Biopharmaceuticals for reducing lipid content in cells

Abstract

The present disclosure relates to a biopharmaceutical for treating and / or preventing atherosclerosis and / or obesity and / or insulin resistance and / or diabetes, the biopharmaceutical comprising 37kDa / 67kDa laminin receptor precursor / high affinity laminin receptor (LRP / LR) and / or its fragments. The biopharmaceutical can be encapsulated by functionalized or non-functionalized nanoparticles, and further can be formulated to include a carrier to provide a pharmaceutical composition for parental or non-parental administration. When used, the biopharmaceutical reduces the lipid content in the target cell. The present disclosure also relates to a method for reducing the lipid concentration in the target cell of a human or animal subject.

Description

Technical Field

[0001] The present disclosure relates to methods for reducing lipid concentrations in target cells of human or animal subjects. The present disclosure extends to the use of biopharmaceuticals in treating and / or preventing atherosclerosis and / or insulin resistance and / or diabetes in human or animal subjects, the biopharmaceuticals comprising (i) 37kDa / 67kDa laminin receptor precursor / high affinity laminin receptor (LRP / LR) and / or fragments thereof, or (ii) transfection agents for expressing LRP / LR. Administration of the biopharmaceuticals reduces lipid content at target cells when used. Background Art

[0002] Atherosclerosis is a chronic, progressive disease involving blockage of arteries and is the leading form of cardiovascular disease (CVD). It is the result of the formation of atherosclerotic plaques, which occlude arteries and reduce blood flow to vital organs.

[0003] Atherosclerosis is greatly affected by oxidative stress, in which reactive oxygen species (ROS) contribute greatly to the body's oxidative state. ROS are formed during various cellular processes, such as cellular respiration. They are produced when oxygen molecules lose electrons, which makes them susceptible to participate in oxidation reactions and thus contribute to oxidative stress (Chen et al., 2003). ROS readily react with low-density lipoprotein (LDL) to form oxidized LDL (oxLDL), a molecule associated with the inflammatory response that leads to the formation of atherosclerotic plaques (Grahame and Schlesinger, 2012).

[0004] Over time, the accumulation of plaques narrows blood vessels and occludes arteries, making it more difficult for the heart to pump, leading to tissue damage and high blood pressure (Tedgui and Mallat, 1999). In addition, these plaques are prone to rupture due to their soft extracellular lipid components. When these plaques rupture, they can cause complete occlusion of the blood vessel, leading to major cardiovascular events such as myocardial infarction or cerebrovascular disease (Tedgui and Mallat, 1999).

[0005] Atherosclerosis is a leading cause of cardiovascular-related death, with current treatments such as ACE inhibitors and beta-blockers aimed at reducing myocardial stress by reducing hypertension caused by vascular occlusion (Conte et al., 2015). However, current treatments are not aimed at removing the atherosclerotic plaque that causes the occlusion, and the only current treatments for atherosclerosis are coronary artery bypass or arteriovenous dilation, both of which are highly invasive. Treating atherosclerosis with drugs that lower blood pressure can prevent organs from receiving adequate oxygen and nutrients because the plaque prevents adequate blood flow, which can lead to eventual organ failure (Conte et al., 2015). Furthermore, in the event of these plaque ruptures, complete occlusion of one or more blood vessels can occur and, depending on the location, can lead to major cardiovascular events such as myocardial infarction and cerebrovascular events.

[0006] Therefore, new innovations are needed that will reduce and / or prevent the formation of plaque.

[0007] Atherosclerosis is often comorbid with obesity, insulin resistance and / or diabetes. A lipid-rich diet often plays a major contributing role. Typically, increased ROS levels occur in obese patients due to mitochondrial damage.

[0008] The molecular mechanism leading to this insulin resistance is triggered by adipose tissue, which is metabolically active in obese individuals, thereby continuously undergoing lipolysis and producing free fatty acids (FFA) (Gavin et al., 2017). These FFAs act as antagonists of insulin (Salpea, 2010) and stimulate glucose production in the liver while inhibiting glucose uptake by skeletal muscle. Increased FFA levels reduce mitochondrial β-oxidation in skeletal muscle cells and hepatocytes. Reduced mitochondrial β-oxidation leads to the accumulation of diacylglycerol (DAG) and long-chain acyl-CoA (LCCoA) (Savage et al., 2007; Salpea, 2012). This in turn induces serine / threonine phosphorylation at the IRS-1 site, thereby inhibiting IRS-1 phosphorylation and activation of phosphatidylinositol 3-phosphate (PI3P) signaling. PI3P regulates the synthesis of glucose transporter 4 (GLUT4) (Savage et al., 2007; Salpea, 2012). Thus, inhibition of PI3P activation in bone results in reduced GLUT4 synthesis and, thus, reduced glucose uptake by skeletal muscle. Inhibition of PI3P activation in hepatocytes results in reduced phosphorylation of forkhead box protein O (FOXO), which in turn leads to increased hepatic gluconeogenesis (Savage et al., 2007). Increased FFA in obese individuals leads to hyperglycemia and induces insulin resistance in patients with type II diabetes (Gavin, 2017; Mejia, 2006). These cellular changes promote the accumulation of specific lipid metabolites (diacylglycerol and / or ceramide) in the liver and skeletal muscle, and the lipid content of cells has been found to be increased in diabetic patients, which is also associated with insulin resistance (Gavin et al., 2017).

[0009] Current treatment options for type 2 diabetes target blood glucose rather than the impaired insulin signaling caused by chronically elevated insulin levels (Ford, 2010). Therefore, new innovations that target impaired insulin signaling are needed.

[0010] The present disclosure seeks to improve upon the shortcomings known in the prior art. Summary of the invention

[0011] According to a first aspect of the present disclosure, there is provided a method for reducing lipid concentration in target cells of a human or animal subject, the method comprising the following steps:

[0012] (i) transfecting cells to produce 37 kDa / 67 kDa laminin receptor precursor / high affinity laminin receptor (LRP / LR) and / or fragments thereof; or

[0013] (ii) providing LRP / LR and / or fragments thereof to the cell,

[0014] Wherein the reduction of lipid concentration in cells treats and / or prevents atherosclerosis and / or obesity and / or insulin resistance and / or diabetes.

[0015] The step of transfecting may include encapsulating a transfection agent for site-specific delivery to the cell, and the step of providing may include encapsulating an LRP / LR for site-specific delivery to the cell.

[0016] The transfection agent and / or LRP / LR and / or fragment of LRP / LR is a biopharmaceutical agent.

[0017] Encapsulation can be accomplished with the aid of nanoparticles to form a delivery vehicle for transfection agents and / or LRP / LR. Nanoparticles can be functionalized with chemical, biochemical or biological moieties to ensure site-specific delivery to cells.

[0018] These moieties can act as ligands to ensure site-specific delivery at the cell.

[0019] The cell may be, but is not limited to, at least one of the following group: vascular endothelial cells, vascular smooth muscle cells, pancreatic cells including alpha (α) cells, beta (β) cells, delta (δ) cells, and gamma (γ) cells.

[0020] The delivery vehicle may be formulated as a pharmaceutical composition, which may further include a pharmaceutical carrier for parenteral or non-parenteral administration to a subject.

[0021] Non-parenteral administration may include, but is not limited to, at least one of the following groups: oral, intranasal, rectal, vaginal, optical, and transdermal administration. Typically, non-parenteral administration may be oral. Parenteral administration may include at least one of intravenous, subcutaneous, and intramuscular administration. Typically, parenteral administration may be intravenous administration.

[0022] The pharmaceutical composition may further include an antioxidant, such that the antioxidant scavenges reactive oxygen species when used at the target cell.

[0023] The pharmaceutical composition may further comprise an active pharmaceutical ingredient (API).

[0024] The subject may be a human, an animal, a reptile, a bird or an amphibian. Typically, the subject may be a human and / or an animal, preferably a human.

[0025] The transfection agent can be pCIneo-moLRP::FLAG plasmid.

[0026] The LRP / LR may comprise a peptide / protein sequence as listed in SEQ ID NO: 1 or SEQ ID NO: 2, or a fragment thereof.

[0027] SEQ ID NO: 1 may be a peptide / protein sequence of human LRP / LR and may have the following sequence: MSGALDVLQMKEEDVLKFLAAGTHLGGTNLDFQMEQYIYKRKSDGIYIINLKRTWEKLLL

[0028] AARAIVAIENPADVSVISSRNTGQRAVLKFAAATGATPIAGRFTPGTFTNQIQAAFREPR

[0029] LLVVTDPRADHQPLTEASYVNLPTIALCNTDSPLRYVDIAIPCNNKGAHSVGLMWWMLAR

[0030] EVLRMRGTISREHPWEVMPDLYFYRDPEEIEKEEQAAAEKAVTKEEFQGEWTAPAPEFTA

[0031] TQPEVADWSEGVQVPSVPIQQFPTEDWSAQPATEDWSAAPTAQATEWVGATTDWS

[0032] SEQ ID NO: 2 may be a peptide / protein sequence of mouse (Mus musculus) LRP / LR and may have the following sequence:

[0033] MSGALDVLQMKEEDVLKFLAAGTHLGGTNLDFQMEQYIYKRKSDGIYIINLKRTWEKLLL

[0034] AARAIVAIENPADVSVISSRNTGQRAVLKFAAATGATPIAGRFTPGTFTNQIQAAFREPR

[0035] LLVVTDPRADHQPLTEASYVNLPTIALCNTDSPLRYVDIAIPCNNKGAHSVGLMWWMLAR

[0036] EVLRMRGTISREHPWEVMPDLYFYRDPEEIEKEEQAAAEKAVTKEEFQGEWTAPAPEFTA

[0037] AQPEVADWSEGVQVPSVPIQQFPTEDWSAQPATEDWSAAPTAQATEWVGATTEWS

[0038] It is understood that LRP / LR is highly conserved and that homologs or fragments of SEQ ID NO: 1 and SEQ ID NO: 2, and / or homologs of fragments may also be utilized to practice the invention as described, illustrated and / or exemplified herein.

[0039] The LRP / LR may comprise a peptide / protein sequence listing having at least 80% homology to the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 or a fragment thereof.

[0040] LRP / LR may include homologues or fragments thereof, as well as homologues of fragments, wherein LRP / LR may include a peptide / protein sequence listing as listed in SEQ ID NO:1 or SEQ ID NO:2.

[0041] The peptide / protein sequence of LRP / LR or its homologue or fragment, or homologue of the fragment, may be combined, bonded, connected, conjugated or associated with an additional protein sequence, amino acid sequence, peptide, protein or antibody. Alternatively and / or additionally, the peptide / protein sequence of LRP / LR may form part of a larger and / or longer peptide / protein sequence. In a specific embodiment of the invention, LRP / LR may be combined, bonded, connected, conjugated or associated with a FLAG protein, so that when used, LRP / LR can be labeled with FLAG. The FLAG protein may include a peptide / protein sequence that includes at least the sequence motif DYKDDDDK (SEQ ID NO: 3). For all embodiments of the present disclosure, in addition to FLAG, other marker proteins are also envisioned.

