., NCBI gene ID: 4973 or Gene sequence: Ensembl: ENSG00000173391. The LOX-1 protein sequence is found at Hugo Gene Nomenclature Committee 8133, Protein Sequence HPRD:04003.
The term LOX-1 can also represent die receptor protein in various species, and with conservative changes in the amino acid or encoding sequences, or with other naturally occurring modifications that may vary among species and between members of the same species, as well as naturally occurring mutations thereof.
The term “soluble LOX-1” or “sLox-1” means that a part of LOX-1 existing in the membrane (usually part of “extracellular domain”) is cleaved (dissociated) and released into the blood. Means (shed) part of LOX-1. In an embodiment, sLox-1 is shown in
Production of sLox-1
In an embodiment, a method of generating, ex vivo production of soluble Lox-1 (sLox-1), comprising introducing a blood sample into a device; adding a coagulation enhancing material in the device before clotting of the blood sample into the device; incubating the device; forming a cultured blood clot in the device; and shedding of the sLox-1 outside the cultured blood clot into the device.
In an embodiment, the blood sample has an anti-coagulant less than 10 wt. %, 9 wt. %, 8 wt. %, 7 wt. %, 6 wt. %, 5 wt. %, 4 wt. %, 3 wt. %, 2 wt. %, 1 wt. %, 0.5 wt. %, 0.1 wt. % or less.
In an embodiment, the blood sample is free from an anti-coagulant factor such as but not limited to heparin. 11111 In an embodiment, the blood sample is an anti-coagulated blood sample (fresh blood).
In an embodiment, the blood sample may contain coagulating enhancing material to accelerate formation of the clot.
In an embodiment, the blood sample may contain an enhancing material to stimulate sLox-1 production, for example comprises a lipopolysaccharide (LPS), a drug, or blood transfusion products (platelets, liposomes or microparticles), etc.
In an embodiment, the clot is incubated at the temperature around 20° C., 25° C., 30° C., 35° C., 37° C., 40° C., 45° C., 50° C. In an embodiment, the clot is incubated inside the device for about 0.5 hour (hr), 1 hr, 1.5 hrs, 2 hrs, 2.5 hrs, 3 hrs, 3.5 hrs, 4 hrs, 5 hrs, 6 hrs, 7 hrs, 8 hrs, 10 hrs, 12 hrs, 24 hrs or more.
In an embodiment, addition of the enhancing material in the blood forms a treated cultured clot. The cultured clot has about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more production of sLox-1 compared to an untreated cultured blood clot free of the coagulation enhancing material.
In an embodiment, addition of the enhancing material in the blood forms a treated cultured clot. The cultured clot has about 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10× or more production of sLox-1 compared to an untreated cultured blood clot free of the coagulation enhancing material.
In an embodiment, the method is configured to shed sLox-1 more than a fresh blood clot.
In an embodiment, during clot culture, the white blood cells are induced to express OLR1, the mRNA encoding Lox-1, and to shed on average 0.6 ng/mL sLox-1 into the serum. This novel technology enables the investigation of the role of donor sex, age, ethnicity, blood type, and other demographics in mechanisms of Lox-1 induction and sLox-1 shedding.
In an embodiment, the cultured clot system can be a precision medicine tool for detecting the capacity to induce sLox-1 appearance in the serum.
In an embodiment, sLox-1 can be readily and reproducibly generated in a serum sample, by placing whole blood from a donor at 37° C. The high levels of sLox-1 produced after just 4 hours of culture at 37° C. suggests that this approach may be used to generate autologous sLox-1 for clinical use. This approach could also be scalable for larger blood volumes from any species, to produce large amounts of bioactive sLox-1.
In an embodiment, our data shows that the thrombus may be a highly important source of Lox-1 expression and sLox-1 shedding.
In an embodiment, the method is configured to shed 0.5 ng to 50 ng of the sLox-1 per ml of the blood sample.
In an embodiment, the cultured clot serum sLox-1 concentration compared to fresh blood plasma is from 0 pg/mL to 10 ng/mL to 20 ng/mL to 30 ng/mL to 40 ng/mL to 50 ng/mL to 70 ng/mL to 100 ng/mL. In an embodiment, the concentration of sLox-1 in the cultured clot serum compared to fresh blood plasma is about 2 times, 3 times, 4 times, 5 times, 7 times, 10 times, 20 times, 50 times or 100 times more.