[0042] It should be understood that the step of transfecting cells to produce 37kDa / 67kDa laminin receptor precursor / high affinity laminin receptor (LRP / LR) and / or fragments can be performed via procedures known in the art, including introducing a transfection agent into the cells. The step of transfecting cells can upregulate LRP / LR to result in overexpression of LRP / LR.

[0043] Exemplary embodiments of fragments of peptide / protein sequence listings are illustrated as SEQ ID NO:4 corresponding to a fragment from 102 to 295 of SEQ ID NO:1 and / or SEQ ID NO:5 corresponding to a fragment from 102 to 295 of SEQ ID NO:2.

[0044] SEQ ID NO:4 may be a peptide / protein sequence of a fragment of human LRP / LR, and may have the following sequence:

[0045] RFTPGTFTNQIQAAFREPR

[0046] LLVVTDPRADHQPLTEASYVNLPTIALCNTDSPLRYVDIAIPCNNKGAHSVGLMWWMLAR

[0047] EVLRMRGTISREHPWEVMPDLYFYRDPEEIEKEEQAAAEKAVTKEEFQGEWTAPAPEFTA

[0048] TQPEVADWSEGVQVPSVPIQQFPTEDWSAQPATEDWSAAPTAQATEWVGATTDWS

[0049] SEQ ID NO:5 may be a peptide / protein sequence of a fragment of mouse LRP / LR and may have the following sequence:

[0050] RFTPGTFTNQIQAAFREPR

[0051] LLVVTDPRADHQPLTEASYVNLPTIALCNTDSPLRYVDIAIPCNNKGAHSVGLMWWMLAR

[0052] EVLRMRGTISREHPWEVMPDLYFYRDPEEIEKEEQAAAEKAVTKEEFQGEWTAPAPEFTA

[0053] AQPEVADWSEGVQVPSVPIQQFPTEDWSAQPATEDWSAAPTAQATEWVGATTEWS.

[0054] The approach is often extended to upregulating LRP / LR expression in target cells.

[0055] According to a second aspect of the present disclosure, a biopharmaceutical for treating and / or preventing atherosclerosis and / or obesity and / or insulin resistance and / or diabetes is provided, the biopharmaceutical comprising 37kDa / 67kDa laminin receptor precursor / high affinity laminin receptor (LRP / LR) and / or fragments thereof, wherein LRP / LR and / or fragments thereof are administered to target cells of a subject in need thereof. Administration of the biopharmaceutical reduces lipid content in the target cells when used.

[0056] The LRP / LR may be encapsulated into a delivery vehicle.

[0057] The delivery vehicle may include nanoparticles. The nanoparticles may be functionalized with chemical, biochemical or biological moieties to ensure site-specific delivery to target cells.

[0058] These moieties can act as ligands to ensure site-specific delivery at target cells.

[0059] The target cell may be, but is not limited to, at least one of the following group: vascular endothelial cells, vascular smooth muscle cells, pancreatic cells including alpha (α) cells, beta (β) cells, delta (δ) cells, and gamma (γ) cells.

[0060] The delivery vehicle may be formulated as a pharmaceutical composition, which may further include a pharmaceutical carrier for parenteral or non-parenteral administration to a subject.

[0061] Non-parenteral administration may include, but is not limited to, at least one of the following groups: oral, intranasal, rectal, vaginal, optical, and transdermal administration. Typically, non-parenteral administration may be oral. Parenteral administration may include at least one of intravenous, subcutaneous, and intramuscular administration. Typically, parenteral administration may be intravenous administration.

[0062] The pharmaceutical composition may further include an antioxidant, such that the antioxidant scavenges reactive oxygen species when used at the target cell.

[0063] The pharmaceutical composition may further comprise an active pharmaceutical ingredient (API).

[0064] The subject may be a human, an animal, a reptile, a bird or an amphibian. Typically, the subject may be a human and / or an animal, preferably a human.

[0065] The LRP / LR may comprise a peptide / protein sequence as listed in SEQ ID NO: 1 or SEQ ID NO: 2 as listed in the first and second aspects above, or a fragment thereof.

[0066] According to a third aspect of the present disclosure, a biopharmaceutical agent is provided, which includes a transfection agent for expressing 37kDa / 67kDa laminin receptor precursor / high affinity laminin receptor (LRP / LR) and / or fragments thereof, wherein the transfection agent is used to treat and / or prevent atherosclerosis and / or obesity and / or insulin resistance and / or diabetes, wherein the transfection agent is used to administer to target cells of a subject in need thereof.

[0067] The transfection agent can be encapsulated into a delivery vehicle.

[0068] The delivery vehicle may include nanoparticles. The nanoparticles may be functionalized with chemical, biochemical or biological moieties to ensure site-specific delivery to target cells.

[0069] These moieties can act as ligands to ensure site-specific delivery at target cells.

[0070] The target cell may be, but is not limited to, at least one of the following group: vascular endothelial cells, vascular smooth muscle cells, pancreatic cells including alpha (α) cells, beta (β) cells, delta (δ) cells, and gamma (γ) cells.

[0071] The delivery vehicle may be formulated as a pharmaceutical composition, which may further include a pharmaceutical carrier for parenteral or non-parenteral administration to a subject.

[0072] Non-parenteral administration may include, but is not limited to, at least one of the following groups: oral, intranasal, rectal, vaginal, optical, and transdermal administration. Typically, non-parenteral administration may be oral. Parenteral administration may include at least one of intravenous, subcutaneous, and intramuscular administration. Typically, parenteral administration may be intravenous administration.

[0073] The pharmaceutical composition may further include an antioxidant, such that the antioxidant scavenges reactive oxygen species when used at the target cell.

[0074] The pharmaceutical composition may further comprise an active pharmaceutical ingredient (API).

[0075] The transfection agent can be pCIneo-moLRP::FLAG plasmid.

[0076] The LRP / LR may comprise a peptide / protein sequence as listed in SEQ ID NO: 1 or SEQ ID NO: 2 as listed in the first and second aspects above, or a fragment thereof.

[0077] According to a fourth aspect of the present disclosure, a pharmaceutical composition is provided, comprising a transfection agent and a carrier for 37kDa / 67kDa laminin receptor precursor / high affinity laminin receptor (LRP / LR) and / or fragments thereof, the pharmaceutical composition being used for treating and / or preventing atherosclerosis and / or obesity and / or insulin resistance and / or diabetes, wherein LRP / LR and / or fragments thereof are used for administration to target cells of a subject in need thereof.

[0078] Pharmaceutical compositions in which LRP / LR are encapsulated provide a delivery vehicle.

[0079] The pharmaceutical composition may further include an antioxidant, such that the antioxidant scavenges reactive oxygen species when used at the target cell.

[0080] The pharmaceutical composition may further comprise an active pharmaceutical ingredient (API).

[0081] The LRP / LR may comprise a peptide / protein sequence as listed in SEQ ID NO: 1 or SEQ ID NO: 2 as listed in the first and second aspects above, or a fragment thereof.

[0082] According to the fifth aspect of the present disclosure, a pharmaceutical composition is provided, which comprises a transfection agent for expressing 37kDa / 67kDa laminin receptor precursor / high affinity laminin receptor (LRP / LR) and / or its fragments, and a carrier, and the pharmaceutical composition is used to treat and / or prevent atherosclerosis and / or obesity and / or insulin resistance and / or diabetes.

[0083] Pharmaceutical compositions in which the transfection agent is encapsulated provide a delivery vehicle.

[0084] The pharmaceutical composition may further include an antioxidant, such that the antioxidant scavenges reactive oxygen species when used at the target cell.

[0085] The pharmaceutical composition may further comprise an active pharmaceutical ingredient (API).

[0086] The subject may be a human, an animal, a reptile, a bird or an amphibian. Typically, the subject may be a human and / or an animal, preferably a human.

[0087] The LRP / LR may comprise a peptide / protein sequence as listed in SEQ ID NO: 1 or SEQ ID NO: 2 as listed in the first and second aspects above, or a fragment thereof.

[0088] The transfection agent can be pCIneo-moLRP::FLAG plasmid.

[0089] According to a sixth aspect of the present disclosure, a method for treating and / or preventing atherosclerosis and / or obesity and / or insulin resistance and / or diabetes is provided, the method comprising the following steps:

[0090] (i) transfecting cells to produce 37 kDa / 67 kDa laminin receptor precursor / high affinity laminin receptor (LRP / LR) and / or fragments thereof; or

[0091] (ii) providing LRP / LR and / or fragments thereof to the cell,

[0092] Such that, in use, a reduction in lipid concentration in target cells is provided wherein atherosclerosis and / or insulin resistance and / or diabetes is treated and / or prevented.

[0093] The transfection agent and / or LRP / LR and / or fragment of LRP / LR is a biopharmaceutical agent.

[0094] The step of transfecting may include encapsulating a transfection agent for site-specific delivery to the cell, and the step of providing may include encapsulating an LRP / LR for site-specific delivery to the cell.

[0095] Encapsulation can be accomplished with the aid of nanoparticles to form a delivery vehicle for transfection agents and / or LRP / LR. Nanoparticles can be functionalized with chemical, biochemical or biological moieties to ensure site-specific delivery to cells.

[0096] These moieties can act as ligands to ensure site-specific delivery at the cell.

[0097] The target cell may be, but is not limited to, at least one of the following group: vascular endothelial cells, vascular smooth muscle cells, pancreatic cells including alpha (α) cells, beta (β) cells, delta (δ) cells, and gamma (γ) cells.

[0098] The delivery vehicle may be formulated as a pharmaceutical composition, which may further include a pharmaceutical carrier for parenteral or non-parenteral administration to a subject.

[0099] Non-parenteral administration may include, but is not limited to, at least one of the following groups: oral, intranasal, rectal, vaginal, optical, and transdermal administration. Typically, non-parenteral administration may be oral. Parenteral administration may include at least one of intravenous, subcutaneous, and intramuscular administration. Typically, parenteral administration may be intravenous administration.

[0100] The pharmaceutical composition may further include an antioxidant, such that the antioxidant scavenges reactive oxygen species when used at the target cell.

[0101] The pharmaceutical composition may further comprise an active pharmaceutical ingredient (API).

[0102] The subject may be a human, an animal, a reptile, a bird or an amphibian. Typically, the subject may be a human and / or an animal, preferably a human.

[0103] The transfection agent can be pCIneo-moLRP::FLAG plasmid.

[0104] The LRP / LR may comprise a peptide / protein sequence as listed in SEQ ID NO: 1 or SEQ ID NO: 2 as listed in the first aspect disclosed herein above, or a fragment thereof.