Lox-1 binds to phosphatidyl serine and to activated platelets which expose phosphatidyl serine to trigger the contact pathway for thrombin generation. It is therefore feasible that sLox-1 binds to platelet phosphatidyl serine, to have a two-fold effect in muffling the contact pathway activation of thrombin and limiting the ability of platelet microparticles to bind to endothelial cells.
Given these collective data, we postulate that thrombus-induced Lox-1 expression shedding of sLox-1 may be a natural protective mechanism whereby sLox-1 binds platelet microparticles to protect endothelial cells from becoming damaged by these pro-thrombogenic vesicles, and maintain hemostasis after traumatic injury (
In an embodiment, cultured clots were spiked with LPS by injecting an LPS solution into the vacutainer tube immediately after blood collection and before clot formation. These LPS/cultured clots shed even more sLox-1 into the serum than the paired cultured clots without LPS, in line with the known effect of LPS and downstream mediators (TNF, IL1A) in stimulating Lox-1 expression. (
In an embodiment, in the cultured clot serum and fresh blood, IL-8 and IL-6 concentrations are analyzed along with sLox-1 concentrations is also analyzed.
In an embodiment, the concentration of cultured clot serum IL-8 compared to fresh blood plasma is from 50 pg/mL to 10 ng/ml to 20 ng/ml to 40 ng/ml to 50 ng/ml to 70 ng/ml to 100 ng/ml.
In an embodiment, the concentration of cultured clot serum IL-8 compared to fresh blood plasma is about 2 times, 3 times, 4 times, 5 times, 7 times, 10 times, 20 times, 50 times or 100 times more.
In an embodiment, the concentration of IL-6 in cultured clot serum and/or fresh blood plasma is from 0 to 100 ng/mL. In an embodiment, the concentration of cultured clot serum IL-6 compared to fresh blood plasma is about 2 times, 3 times, 4 times, 5 times, 7 times, 10 times, 20 times, 50 times or 100 times more.
In an embodiment, the device may be used to screen blood donors for sLox-1 levels in order to produce lots of blood plasma or serum with “high” and “low” sLox-1 levels, for specific clinical applications where the level of sLox-1 could affect the clinical outcome, for example, inhibition of coagulation in organ transplantation, pro-coagulant activity for survival from hemorrhage.
In an embodiment, the method is configured to produce an autologous sLox-1.
In an embodiment, the cultured clot system is used as a novel diagnostic blood test that mimics a thrombus. The ex vivo thrombus device was optimized with blood samples from a cohort of 37 consenting healthy donors with diverse demographics (male/female, 18-61 years old, smoking or non-smoking; Caucasian/Hispanic/Latino/Asian/African-American; blood types ABO, Rh+/−, Lewis a/b/null, Duffy a/b/null, secretor/non-secretor).
The RNAseq study revealed that OLR1, the mRNA encoding Lox-1, is significantly induced in the ex vivo thrombus relative to fresh blood and fresh blood clots (
Among 21 different healthy donors, 4 donors under 30 years old showed unexpectedly high baseline plasma sLox-1 levels (1.1-1.9 ng/mL). In addition, all healthy donors showed a personalized level of clot-induced sLox-1 shedding into cultured clot serum that was reproducible in blood draws from the same donors on different occasions.
Table 1 provides Donor Demographics for RNA sequencing study.
In an embodiment, sLox-1 becomes elevated in blood plasma during thrombogenic viral infection.
In an embodiment, sLox-1 is a biomarker.
In an embodiment, present invention helps to detect cell apoptosis, scramblase activity, flippase activity, ADAM17 activity, ADAM10 activity, alpha secretase activity, sLox-1 sheddase activity, tumor necrosis factor activation.
In an embodiment, our novel data show that release of sLox-1 from the cultured clot is associated with those cells (neutrophils) that have a profile of PMN-MDSCs, which are known to drive cancer progression and inhibit immune defense against infection.