[0105] According to a seventh aspect of the present disclosure, there is provided the use of (i) 37kDa / 67kDa laminin receptor precursor / high affinity laminin receptor (LRP / LR) and / or fragments thereof, or (ii) a transfection agent for expressing LRP / LR in the manufacture of a pharmaceutical composition for treating and / or preventing atherosclerosis and / or obesity and / or insulin resistance and / or diabetes.

[0106] According to an eighth aspect of the present disclosure, there is provided a pharmaceutical composition for site-specific delivery of a biopharmaceutical to a specific target site in a human or animal body, the pharmaceutical composition comprising:

[0107] A biopharmaceutical agent encapsulated by a carrier matrix such that, in use, the carrier matrix facilitates site-specific delivery of the biopharmaceutical agent and is accompanied by preventing its degradation prior to reaching or at the target site,

[0108] The biopharmaceutical agent is at least one of the following groups: (i) 37kDa / 67kDa laminin receptor precursor / high affinity laminin receptor (LRP / LR) and / or fragments thereof, or (ii) a transfection agent for expressing LRP / LR, and (iii) an anti-LRP / LR specific antibody.

[0109] The biopharmaceutical agent may additionally or alternatively be any means for up-regulating or down-regulating LRP / LR expression in a human or animal. The biopharmaceutical agent may be a nucleic acid. The biopharmaceutical agent may be, for example, siRNA and / or shRNA.

[0110] The carrier matrix may be a polymeric carrier matrix.

[0111] The polymer carrier matrix may include polymer nanoparticles.

[0112] Polymer nanoparticles may include synthetic or natural polymers.

[0113] The polymer nanoparticles may be biodegradable to provide a reduced risk of immunogenic response to the polymer nanoparticles when used.

[0114] The polymer nanoparticles may be biocompatible to mitigate the risk of any immunogenic response to the polymer nanoparticles.

[0115] The polymer nanoparticles may be stimuli-responsive, such that in use the nanoparticle undergoes a conformational change when exposed to certain stimuli to facilitate delivery of the biopharmaceutical to its target site. The nanoparticles may respond to stimuli including, for example: pH, temperature, and electrical current.

[0116] Polymeric nanoparticles can be hydrophobic or hydrophilic, depending on the specific target site.

[0117] The polymer nanoparticles may be cross-linked. Typically, a cross-linking agent is used for cross-linking. However, cross-linking may be performed by ultraviolet (UV) irradiation.

[0118] The polymer nanoparticles can be lyophilized. Lyophilization typically provides porosity to facilitate diffusion of the biopharmaceutical from the nanoparticle capsule and to the target site.

[0119] The polymer nanoparticles may be any one or more selected from, but not limited to, the following groups: acrylic resin, gum arabic, carrageenan, cellulose, hydroxypropyl cellulose (HPC), methyl cellulose (MC), hydroxypropyl methyl cellulose (HPMC), lactic acid-glycolic acid copolymer (PLGA), chitin, pectin, pullulan, natural rubber, polyethylene, polypropylene, polystyrene, polyamide, polyacrylonitrile, polyvinyl chloride, polyvinyl alcohol (PVA), polyethylene glycol (PEG), polyethylene oxide (PEO), poly (D-lactide) (PDLA), polylactic acid (PLLA), polygalacturonate, methyl cellulose (polyacetal), poly (ε-caprolactone), phospholipids, polysaccharides, polyanionic polysaccharides, carboxymethyl cellulose , carboxymethyl amylose, chondroitin 6-sulfate, dermatan sulfate, heparin, heparin sulfate, poly(hydroxyethyl methacrylate), collagen, fibrinogen, albumin, fibrin, acrylamide, hydroxypropyl methacrylamide copolymer, polyacrylamide, poly(N-isopropyl acrylamide) (pNIPAAm), polyvinyl pyrrolidone, poly(methacrylic acid-g-glycol), poly(N-2-hydroxypropyl methacrylamide), poly(glycolic acid) (PGA), poly(lactic acid) (PLA), chitosan, poly(2-hydroxyethyl methacrylate) (HEMA), polyphazene, phosphorylcholine, hyaluronic acid (HA), hydroxyethyl methacrylate (HEMA), methylene-bis-acrylamide poly(methacrylate) (PEMA), poly(propylene glycol fumarate) (PF), poly(glucose ethyl methacrylate) (PGEMA), poly(hydroxybutyrate) (PHB), poly(hydroxyethyl methacrylate) (PHEMA), poly(hydroxypropyl methacrylamide) (PHPMA), poly(methyl methacrylate) (PMMA), poly(N-vinyl pyrrolidone) (PNVP), poly(propylene oxide) (PPO), poly(vinyl acetate) (PVAc), poly(ethylene imine) (PEI), poly(ethyl methacrylate) (PEMA), poly(propylene glycol fumarate) (PF), poly(glucose ethyl methacrylate) (PGEMA), poly(hydroxybutyrate) (PHB), poly(hydroxyethyl methacrylate) (PHEMA), poly(hydroxypropyl methacrylamide) (PHPMA), poly(methyl methacrylate) (PMMA), poly(N-vinyl pyrrolidone) (PNVP), poly(propylene oxide) (PPO), poly(vinyl acetate) (PVAc), poly(ethylene enamine), chondroitin sulfate, dextran sulfate, polylysine, gelatin, carboxymethyl chitin, dextran, agarose, pullulan, polyester, PEG-PLA-PEG, PEG-PLGA-PEG, PEG-PCL-PEG, PLA-PEG-PLA, poly(PF-co-EG), poly(PEG / PBO-terephthalate), PEG-bis-(PLA-acrylate), PEG6CD, PEG-g-poly(AAm-co-vinylamine), poly(NIPAAm-co-AAc), poly(NIPAAm-co-EMA), PNVP, poly(MMA-co-HEMA), poly(AN-co-allyl sulfonate), poly(biscarboxy-phenoxy-phosphazene),Poly(GEMA-sulfate), poly(PEG-co-peptide), alginate-g-(PEO-PPO-PEO), poly(PLGA-co-serine), collagen-acrylate, alginate, alginate-acrylate, poly(HPMA-g-peptide), HA-g-NIPAAm and poly(vinyl methyl ether) (PVME) and / or derivatives of any one or more of the above.

[0120] In an exemplary embodiment of the present disclosure, the polymer nanoparticles may be poly(lactic-co-glycolic acid) (PLGA) nanoparticles.

[0121] PLGA is biodegradable, biocompatible, and can cross the blood-brain barrier (BBB).

[0122] The nanoparticles may be functionalized with a first functional group. The first functional group may be, but is not limited to, at least one of the following group: a chemical, a biochemical, and a biological moiety.

[0123] The chemical moieties may include organic, inorganic, or a combination of organic and inorganic moieties.

[0124] The biochemical moiety may include, but is not limited to, at least one of the following groups: amino acids, peptides, polypeptides, oligopeptides, proteins, enzymes, antibodies, and RNA or DNA sequences encoding any of the above.

[0125] Typically, the first functional group acts as a ligand when used to facilitate site-specific delivery of the pharmaceutical composition. It should be understood that the functional group may be different depending on the target site. When used, the first functional group is bonded, connected, conjugated or associated with the target site.

[0126] Additionally or alternatively, the biopharmaceutical is provided to include at least one second functional group.

[0127] The second functional group may be, but is not limited to, at least one of the following group: chemical, biochemical, and biological moieties. In this way, not only nanoparticles will aid in site-specific delivery, but also biopharmaceuticals will aid in site-specific delivery.

[0128] The chemical moieties may include organic, inorganic, or a combination of organic and inorganic moieties.

[0129] The biochemical moiety may include, but is not limited to, at least one of the following groups: amino acids, peptides, polypeptides, oligopeptides, proteins, enzymes, antibodies, and RNA or DNA sequences encoding any of the above. When used, the second functional group is bonded, connected, conjugated, or associated with the target site.

[0130] The specific target site may be a cell including but not limited to at least one of the following group: brain cells, vascular endothelial cells, vascular smooth muscle cells, pancreatic cells including alpha (α) cells, beta (β) cells, delta (δ) cells and gamma (γ) cells.

[0131] The pharmaceutical composition may further include a pharmaceutical carrier for parenteral or non-parenteral administration to a subject.

[0132] Non-parenteral administration may include, but is not limited to, at least one of the following groups: oral, intranasal, rectal, vaginal, optical, and transdermal administration. Typically, non-parenteral administration may be oral. Parenteral administration may include at least one of intravenous, subcutaneous, and intramuscular administration. Typically, parenteral administration may be intravenous administration.

[0133] The pharmaceutical composition may further include an antioxidant, such that the antioxidant scavenges reactive oxygen species when used at the target site.

[0134] The pharmaceutical composition may further include an active pharmaceutical ingredient (API).The active pharmaceutical ingredient (API) may be included to treat and / or prevent co-morbid conditions such as, but not limited to, inflammation and pain.

[0135] The API may be at least one of the following group, but is not limited to: enfuvirtide; octreotide; cyclosporine; insulin; glucagon; glucagon-like peptide-1 (GLP-1); antibiotics, including ciprofloxacin or other fluoroquinolones; peptide antibiotics, such as polymyxin and colistin; bovine serum albumin (BSA); felodipine, nimodipine; interferon beta; salmon calcitonin; eel calcitonin; chicken calcitonin; rat calcitonin; human calcitonin; porcine calcitonin or any genetic variant of calcitonin; parathyroid hormone; parathyroid hormone analog PTH 1-31NH2; parathyroid hormone analog PTH 1-34NH2; insulin of any genetic variant; vasopressin; desmopressin; buserelin; luteinizing hormone-releasing factor; erythropoietin; tissue plasminogen activator; human growth factor; adrenocorticototropin; various interleukins; encephalin; etanercept; adalimumab; rituximab; infliximab; abatacept; traztuzumab; feglymycin; heparin; and all known vaccines.

[0136] The API may also be an analgesic, such as acetaminophen or a nonsteroidal anti-inflammatory drug (NSAID). NSAIDs include aspirin (Anacin, Ascriptin, Bayer, Bufferin, Ecotrin, Excedrin), choline and magnesium salicylate (CMT, Tricosal, Trilisate), choline salicylate (Arthropan), celecoxib (Celebrex), diclofenac potassium (Cataflam), diclofenac sodium (Voltaren, Voltaren XR), diclofenac sodium with misoprostol (Arthrotec), diflunisal (Dolobid), etodolac (Lodine, Lodine XL), fenoprofen calcium (Nalfon), flurbiprofen (Ansaid), ibuprofen (Advil, Motrin, Motrin IB, Nuprin), indomethacin (Indocin, Indocin SR), ketoprofen (Actron, Orudis, Orudis KT, Oruvail), magnesium salicylate (Arthritab, Bayer Select, Doan's Pills, Magan, Mobidin, Mobogesic), meclofenamic acid sodium (Meclomen), mefenamic acid (Ponstel), meloxicam (Mobic), nabumetone (Relafen), naproxen (Naprosyn, Naprelan), naproxen sodium (Aleve, Anaprox), oxaprozin (Daypro), piroxicam (Feldene), rofecoxib (Vioxx), salsalate (Amigesic, Anaflex 750, Disalcid, Marthritic, Mono-Gesic, Salflex, Salsitab), sodium salicylate (various generic drugs), sulindac (Clinoril), tolmetin sodium (Tolectin), and valdecoxib (Bextra).