The term “biomarker” as described in this specification includes any physiological molecular form, or modified physiological molecular form, isoform, pro-form, naturally occurring forms or naturally occurring mutated forms of sLOX-1, expressed on the cell surface, unless otherwise specified. Other biomarkers that may be useful to detect cell apoptosis, scramblase activity, flippase activity, ADAM17 activity, ADAM10 activity, alpha secretase activity, sLox-1 sheddase activity, tumor necrosis factor activation It is understood that all molecular forms useful in this context are physiological, e.g., naturally occurring in the species. Preferably the peptide fragments obtained from the biomarkers are unique sequences. However, it is understood that other unique fragments may be obtained readily by one of skill in the art in view of the teachings provided herein.
By “isoform” or “multiple molecular form” is meant an alternative expression product or variant of a single gene in a given species, including forms generated by alternative splicing, single nucleotide polymorphisms, alternative promoter usage, alternative translation initiation small genetic differences between alleles of the same gene, and posttranslational modifications (PTMs) of these sequences.
“Gene Ontology” pathways induced in the cultured clot samples from 10 different (diverse) human donors that are induced by factors that are released from platelets (Transforming Growth Factor Beta). We also see pathways induced in the cultured clots from 5 different donors treated with lipopolysaccharide—that are activated by lipopolysaccharide, so the system is definitely working.
Table 2 shows GO pathways upregulated and downregulated in the cultured clotted blood.
In an embodiment, cultured clot device could be used to screen natural products derived from a host of microbial sources, yeast, bacteria, and screen for donor-specific inflammatory activity.
In an embodiment, a precision medicine tool is developed that detects Lox-1 induction in an ex vivo thrombus and shedding of soluble Lox-1 (sLox-1) into the serum. The tool consists of a red vacutainer tube with as little as 0.5 mL and more ideally 3 or 4 mL of peripheral whole venous blood, that is placed for 4 hours at 37° C.
The data suggest that sLox-1 generated from a cultured clot may have cardioprotective activities. The cultured clot system may be used to create a sLox-1 based therapeutic that could prevent trauma-induced cardiomyopathy and diminish symptoms of acute coronary syndrome.
Data were generated that suggests cultured clot serum has an inhibitory effect on blood coagulation that may be due to accumulation of sLox-1 in the serum (
In an embodiment, sLox-1 may serve as a novel cardioprotective agent or new biological anti-coagulant.
In an embodiment, sLox-1 may serve as a novel anti-thrombogenic agent. Anti-thrombogenic refers to a process of delaying clotting of blood or delaying activation of pro-platelets or platelets in peripheral blood.
In an embodiment, anti-thrombogenic agent is served in an Anti-thrombogenic amounts. The term, “Anti-thrombogenic amounts”, without implying any limitation, “anti-thrombogenically effective” amount encompasses an amount that reduces some aspect of blood clotting to less than 100% of a maximal value, to less than 95% of a maximal value, to less than 90% of a maximal value, to less than 85% a maximal value, to less than 80%, to less than 75%, to less than 70%, to less than 65%, to less than 60%, to less than 55%, to less than 50%, to less than 40%, to less than 30%, to less than 20%, to less than 10%, to less than 5%, to less than 2%, to less than 1%, of the maximal value, and so on.
A “personalized pharmaceutical” shall mean specifically tailored therapies for one individual patient that will only be used for therapy in such individual patient, including actively personalized slox-1 using autologous patient's blood.
As used herein in its broadest meaning, the term “preventing” or “prevention” refers to preventing the disease or condition from occurring in a subject which has not yet been diagnosed as having it or which does not have any clinical symptoms.
As used herein, the term “treating” or “treatment”, as used herein, means reversing, alleviating, or inhibiting the progress of the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. A “therapeutically effective amount” is intended for a minimal amount of active agent which is necessary to impart therapeutic benefit to a subject. For example, a “therapeutically effective amount” to a patient is such an amount which induces, ameliorates, stabilises, slows down the progression or otherwise causes an improvement in the pathological symptoms, disease progression or physiological conditions associated with or resistance to succumbing to a disorder.
In an embodiment, sLox-1 could be used as a pharmaceutical composition.
As used herein, a “pharmaceutical composition” is a composition suitable for administration to a human being in a medical setting. Preferably, a pharmaceutical composition is sterile and produced according to GMP guidelines.