[0137] The pharmaceutical composition may further comprise a cytochrome P450 3A4 (CYP3A4) inhibitor. The cytochrome P450 3A4 (CYP3A4) inhibitor may be selected from the group consisting of polyethylene glycol, polyamines, polymethyl methacrylate and derivatives thereof, wherein the inhibitor is present in an effective amount to substantially inhibit the pro-systemic metabolism of the biopharmaceutical and / or API, thereby increasing the bioavailability of the biopharmaceutical and / or API.

[0138] The pharmaceutical composition may further comprise a P-glycoprotein (P-gp) efflux pump inhibitor.

[0139] In certain embodiments, where the pharmaceutical composition is formulated for oral delivery, the pharmaceutical composition may further include a coating around it, preferably an enteric coating. The coating prevents degradation of the biopharmaceutical in the stomach during use.

[0140] The coating may include a cytochrome P450 3A4 (CYP3A4) inhibitor and / or a P-glycoprotein (P-gp) efflux pump inhibitor.

[0141] The transfection agent can be pCIneo-moLRP::FLAG plasmid.

[0142] It will be appreciated that other transfection agents may be used. It is well known that FLAG is a special tag and may be replaced with other tags if desired.

[0143] Anti-LRP / LR specific antibodies may include igG1-iS18. It should be understood that igG1-iS18 ​​is commercially available.

[0144] The LRP / LR may comprise a peptide / protein sequence as listed in SEQ ID NO: 1 or SEQ ID NO: 2, or a fragment thereof.

[0145] The LRP / LR may comprise a peptide / protein sequence listing having at least 80% homology to the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 or a fragment thereof.

[0146] LRP / LR may include homologues or fragments thereof, as well as homologues of fragments, wherein LRP / LR may include a peptide / protein sequence listing as listed in SEQ ID NO:1 or SEQ ID NO:2.

[0147] SEQ ID NO: 1 may be a peptide / protein sequence of human LRP / LR, and may have the following sequence:

[0148] MSGALDVLQMKEEDVLKFLAAGTHLGGTNLDFQMEQYIYKRKSDGIYIINLKRTWEKLLL

[0149] AARAIVAIENPADVSVISSRNTGQRAVLKFAAATGATPIAGRFTPGTFTNQIQAAFREPR

[0150] LLVVTDPRADHQPLTEASYVNLPTIALCNTDSPLRYVDIAIPCNNKGAHSVGLMWWMLAR

[0151] EVLRMRGTISREHPWEVMPDLYFYRDPEEIEKEEQAAAEKAVTKEEFQGEWTAPAPEFTA

[0152] TQPEVADWSEGVQVPSVPIQQFPTEDWSAQPATEDWSAAPTAQATEWVGATTDWS

[0153] SEQ ID NO: 2 may be a peptide / protein sequence of mouse (Mus musculus) LRP / LR and may have the following sequence:

[0154] MSGALDVLQMKEEDVLKFLAAGTHLGGTNLDFQMEQYIYKRKSDGIYIINLKRTWEKLLL

[0155] AARAIVAIENPADVSVISSRNTGQRAVLKFAAATGATPIAGRFTPGTFTNQIQAAFREPR

[0156] LLVVTDPRADHQPLTEASYVNLPTIALCNTDSPLRYVDIAIPCNNKGAHSVGLMWWMLAR

[0157] EVLRMRGTISREHPWEVMPDLYFYRDPEEIEKEEQAAAEKAVTKEEFQGEWTAPAPEFTA

[0158] AQPEVADWSEGVQVPSVPIQQFPTEDWSAQPATEDWSAAPTAQATEWVGATTEWS

[0159] It is understood that LRP / LR is highly conserved and that homologs or fragments of SEQ ID NO: 1 and SEQ ID NO: 2, and / or homologs of fragments may also be utilized to practice the present disclosure as described, illustrated and / or exemplified herein.

[0160] The peptide / protein sequence of LRP / LR or a homologue or fragment thereof, or a homologue of a fragment, may be bound, bonded, linked, conjugated or associated with an additional protein sequence, amino acid sequence, peptide, protein or antibody.

[0161] Alternatively and / or additionally, the peptide / protein sequence of LRP / LR may form part of a larger and / or longer peptide / protein sequence. In a particular embodiment of the invention, LRP / LR may be bound, bonded, connected, conjugated or associated with a FLAG protein, such that, when used, LRP / LR may be tagged with FLAG. The FLAG protein may include a peptide / protein sequence that includes at least the sequence motif DYKDDDDK (SEQ ID NO: 3). Applicants envision the use of other tags.

[0162] Exemplary embodiments of fragments of peptide / protein sequence listings are illustrated as SEQ ID NO:4 corresponding to a fragment from 102 to 295 of SEQ ID NO:1 and / or SEQ ID NO:5 corresponding to a fragment from 102 to 295 of SEQ ID NO:2.

[0163] SEQ ID NO:4 may be a peptide / protein sequence of a fragment of human LRP / LR, and may have the following sequence:

[0164] RFTPGTFTNQIQAAFREPR

[0165] LLVVTDPRADHQPLTEASYVNLPTIALCNTDSPLRYVDIAIPCNNKGAHSVGLMWWMLAR

[0166] EVLRMRGTISREHPWEVMPDLYFYRDPEEIEKEEQAAAEKAVTKEEFQGEWTAPAPEFTA

[0167] TQPEVADWSEGVQVPSVPIQQFPTEDWSAQPATEDWSAAPTAQATEWVGATTDWS

[0168] SEQ ID NO:5 may be a peptide / protein sequence of a fragment of mouse LRP / LR and may have the following sequence:

[0169] RFTPGTFTNQIQAAFREPR

[0170] LLVVTDPRADHQPLTEASYVNLPTIALCNTDSPLRYVDIAIPCNNKGAHSVGLMWWMLAR

[0171] EVLRMRGTISREHPWEVMPDLYFYRDPEEIEKEEQAAAEKAVTKEEFQGEWTAPAPEFTA

[0172] AQPEVADWSEGVQVPSVPIQQFPTEDWSAQPATEDWSAAPTAQATEWVGATTEWS. BRIEF DESCRIPTION OF THE DRAWINGS

[0173] Embodiments of the present disclosure will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0174] Figure 1 Shown is the overexpression of LRP::FLAG in THP-1 cells in Example 1;

[0175] Figure 2 It was shown that overexpression of LRP::FLAG in Example 1 significantly increased telomerase activity in oxLDL-treated and untreated THP-1 cells;

[0176] Figure 3 It is shown that in Example 1, LRP::FLAG overexpression increases the cell viability of oxLDL-treated THP-1 cells;

[0177] Figure 4 The DNA extracted in Example 1 and separated by agarose gel electrophoresis to determine the effect of LRP::FLAG overexpression on DNA fragmentation is shown;

[0178] FIG5 shows that in Example 1, overexpression of LRP::FLAG significantly reduces the uptake of oxLDL lipids by THP-1 cells;

[0179] Figure 6 It is shown that in Example 2, the overexpression of LRP::FLAG in THP-1 cells was detected by western blotting;

[0180] Figure 7 It is shown that in Example 2, overexpression of LRP::FLAG significantly increases telomerase activity in THP-1 cells simulating a diabetic state;

[0181] FIG8 shows that overexpression of LRP::FLAG significantly reduces lipid content in THP-1 cells simulating type II diabetes in Example 2; and

[0182] Fig. 9 It is shown that in Example 2, LRP::FLAG overexpression induces an increase in glucose uptake in THP-1 cells that mimics a diabetic state. DETAILED DESCRIPTION

[0183] The content of the above invention summary is repeated in full by reference here to avoid unnecessary repetition. However, non-limiting aspects of the present disclosure are provided herein to include a biopharmaceutical for treating and / or preventing atherosclerosis and / or obesity and / or insulin resistance and / or diabetes, the biopharmaceutical comprising 37kDa / 67kDa laminin receptor precursor / high affinity laminin receptor (LRP / LR) and / or fragments thereof, wherein LRP / LR and / or fragments thereof are used for administration to target cells of a subject in need thereof, and wherein the target cells are at least one of the following groups: vascular endothelial cells, vascular smooth muscle cells, pancreatic cells including α (α) cells, β (β) cells, δ (δ) cells and γ (γ) cells. When used, the biopharmaceutical reduces the lipid content in the target cells.

[0184] An example of one aspect includes a biopharmaceutical for treating and / or preventing atherosclerosis, the biopharmaceutical comprising 37kDa / 67kDa laminin receptor precursor / high affinity laminin receptor (LRP / LR) and / or fragments thereof, wherein the LRP / LR and / or fragments thereof are administered to a target cell of a subject in need thereof, and wherein the target cell is at least one of the following groups: a vascular endothelial cell and a vascular smooth muscle cell. When used, the biopharmaceutical reduces the lipid content in the target cell.

[0185] Another example of one aspect includes a biopharmaceutical for treating and / or preventing obesity and / or insulin resistance and / or diabetes, the biopharmaceutical comprising 37kDa / 67kDa laminin receptor precursor / high affinity laminin receptor (LRP / LR) and / or fragments thereof, wherein the LRP / LR and / or fragments thereof are administered to a target cell of a subject in need thereof, and wherein the target cell is at least one of the following group: a pancreatic cell comprising an alpha (α) cell, a beta (β) cell, a delta (δ) cell, and a gamma (γ) cell. When used, the biopharmaceutical reduces lipid content in the target cell.

[0186] The LRP / LR is typically encapsulated into a delivery vehicle comprising nanoparticles, preferably the nanoparticles are functionalized with chemical, biochemical or biological moieties to ensure site-specific delivery to the target cells, wherein the moieties act as ligands to ensure site-specific delivery at the target cells.

[0187] The delivery vehicle is typically formulated as a pharmaceutical composition including a pharmaceutical carrier for parenteral or non-parenteral administration to a subject.

[0188] The pharmaceutical composition may further comprise an active pharmaceutical ingredient (API). The API may treat atherosclerosis and / or obesity and / or insulin resistance and / or diabetes and / or comorbid conditions thereof.

[0189] The pharmaceutical composition may further include an antioxidant, such that the antioxidant scavenges reactive oxygen species when used at the target cell.

[0190] The LRP / LR typically comprises a peptide / protein sequence listing as set forth in SEQ ID NO:1 or SEQ ID NO:2, or a fragment thereof as set forth in SEQ ID NO:4 or SEQ ID NO:5.

[0191] LRP / LR can form part of a transfection agent for expressing 37kDa / 67kDa laminin receptor precursor / high affinity laminin receptor (LRP / LR) and / or fragments thereof, preferably the transfection agent is a pCIneo-moLRP::FLAG plasmid. In other words, the transfection agent can be used to upregulate LRP / LR.