The pharmaceutical compositions comprise the protein extracted according to an embodiment of the invention, either in the free form or in the form of a pharmaceutically acceptable salt. As used herein, “a pharmaceutically acceptable salt” refers to a derivative of the protein extracted according to an embodiment of the invention, wherein the protein is modified by making acid or base salts of the agent. For example, acid salts are prepared from the free base (typically wherein the neutral form of the drug has a neutral —NH2 group) involving reaction with a suitable acid. Suitable acids for preparing acid salts include both organic acids, e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methane sulfonic acid, ethane sulfonic acid, p-toluene sulfonic acid, salicylic acid, and the like, as well as inorganic acids, e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid phosphoric acid and the like. Conversely, preparation of basic salts of acid moieties which may be present on a peptide are prepared using a pharmaceutically acceptable base such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine or the like.
In an embodiment, composition is used for parenteral administration, such as subcutaneous, intradermal, intramuscular or oral administration. For this, the proteins extracted according to an embodiment or other molecules are dissolved or suspended in a pharmaceutically acceptable, preferably aqueous carrier. In addition, the composition can contain excipients, such as buffers, binding agents, blasting agents, diluents, flavors, lubricants, etc. The proteins can also be administered together with immune stimulating substances, such as cytokines. An extensive listing of excipients that can be used in such a composition can be, for example, taken from A. Kibbe, Handbook of Pharmaceutical Excipients (Kibbe, 2000). The composition can be used for a prevention, prophylaxis and/or therapy of coagulopathy and/or cardiopathy.
In an embodiment, the pharmaceutical composition is administered in an therapeutically effective amount.
As used herein, the term “therapeutically effective amount” refers to an amount that is sufficient or effective to prevent or treat (delay or prevent the onset of, prevent the progression of, inhibit, decrease or reverse) a disease or condition described or contemplated herein, including alleviating symptoms of such disease or condition. As used herein, the term “effective amount” or “therapeutically effective amount” of a compound is that amount of compound that is sufficient to provide a beneficial effect to the subject to which the compound is administered. Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
In an embodiment, device may be used to screen blood donors for sLox-1 levels in order to produce lots of blood plasma or serum with “high” and “low” sLox-1 levels, for specific clinical applications where the level of sLox-1 could affect the clinical outcome, for example, inhibition of coagulation in organ transplantation, pro-coagulant activity for survival from hemorrhage, or to discriminate between low platelets arising from inadequate platelet production or platelet activation in the circulation which according to this invention is expected to stimulate sLox-1 shedding into the blood plasma.
In an embodiment, the device may be as shown in
In an embodiment, cultured clot device to screen agents that promote or inhibit one or more of the following: cell apoptosis, scramblase activity, flippase activity, ADAM17 activity, ADAM10 activity, alpha secretase activity, sLox-1 sheddase activity, tumor necrosis factor activation.
In an embodiment, the device has a means of heating and a tube. The tube could be Vacutainer tube. The heating could be an inbuilt heating technique. The heating method could be portable.
In an embodiment, the device is capable to maintain the temperature at around 37° C. The temperature could be maintained for less than 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 24 hours, 48 hours or more.
In an embodiment, the device may have a cooling mechanism to decrease the temperature at a desired cooling temperature such as but not limited around 4° C. In an embodiment, the device is capable to decrease the temperature at around 4° C. automatically or manually after a specified time of usage for example: 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 24 hours, 48 hours or more.
In an embodiment, as shown in
In an embodiment, device could detect other soluble factors (i.e., high IL8 to document cell viability, no IL6 to document freedom from infection, TNF, IL1A, IL8, VEGF, resolvins, oxylipids, etc or other factors upregulated in cultured clots) for concentration and/or bioactivity.
In an embodiment, the device is as described in US20220133192A1.
All references, including granted patents and patent application publications, referred herein are incorporated herein by reference in their entirety.
This application claims the benefit under 35 U.S.C § 119 of U.S. Provisional Application 63/344,258 filed on 20 May 2022, titled as “METHOD FOR INDUCING AND DETECTING SOLUBLE LOX-1 (sLOX-1) IN CULTURED BLOOD CLOTS”, which is hereby incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63344258 | May 2022 | US |