[0192] The nanoparticles are typically polymer nanoparticles and are biodegradable to provide a reduced risk of an immunogenic response to the polymer nanoparticles when used, and further wherein the polymer nanoparticles are biocompatible to mitigate the risk of any immunogenic response to the polymer nanoparticles, and further wherein the polymer nanoparticles are one or more selected from the group consisting of acrylic resin, gum arabic, carrageenan, cellulose, hydroxypropyl cellulose (HPC), methyl cellulose (MC), hydroxypropyl methyl cellulose (HPMC), lactic acid-glycolic acid copolymer (PLGA), chitin, pectin, pullulan, natural rubber, polyethylene, polypropylene, polystyrene, polyamide, polyacrylonitrile, polyvinyl chloride olefins, polyvinyl alcohol (PVA), polyethylene glycol (PEG), polyethylene oxide (PEO), poly (D-lactide) (PDLA), polylactic acid (PLLA), polygalacturonate, methylcellulose (polyacetal), poly (ε-caprolactone), phospholipids, polysaccharides, polyanionic polysaccharides, carboxymethyl cellulose, carboxymethyl amylose, chondroitin 6-sulfate, dermatan sulfate, heparin, heparin sulfate, poly (hydroxyethyl methacrylate), collagen, fibrinogen, albumin, fibrin, acrylamide, hydroxypropyl methacrylamide copolymer, polyacrylamide, poly (N-isopropylacrylamide) (pNIPAAm), polyvinyl pyrrolidone, poly (methacrylic acid-g-ethylene glycol), poly (N-2-hydroxypropyl methyl Acrylamide), poly(glycolic acid) (PGA), poly(lactic acid) (PLA), chitosan, poly(2-hydroxyethyl methacrylate) (HEMA), polyphazene, phosphorylcholine, hyaluronic acid (HA), hydroxyethyl methacrylate (HEMA), methylene-bis-acrylamide (MBAAm), poly(acrylic acid) (PAAc), polyacrylamide (PAAm), polyacrylonitrile (PAN), polybutylene oxide (PBO), polycaprolactone (PCL), poly(ethyleneimine) (PEI), poly(ethyl methacrylate) (PEMA), propylene glycol fumarate (PF), poly(glucose ethyl methacrylate) (PGEMA), poly(hydroxybutyrate) (PHB) , poly(hydroxyethyl methacrylate) (PHEMA), poly(hydroxypropyl methacrylamide) (PHPMA), poly(methyl methacrylate) (PMMA), poly(N-vinyl pyrrolidone) (PNVP), poly(propylene oxide) (PPO), poly(vinyl acetate) (PVAc), poly(vinylamine), chondroitin sulfate, dextran sulfate, polylysine, gelatin, carboxymethyl chitin, dextran, agarose, pullulan, polyester, PEG-PLA-PEG, PEG-PLGA-PEG, PEG-PCL-PEG, PLA-PEG-PLA, poly(PF-co-EG), poly(PEG / PBO-terephthalate), PEG-bis-(PLA-acrylate),PEG6CD, PEG-g-poly(AAm-co-vinylamine), poly(NIPAAm-co-AAc), poly(NIPAAm-co-EMA), PNVP, poly(MMA-co-HEMA), poly(AN-co-allyl sulfonate), poly(dicarboxy-phenoxy-phosphazene), poly(GEMA-sulfate), poly(PEG-co-peptide), alginate-g-(PEO-PPO-PEO), poly(PLGA-co-serine), collagen-acrylate, alginate, alginate-acrylate, poly(HPMA-g-peptide), HA-g-NIPAAm and poly(vinyl methyl ether) (PVME) and / or derivatives of any one or more of the above. . ,

[0193] Typically, the delivery vehicle is formulated for oral delivery and comprises a coating at least partially surrounding it comprising a cytochrome P450 3A4 (CYP3A4) inhibitor selected from the group consisting of polyethylene glycol, polyamines, polymethyl methacrylate, and derivatives thereof, and wherein the coating further comprises a P-glycoprotein (P-gp) efflux pump inhibitor.

[0194] Substantially as described herein, transfection agents and / or LRP / LR and / or fragments of LRP / LR are examples of biopharmaceutical agents useful as part of the present disclosure. It should be understood that LRP / LR is highly conserved, and that homologs or fragments of SEQ ID NO: 1 and SEQ ID NO: 2, and / or homologs of fragments may also be utilized in order to practice the present disclosure described, illustrated and / or exemplified herein.

[0195] Substantially identical sequences can also be used. As used herein, substantially identical sequences are amino acid or nucleotide sequences that differ from a reference sequence only by one or more conservative substitutions, or by one or more non-conservative substitutions, deletions or insertions at sequence positions that do not destroy or substantially reduce the activity of one or more of the expressed polypeptides or polypeptides encoded by the nucleic acid molecule.

[0196] In the examples below, the transfection agent is pCIneo-moLRP::FLAG plasmid. It should be understood that the applicant envisions another transfection agent for upregulating LRP / LR expression in target cells. Detailed example embodiments of the present disclosure that do not limit the scope of the present disclosure are provided below as Examples 1 and 2.

[0197] Example

[0198] Example 1 - Atherosclerosis

[0199] Materials and methods

[0200] Cell culture of THP-1 (human leukemic mononuclear cells)

[0201] Human monocytic cell line (THP-1) was used for all experimental procedures performed. This cell line was selected because it can easily absorb oxLDL (oxidized low-density lipoprotein) and become foam cells, which are the main components of atherosclerotic plaques. RPMI medium with 15% fetal bovine serum (FBS), 1% MEM non-essential amino acids was used to provide essential nutrients for culturing untransfected and transfected THP-1 cells. In addition, 2% penicillin / streptomycin was used to prevent bacterial contamination. THP-1 cells grow both adherently and in suspension, and therefore require additional culture medium to grow; therefore, these cells were cultured in 5ml flasks using 15ml culture medium. Cells were stored in an incubator pre-set to 37°C and with 5% CO2 to simulate the in vivo conditions present in the human body. Culture medium replacement was performed two to three times a week. In addition, when the flask reached approximately 70-80% confluence, a 1:10 split was performed.

[0202] For culture medium replacement and cell passage, use cell scraper to scrape flask gently, and culture medium is placed in centrifuge tube, and centrifuges 10 minutes with 4000rpm.Discard old culture medium, and precipitation is resuspended in the Dulbecco's phosphate buffered saline (D-PBS) of 2ml, to remove debris, and centrifuges 10 minutes again with 4000rpm.Then precipitation is resuspended in the culture medium of 1ml, and if converging is lower than 70%, then whole 1ml is put back in flask.In the flask that converges, cell is split with the ratio of 1:10.By remaining cell precipitation and be resuspended in the 90%FBS with 10%DMSO, and freeze at-80 ℃ as reserve.

[0203] Overexpression of LRP::FLAG and treatment of THP-1 cells

[0204] To overexpress LRP::FLAG, THP-1 cells were transfected with pCIneo-moLRP::FLAG plasmid using the Xfect transfection kit and protocol. A flask confluent at approximately 70% was selected for transfection. In brief, Xfect reaction buffer was added to 5 μg of pCIneo-moLRP::FLAG plasmid to prepare 100 μl samples. Thereafter, 1.5 μl of Xfect polymer was added to the mixture, and the reagent was incubated at room temperature for 10 minutes to allow the formation of nanoparticle complexes. The reaction mixture was then added dropwise to the flask. Cells were treated once with 10 μl of geneticin (50 mg / ml) every 3 weeks to promote transcription of the plasmid and ensure that transfection had occurred.

[0205] To induce atherosclerotic plaque formation, THP-1 cells were treated with oxLDL (Bio-Rad). Briefly, untransfected and transfected cells were seeded into 6, 48 or 96 well plates and allowed to recover and grow for 24-48 hours. When the cells reached 70-80% confluence, a treatment consisting of 50 μg / μl of oxLDL was added to each well of the respective plates, and the cells were incubated for 48 hours. Thereafter, cells from each well were harvested as a pellet or the experiment was performed in the well. For each experiment, the following treatments were used: untreated untransfected, untreated transfected, treated untransfected, and treated transfected.

[0206] Bicinchoninic acid TM (BCA) protein assay

[0207] Perform BCA to measure the concentration of protein extracted from each cell treatment, and ensure the equal loading of Western blotting. For this technology, scrape the cells in the flask of about 70% confluence, and centrifuge at 10000rpm for 10 minutes. Then the precipitation is resuspended in RIPA lysis buffer (50mM Tris-HCl, 50mM NaCl, 2mM EDTA and 0.1% SDS are mixed to form by pH 7.4). The SDS in the buffer makes the extracted protein linear, and provides a negative charge proportional to the protein molecular weight. Next, each lysate of 5 μl is loaded into 96-well plates with 20 μl distilled water. In addition, a set of standards (0 mg / ml, 0.2 mg / ml, 0.4 mg / ml, 0.6 mg / ml, 0.8 mg / ml and 1 mg / ml) of 25 μl of load composed of a serial dilution (1:10) of bovine serum albumin (BSA). This was done to construct a standard curve for determining the protein concentration of unknown lysates. Afterwards, 200 μl of a mixture of bicinchoninic acid and copper sulfate (930 μl:30 μl) was added to each well and the plate was incubated at 37°C for 30 minutes. This allowed time for the peptide bonds to transfer Cu 2+ Reduction to Cu + , in order to produce a color change proportional to the amount of protein present. A Sunrise Teccan ELISA plate reader was used to measure the color change at 562 nm and the concentration of the samples was obtained from the standard curve.

[0208] Western blotting and SDS-PAGE

[0209] Immunodetection and quantification of proteins use SDS PAGE and Western blotting. This is a mature technology that is simple to use and particularly useful in this study because it allows detection of LRP::FLAG to confirm transfection. In addition, it is useful for detecting changes in CVD markers in cells that overexpress LRP::FLAG, and therefore it is selected over other protein detection techniques. The disadvantages of this technology are the possibility of non-specific binding of antibodies, the length of the program, and the influence of pollutants. This technology utilizes sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which is then blotted on a PVDF membrane and detected with a specific antibody complex targeting the protein of interest. The LRP::FLAG and β-actin loading controls extracted from THP-1 cells were diluted to a final concentration of 100 μl / ml. This allows equal and accurate separation of proteins during SDS-PAGE.

[0210] SDS-PAGE

[0211] SDS-PAGE is performed in order to linearize and separate different proteins according to their molecular mass. The SDS in the RIPA buffer binds proportionally to the size of the proteins, giving them an equal charge-to-mass ratio and allowing them to be separated based solely on their molecular size. This allows proteins of known molecular weight to be accurately detected by using molecular weight markers. A 10% polyacrylamide gel is prepared in two parts. First, a separation gel is prepared by mixing together 4.8 ml of distilled water, 2.5 ml of 40% acrylamide, 2.5 ml of separation buffer (187 g Tris base at pH 8.8 and 0.4% SDS in 1 liter of distilled water), 100 μl of 10% SDS and 100 μl of 3% APS and 5 μl of TEMED. This is then added to the plate and allowed to polymerize. Thereafter, a stacking gel was prepared by adding 3.65 ml of distilled water to 625 μl of 40% acrylamide, 625 μl of stacking buffer (60.5 g Tris base at pH 6.8 and 4% SDS in 1 liter of distilled water), 50 μl of 10% SDS and 50 μl of APS and 5 μl of TEMED. This was added on top of the separating gel, a comb was inserted and the gel was polymerized. The stacking gel allows the proteins to accumulate as a single band at the bottom of the wells, while the separating gel allows the different proteins to be separated as single bands across the gel. The extracted lysate was then mixed with the loading dye, and the samples were incubated at 95° C. for 5 minutes in order to allow the dye to intercalate into the proteins. Thereafter, 5 μl of each sample and molecular weight markers were loaded into the wells. The proteins were separated in running buffer at 120 V until the dye front was about 1 cm from the bottom of the gel (about 45 minutes).

[0212] Western blotting

[0213] After protein separation, they are blotted onto PVDF membranes where they can be incubated with suitable antibodies (Table 5.4.1). First, the membrane is placed in methanol (to activate) and then placed in transfer buffer with blotting paper (to balance). Thereafter, the membrane with the gel on top is placed between blotting papers, which are placed in a semi-dry transfer blotting device and electroblotted for 45 minutes at 120V. Once the transfer is complete, the PVDF membrane is blocked with 10ml of 3% BSA to prevent nonspecific antibody binding. The membrane is then incubated overnight at 4°C with suitable primary antibodies (Table 5.4.1). Thereafter, it is washed five times with PBS-Tween for 5 minutes each time. If applicable, the membrane is incubated with appropriate secondary antibodies (Table 5.4.1) and incubated for one hour at room temperature. It is then washed five more times. Next, the membrane was incubated with 4 ml of Clarity Western ECL substrate (Bio-Rad) for 1 min in the dark and then observed with a ChemiDock imaging system (Bio-Rad) that captured images. Densitometric analysis allowed quantification of protein levels. However, delayed delivery of oxLDL treatment together with continued contamination resulted in insufficient time to perform densitometric analysis and quantify extracted proteins.

[0214] Table 5.4.1: List of primary and secondary antibodies used in western blotting protocols.

[0215]

[0216] Detection of telomerase activity

[0217] Telomere shortening is a hallmark of CVD and is primarily caused by reduced telomerase activity. qPCR was used to assess the effect of LRP::FLAG overexpression on telomerase activity in differentially treated cells. Telomerase detection kit (Merck Millipore TM) is used to extract active telomerase and allow the telomere substrate to extend in order to provide a relative indication of telomerase activity. This technique is very accurate in determining telomerase activity because it extends the substrate relative to the amount of protein available, so this technique was chosen. Cells were initially seeded into 6-well plates and treated with oxLDL. After 48 hours, the wells were precipitated and resuspended in 200 μl of Chaps lysis buffer. The samples were then incubated at 4°C for 30 minutes to extract protein. Thereafter, the samples were centrifuged at 12000 rpm for 20 minutes at 4°C, and the protein concentration was determined using a Nanodrop ND-1000 spectrophotometer (Thermo Scientific). The protein samples were then diluted to 500 ng / μl, and 30 μl of the protein samples were incubated at 100°C for 20 minutes to provide a negative control for heat treatment, in which telomerase was inactivated. Controls and reaction mixtures were prepared using PCR grade water, Taq polymerase, and control or reaction mixtures. In a 96-well plate, the control mixture was added to the heat-treated samples and the chaps negative control, while the reaction mixture was added to all protein samples, standards and positive controls. The plate was then covered with a cover glass and placed in the AryaqPCR machine. The following parameters were used: an initial 10-minute denaturation step at 95°C, followed by 45 amplification cycles, each consisting of: denaturation at 95°C for 10 seconds, annealing at 58°C for 10 seconds and extension at 72°C for 60 seconds. Data were normalized to the standard curve and analyzed using the Student's t-test.

[0218] Cell counting

[0219] In order to obtain accurate data, it is necessary to determine the number of cells so that equal amounts of cells are inoculated for determinations such as MTT determinations and oil red staining. A hemocytometer is used to determine the number of cells required for inoculation. In short, cells are scraped and 10 μl of cell suspension is mixed with 10 μl of filtered trypan blue. Cells are then counted with a hemocytometer and the cell concentration is determined using the following equation:

[0220]

[0221] The cell concentration was then used to seed equal concentrations of cells into each well of the plate used in order to ensure that the data obtained were accurate.

[0222] Detection of changes in cell viability in cells overexpressing LRP::FLAG

[0223] The MTT cell viability assay is a well-established technique for determining cell viability. This assay utilizes MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), a chemical that is converted to insoluble formazan in the mitochondria of living cells (van Meerloo et al., 2011). Cells treated with oxLDL undergo apoptosis when they become foam cells (Wintergerst et al., 2000), so testing cell viability can determine whether overexpression of LRP::FLAG rescues cells from apoptosis. For this assay, 8000 cells were seeded into a 48-well plate, treated with 50 μg / μl of oxLDL and incubated for 48 hours. Thereafter, 7 mg of MTT (Duchefa Biochemie) was dissolved in 1.5 ml of PBS and then filter sterilized. Next, 100 μl of a 0.5 mg / ml MTT solution was added to each well, and the plate was incubated at 37°C for approximately 3 hours. Finally, the formed formazan crystals were dissolved in 250 μl DMSO and the color change was detected at 590 nm using a Sunrise Teccan ELISA plate reader. The data obtained were analyzed using Student's t-test and then plotted into a histogram.

[0224] Assessment of lipid content in cells overexpressing LRP::FLAG

[0225] In treated cells, uptake of oxLDL results in an increase in lipid content in the cells due to the lipid portion of the lipoprotein (Grahame and Schlesinger, 2012). In addition, oxLDL promotes the cellular uptake of cholesterol and triglycerides (Batt et al., 2004). Therefore, assessing lipid content in cells provides possible insights into how overexpression of LRP::FLAG affects oxLDL uptake and subsequent atherosclerotic plaque formation. The Oil Red O lipid staining kit of is used to stain lipids in cells, allowing for quantitative measurement of oxLDL present in cells. This technique is a quick and simple technique that accurately determines lipid content. In addition, the dye is extracted to quantify the amount of lipids present, giving a more accurate indication of the effect of LRP::FLAG overexpression. The disadvantage of this technique is that the large amount of washing required can remove some cells, resulting in skewed data. This technique is selected over other techniques because it provides qualitative and quantitative data. Once the number of cells is obtained using a hemocytometer, 16,000 transfected and untransfected cells are seeded into a 96-well plate, where appropriate wells are treated with oxLDL and incubated for 48 hours. Thereafter, the cells are washed twice with 100 μl of PBS and then incubated with 10% formalin at room temperature for 45 minutes to fix them. After the incubation period, the cells are washed twice with distilled water and then incubated with 60% isopropanol for 5 minutes. Next, a working oil red solution of 3 parts of stock and 2 parts of distilled water is prepared, and 200 μl is added to each well and incubated for 15 minutes. The cells were washed 3 times with water and images were taken with an inverted optical microscope. In addition, the dye was extracted using 100% isopropanol and quantified at 490 nm to accurately determine changes in lipid content. The data obtained were analyzed via a Student's t-test and plotted as a histogram.

[0226] Statistical analysis

[0227] All data obtained during the experiment were subjected to rigorous statistical analysis using Microsoft Excel 2010 to ensure the relevance and significance of the data. In addition, each experiment was performed at least three times to ensure the accuracy of the data and to calculate the standard deviation. The two-tailed Student's t-test was used for the analysis of 95% confidence intervals. Values ​​with *p<0.05 were considered statistically significant, while values ​​with **p<0.01 and ***p<0.001 were considered highly significant.

[0228] result

[0229] Overexpression of LRP::FLAG in transfected THP-1 cells was confirmed.

[0230] To determine whether LRP / LR has a potential role in CVD, THP-1 cells were stably transfected with pCIneo-moLRP::FLAG plasmid to overexpress LRP::FLAG in THP-1 cells. After transfection, Western blotting was performed to confirm the overexpression of LRP::FLAG ( Figure 1). β-actin (loading control) is present in untransfected and transfected THP-1 cells (lanes 1-8), while LRP::FLAG is only present in transfected cells (lanes 5-8), indicating successful transfection and overexpression of LRP::FLAG. Overexpression of LRP::FLAG by pCIneo-LRP::FLAG plasmid was evaluated in transfected (lanes 1-4) and untransfected (lanes 5-8) THP-1 cells. LRP::FLAG was detected using mouse anti-FLAG primary antibody and mouse anti-IgG-HRP secondary antibody, while β-actin was detected using mouse anti-actin-peroxidase antibody.

[0231] Overexpression of LRP::FLAG significantly increased telomerase activity in transfected THP-1 cells treated with oxLDL

[0232] A key factor in CVD is severe shortening of telomeres, which occurs due to high levels of oxidative stress that impede telomerase activity. To determine whether overexpression of LRP::FLAG subsequently affects telomerase activity, qPCR was performed on transfected and non-transfected THP-1 cells treated with oxLDL. A significant decrease of 57.43% in telomerase activity was observed in non-transfected treated cells relative to untreated non-transfected THP-1 cells. In addition, when compared to non-transfected and untreated THP-1 cells, increases in telomerase activity of 272.72% and 283.47% were observed in transfected non-treated and treated cells, respectively ( Figure 2 ).

[0233] The graph depicts the change in relative telomerase activity after LRP::FLAG overexpression. For plotting the graph, the data were normalized. When treated with oxLDL, a significant decrease of 57.43% (**p<0.01) in telomerase activity was found in non-transfected cells (blue), while a significant increase in telomerase activity in cells overexpressing LRP::FLAG (red) was observed, independent of oxLDL treatment (**p<0.01). Cell transfection significantly increased telomerase activity by 3.8-fold, indicating that LRP::FLAG may play a role in maintaining telomerase activity in the setting of CVD.

[0234] Overexpression of LRP::FLAG significantly increases cell viability in THP-1 cells treated with oxLDL

[0235] One of the hallmarks of oxLDL uptake in cells is induction of apoptosis, and thus reduction of cell viability. Cells were treated with oxLDL to simulate an atherosclerotic environment and to study the effects of overexpressing LRP::FLAG on CVD. In order to assess the effects of overexpressing LRP::FLAG on cell viability, MTT cell viability assays were performed using transfected and untransfected THP-1 cells. Between untreated and oxLDL-treated untransfected THP-1 cells, it was found that cell viability was significantly reduced by 45.53%. When cells were transfected, cell viability remained at approximately normal (untransfected untreated) levels regardless of treatment ( Figure 3 ). The graph depicts changes in cell viability when cells are treated and / or transfected. Untransfected cells treated with oxLDL (blue) show a significant 1.8-fold decrease in cell viability (***p<0.001). When cells were transfected (red), no significant decrease in cell viability was observed after oxLDL treatment, indicating that LRP::FLAG rescues cells from oxLDL-induced cytotoxicity.

[0236] Effect of LRP::FLAG overexpression on DNA fragmentation

[0237] Atherosclerotic plaque formation occurs when monocytes become foam cells through apoptosis. During apoptosis, DNA is fragmented and the fragmented DNA produces a smear when separated by agarose gel electrophoresis. Therefore, changes in DNA fragmentation detected by agarose gel electrophoresis were used to evaluate the role of LRP::FLAG overexpression in DNA fragmentation. However, the DNA is still trapped within the pores of the gel ( Figure 4 ). Transfected treated cells were expected to have reduced smearing when compared to untransfected treated cells, however due to time constraints this experiment could not be repeated.

[0238] DNA extracted from untransfected untreated (lanes 2-4), untransfected oxLDL treated (lanes 5-7), transfected untreated (lanes 8-10), and transfected oxLDL treated (lanes 11-13) were all trapped in the wells. This may occur due to genomic DNA fragments being too large for the gel, resulting in DNA retention in the wells. The use of restriction enzymes may have solved this problem. In addition, the sensitivity of this technique is low, so any fragments that leave the wells may not be detected due to the low concentration of extracted DNA.

[0239] Overexpression of LRP::FLAG significantly reduces lipid content in THP-1 cells treated with oxLDL

[0240] Atherosclerotic plaque formation requires monocytes to absorb oxLDL, which causes an increase in cellular lipid content. This main sign was studied using oil red lipid staining to assess whether LRP::FLAG overexpression plays a role in atherosclerotic plaque formation. Assays show that, when compared with untreated untransfected cells, oxLDL treatment causes an increase in lipid content in untransfected cells (red staining) (Fig. 5Aa-b). In addition, compared with untransfected and untreated cells, transfection causes a decrease in lipid content in THP-1 cells and untreated cells treated with oxLDL (Fig. 5Aa, c and d). In addition, the quantitative staining shows that the lipid content between untreated and untransfected THP-1 cells treated significantly increases. The significant reduction in lipid content was observed between untransfected treated and transfected treated cells, and the untreated cells of transfection returned to normal (untreated untransfected THP-1 cells) lipid levels (Fig. Figure 5B ).

[0241] Figure A qualitatively illustrates the differences observed when THP-1 cells are treated and / or transfected. The selected images represent the staining present in all 6 wells. The images show that the red staining of the untransfected cells (b) treated with oxLDL increases compared to untransfected and untreated cells (a). Regardless of how (c and d) are treated, the transfected cells all show a reduction in oil red staining. Figure B shows a quantitative representation of the stained cells. The untransfected cells (blue) treated with oxLDL significantly increase the intracellular oxLDL lipid concentration (**p<0.01). Transfection causes a significant reduction in the lipid content in the cell (**p<0.01), returning the concentration to the untransfected and untreated cell level (red).

[0242] Discussion

[0243] It is well known that monocytes prone to oxLDL uptake have significantly reduced telomere length, and these monocytes have been shown to have reduced telomerase (Fitzpatrick et al., 2007). It has also been shown that once plaques are formed, telomerase activity is upregulated, followed by an increase in telomere length, but without a significant effect on plaques (Huzen et al., 2011). Therefore, upregulation of LRP / LR is not expected to have any effect on plaques. Therefore, it is surprising and unexpected that upregulation of telomerase activity by using LRP::FLAG leads to a reduction in atherosclerotic plaque formation and rescues cells from oxLDL-induced apoptosis.

[0244] Overexpression of LRP::FLAG in THP-1 cells.

[0245] Human leukemic mononuclear cells (THP-1) cells are used because of their ability to easily absorb oxLDL and become foam cells. To evaluate the function of LRP::FLAG in these cells, pCIneo-moLRP::FLAG constructs were used to transfect these cells, and transfection was confirmed by Western blotting. Western blotting showed that LRP::FLAG was expressed in transfected THP-1 cells, but not in untransfected cells ( Figure 1 ).

[0246] Effect of LRP::FLAG on telomerase activity

[0247] After confirmation of transfection, the effect of LRP::FLAG overexpression on telomerase activity in a cardiovascular cell (atherosclerosis) culture model was assessed.

[0248] Telomerase activity is low in monocytes due to the fact that these cells have a low turnover rate (Demissie et al., 2006). As a result, when oxLDL is taken up by these cells, telomeres are damaged and eroded. In addition, telomerase activity is reduced due to the induction of an oxidative state in the cells, resulting in the recruitment of the hTERT subunit to the mitochondria (Cong et al., 2002). This prevents it from acting on the telomerase located in the nucleus. qPCR analysis was used to assess how overexpression of LRP::FLAG affects telomerase activity. The results confirmed that relative telomerase activity was upregulated in transfected THP-1 cells ( Figure 2 Indeed, when untransfected THP-1 cells were treated with oxLDL, a significant decrease in relative telomerase activity was seen ( Figure 2 ).

[0249] These studies show that oxLDL reduces telomerase activity in a concentration-dependent manner. Without being limited to theory, this may be due to inactivation of PI3K / Akt via dephosphorylation. Interestingly, it was further shown that when THP-1 cells were transfected, telomerase activity increased dramatically regardless of treatment. Without being limited to theory, this may be due to LRP::FLAG inducing increased hTERT expression. Since hTERT is a limiting factor for telomerase, an increase in hTERT concentration may be the cause of increased telomerase activity. However, since hTERT has an additional telomere effect, an increase in its concentration may not be the only cause of increased telomerase activity (Cong et al., 2002).

[0250] The increase in telomerase activity was found to be sufficient to reverse the effects of oxLDL treatment. Furthermore, a 3.8-fold increase in telomerase activity was observed between untransfected untreated cells and transfected treated cells ( Figure 2), and was evident in both untreated and treated transfected THP-1 cells. The observed upregulation of telomerase activity would result in increased telomere length, thereby counteracting the observed shortening of telomere length associated with atherosclerosis. This results in a reduction in plaque formation and / or reversal and / or prevention of atherosclerosis.

[0251] Effect of LRP::FLAG on cell viability

[0252] To determine the role of upregulated telomerase activity in atherosclerosis, the MTT assay was used to assess cell viability. Cell viability of untransfected THP-1 cells treated with oxLDL was significantly reduced by 1.8-fold ( Figure 3 This is consistent with the literature showing that oxLDL induces apoptosis by rapidly activating the caspase-3 apoptotic pathway (Wintergerst et al., 2000).

[0253] In the untreated cells of transfection, cell viability remains similar to the untreated cells of non-transfection, which indicates that transfection does not cause excessive proliferation. This indicates that LRP::FLAG overexpression does not cause cells to become tumorigenic, which is surprising. Similarly, the cell viability between the untreated cells of non-transfection and the cells of transfection processing does not change significantly, which indicates that transfection saves cells from the apoptosis induced by oxLDL. Because monocytes undergo apoptosis and become foam cells, it is essential to save them from apoptosis to prevent atherosclerotic plaques from forming. The increase in cell viability can be due to the increase in telomerase activity shown previously, which causes telomere expansion.

[0254] Interestingly, a slight increase in cell viability was detected in transfected treated cells ( Figure 3 ). Although insignificant, this increase may be due to oxLDL-induced cell hyperproliferation. Without being limited to theory, it is shown that transfection can increase the cell viability of oxLDL-treated cells by rescuing cells from oxLDL-induced apoptosis. This suggests that LRP::FLAG has a role in preventing / reducing the effects of atherosclerosis in cardiovascular (particularly atherosclerosis) cell culture models.

[0255] Effect of LRP::FLAG on lipid content.

[0256] A major hallmark of atherosclerosis is an increase in lipid content in cells. This occurs when the lipid content of oxLDL is taken up by cells, increasing cellular cholesterol and triglyceride content by upregulating LOX-1 and ACAT1,2 (Batt et al., 2004). To further demonstrate that overexpression of LRP::FLAG and subsequent increase in telomerase activity plays a role in atherosclerosis or cardiovascular disease, cellular lipid content was assessed.

[0257] To assess this, oil red lipid staining was used to obtain qualitative and quantitative measurements of lipid content (Figure 5). Since lipid content in atherosclerotic plaques increases, a decrease in lipid content provides evidence that transfection helps to reverse and / or prevent atherosclerotic plaque formation. It was found that treatment with oxLDL did result in an increase in lipid content in untransfected THP-1 cells. This corresponds to the literature showing that oxLDL uptake results in an increase in lipid content due to the lipid components of oxLDL and the subsequent lipid uptake resulting therefrom (Batt et al., 2004). When treated with oxLDL, transfected cells showed a reduction in lipid content that was significantly lower than untransfected treated cells (11%). The possible reason is that LRP::FLAG can play a role in increasing lipid metabolism in cells.

[0258] in conclusion

[0259] In summary, it is shown that in the THP-1 atherosclerosis cell culture model, overexpression of LRP::FLAG increases telomerase activity. In addition, the overexpression of LRP::FLAG increases the viability of cells treated with oxLDL. In addition, it is shown that transfection can save the cells treated with oxLDL from the increase in lipid content characteristically observed in atherosclerotic plaques. Oil red staining assay reveals that the overexpression of LRP::FLAG in cells treated with oxLDL or untreated cells causes a significant reduction in cellular lipid content, which is usually increased in atherosclerosis. These findings show that LRP / LR may play a role in atherosclerosis, and overexpression of LRP::FLAG can provide potential new therapeutic treatments to reduce or hinder atherosclerotic plaque formation.

[0260] It has been shown that increasing telomere length after plaque formation has no effect on the plaque itself. Therefore, upregulating LRP / LR is not expected to have any effect on plaque and / or its formation. Therefore, it is unexpected and surprising that upregulating telomerase activity by providing LRP / LR to cells and / or upregulating LRP / LR leads to a decrease in lipid content and / or rescues cells from oxLDL-induced apoptosis.

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[0302] Example 2 – Obesity, insulin resistance and / or diabetes:

[0303] Materials and methods

[0304] Cell culture

[0305] The THP-1 human leukemic monocytic cell line was used for Example 2 because when treated with high glucose (HG), it activates monocytes and induces the expression of tumor necrosis factor (TNF)-α via oxidant stress and nuclear factor-kB transcription factor (Shanmugum, 2003). This cell line (monocytes) normally grows in suspension but becomes adherent to endothelial cells, leading to endothelial dysfunction in individuals with type II diabetes when maintained on high glucose factors (Shanmugum, 2003). THP-1 cells were cultured in RMPI (Thermofisher Scientific) supplemented with 10% FBS (sigma) to improve growth, 2% penicillin-streptomycin (Thermofisher Scientific) to prevent bacterial contamination, and 1% MEM non-essential amino acids (lonza). To induce a diabetic state, THP-1 cells were maintained in 20 mM glucose prior to use in downstream experimental procedures. This resulted in four different conditions of THP-1 cells: (i) untreated non-transfected cells, (ii) untreated pCIneo-moLRP::FLAG transfected cells, (iii) glucose-treated non-transfected cells, and (iv) glucose-treated pCIneo-moLRP::FLAG transfected cells.

[0306] These cells are cultivated in 5% CO2 at 37 ℃ to simulate in vivo conditions. Cells are cultivated by changing the culture medium twice a week to supplement nutrition. When needed (reaching 70-80% confluence), THP-1 cells are also subcultured and inoculated with suitable density for downstream experimental procedures. The inoculation and subculture of cells involve scraping cells and centrifuging at 4500rpm for 10 minutes. The culture medium is then discarded and the cells are subsequently resuspended with 2ml phosphate buffered saline (PBS) to remove excess culture medium. The supernatant is then discarded and the cell pellet is resuspended in fresh cell culture medium with 4500rpm centrifugation for 10 minutes. Thereafter, the cells are resuspended with fresh culture medium and transferred back to the flask.

[0307] Transfection

[0308] Cells were transfected with pCIneo-moLRP-FLAG plasmid to induce overexpression of LRP::FLAG protein. This program shows that overexpression of LRP::FLAG increases telomerase activity and reduces lipid content in cells, and thus reduces diabetic status. Xfect transfection reagent (Clontech) was then used to perform the transfection procedure. Xfect reaction buffer was added to 5 μg of pCIneo-moLRP::FLAG plasmid to a total volume of 100 μl. 1.5 μl of Xfect polymer was then added and incubated at room temperature for 10 minutes. This was done to allow the formation of a complex of nanoparticles. Thereafter, this Xfect transfection reagent was gently added to the cells in the flask. The cells were then incubated for 72 hours at 37°C with a master mix comprising pCIneo-moLRP-FLAG constructs and Xfect reagents. This allows transcription and translation of LRP::FLAG, resulting in overexpression of LRP::FLAG protein in THP-1 cells. To ensure that cells continue to transcribe the LRP::FLAG plasmid, cells were occasionally treated with 50 μg / ml geneticin. Transfected cells were then used for downstream experimental procedures.

[0309] Cell counting

[0310] Cell counting is performed to ensure that the same number of cells are used for downstream experiments. The procedure involves staining the cells with filtered trypan blue (0.4%). This allows dead cells to be stained blue, while live cells remain unstained because their membranes are still intact and therefore selective for compounds that pass through the membrane. About 10 μl of sample is used and mixed with 10 μl of trypan blue. Thereafter, 10 μl of this solution is placed on a hemocytometer and the average number of live and dead cells in 16 small squares is counted under a microscope. To calculate the total number of live cells, the following formula is used:

[0311]

[0312] The cell concentration was then used to seed equal concentrations of cells into each well of the plate used in order to ensure that the data obtained were accurate.

[0313] Preparation of protein lysate

[0314] The cells were scraped to facilitate cell separation. The cells were then centrifuged at 6600rpm for 15 minutes. The culture medium was discarded and the pellet was resuspended in PBS and centrifuged. The supernatant was then discarded and the pellet was resuspended in 210μl of non-denaturing (CHAPS) lysis buffer and incubated at 4°C for 30 minutes. It was then centrifuged at 12000rpm for 20 minutes at 4°C. The supernatant containing the protein extract was then transferred to a new Eppendorf tube and the pellet containing the cell debris was discarded. The protein lysate was used for downstream applications, including telomerase activity and glucose assays. For telomerase activity, the extracted protein lysate was quantified by Nanodrop spectroscopy, NanodropND-1000, to ensure that the sample was diluted to an equal concentration of 500ng / μl protein.

[0315] Bicinchoninic acid TM (BCA) Protein Assay:

[0316] BCA is performed to ensure equal concentrations of protein for downstream experiments such as SDS-PAGE and Western blotting. This procedure is used over other protein assays because the peptide backbone in the BCA assay also contributes to the color development, helping to minimize variability caused by differences in protein composition. It is also very sensitive and is not affected by different protein compositions.

[0317] The procedure involves resuspending the cell pellet obtained from the confluent flask (70-80% confluent) with 100 μl of RIPA buffer. RIPA buffer is used to lyse cells so as to harvest cell lysate, because RIPA buffer contains SDS, which linearizes the protein and then gives a total negative charge. Thereafter, 5 μl of the extracted lysate is diluted with 20 μl of distilled water, and loaded in 96-well plates in triplicate. 0, 0.2, 0.4, 0.6, 0.8 and 1 mg / ml of BCA standards are prepared, and each standard of 25 μl is loaded on the plate in duplicate. Subsequently, 200 μl of BCA is added to each well, and then incubated at 37°C for 30 minutes. Then the plate is read at 562nm on an ELISA plate reader. Then a standard curve is drawn, and it is used to infer the protein concentration of the sample. The sample is then diluted to a final concentration of 2 mg / ml, which is then used in downstream experiments, such as SDS PAGE and Western blotting. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting

[0318] SDS-PAGE and Western blotting were used to analyze the presence and amount of proteins in transfected and non-transfected THP-1 cells.

[0319] This experimental procedure was used to confirm the overexpression of LRP::FLAG. This was performed between transfected untreated, non-transfected untreated, transfected treated and non-transfected treated THP-1 cells. It was achieved by separating the proteins based on size using SDS-PAGE and western blotting using primary and secondary antibodies for specific protein detection.

[0320] SDS-PAGE

[0321] The extracted lysate was diluted to 2 mg / ml with RIPA buffer. RIPA contains SDS, which linearizes proteins by adding a negative charge proportional to their size and shape. Since the extracted sample is given an overall negative charge, it separates into bands based on size by the migration of proteins from the anode to the cathode when exposed to an electric current. Since the molecular weights of LRP::FLAG and β-actin are ±40 kDa and 42 kDa, respectively, the samples were loaded on a 10% (w / v) polyacrylamide gel.

[0322] SDS-PAGE consists of a separation and stacking gel. The separation gel is prepared by adding 2.5 ml of 40% acrylamide containing 4.8 ml of distilled water, 2.5 ml of separation buffer, 100 μl of 10% APS and 5 μl of TEMED. The separation gel is then poured between the gel casting plates and covered with isopropanol to remove bubbles, as bubbles prevent polymerization. It is then polymerized. Thereafter, a stacking gel is prepared by mixing 625 μl of 40% acrylamide with 3.65 ml of distilled water, 625 μl of stacking buffer, 50 μl of APS and 5 μl of TEMED. The isopropanol is then discarded and the stacking gel is poured on the polymerized separation gel. The comb is then inserted and the gel is polymerized. Once the gel is polymerized, the plate is placed in the electrophoresis instrument. The running buffer is then added and the comb is removed. Thereafter, the 2 mg / ml extracted lysate obtained in Section 3.4 is mixed with 1-fold loading dye and then heated at 95° C. for 5 minutes. The heated samples were vortexed and 5 μl of sample was loaded into the gel wells along with 2 μl of molecular weight markers. The gel was separated at 120 V for 45 minutes (until the dye front was 1 cm from the bottom of the gel).

[0323] Western blotting

[0324] Electrophoretic transfer was applied to the SDS-PAGE gel in order to transfer the separated proteins from the gel to the PVDF membrane. Initially, the PVDF membrane was soaked and activated in methanol for 2 minutes. Thereafter, the PVDF membrane was assembled onto the electroblotting equipment, and the gel was placed on top of the membrane, and electroblotting was performed at 120V for 45 minutes. The PVDF membrane was then blocked for 1 hour using 10ml of 3% BSA blocking solution to prevent nonspecific binding of the primary antibody. Thereafter, the PVDF membrane was incubated overnight at 4°C with 2μl of primary antibody specific to the protein of interest. Unbound primary antibodies were washed away with 5 10ml PBS Tween washes for 5 minutes each. Then 2μl of anti-human secondary antibodies conjugated to HRP enzyme were added in 10ml of 3% BSA at room temperature for 1 hour. After incubation, 5 10ml PBS Tween washes were performed for 5 minutes each to remove unbound secondary antibodies. A chemiluminescent substrate was subsequently added, and its signal was detected and analyzed using Biorad Chemi-doc software. Densitometry was performed to quantify protein levels using ImageJTM software for non-transfected cells and pCIneo-moLRP::FLAG transfected cells.

[0325] Evaluation of telomerase activity in cells overexpressing LRP::FLAG using qPCR

[0326] Quantitative / Real-time PCR

[0327] Telomerase activity was measured in transfected untreated, non-transfected untreated, transfected treated, and non-transfected treated THP-1 cells to determine whether overexpression of LRP::FLAG enhances telomerase activity in a diabetic setting. Telomerase activity assay kit: TRAPeze was used according to the manufacturer's protocol. Telomerase activity was determined by real-time qPCR using a half-reaction.

[0328] The telomerase activity detection kit consists of a buffer, amplification primers and two enzyme systems, which enable fluorescence detection by real-time qPCR. In order for the extracted telomerase to add the telomeric repeat sequence to the 3' end of the substrate, 2μl of the extracted lysate (containing telomerase) obtained from Section 3.2 was incubated with 12.5μl of the reaction mixture in a 96-well plate at 37°C. The reaction mixture was also added to the standard and positive control. Heat-inactivated telomerase was used as a control, and water and chaps were used as negative controls. About 2μl of these controls were added to the 96-well plate and incubated with 12.5μl of the control mixture at 37°C. The heat-inactivated control was performed by incubating 30μl of the extracted lysate at 100°C for 20 minutes. The reaction mixture was completed by adding 2.5μl of 5X TRAPeze RT reaction mixture, 0.2μl Taq polymerase and 8.8μl of PCR-grade water to each well. At the same time, the control mixture was completed by adding 2.5 μl of 5X TRAPeze RT control reaction mixture, 0.2 μl Taq polymerase and 7.8 μl of PCR grade water to each well. The telomerase activity of THP-1 cells was performed in duplicate (3 biological repeats and 2 technical repeats) and analyzed using the Roche LightCycler 480 running 45 cycles. The data generated were normalized to the standard curve generated by the serially diluted TSR8 samples. Data were analyzed using the Student's t test.

[0329] Lipid content was assessed using red oil staining in cells overexpressing LRP::FLAG.

[0330] Oil red staining

[0331] Detect lipid content in THP-1 cells to determine whether the overexpression of LRP::FLAG reduces lipid content (diabetic state) in transfected cells simulating type II diabetes. As described in Section 3.1, 8000 cells were inoculated per well in a 96-well plate. For all oil red dye components used in this program, a volume of 100 μl was used per well. Gently remove the culture medium and wash the cells once with PBS. Thereafter, formalin was added to the cells and incubated at room temperature for 45 minutes to fix the cells on the plate. During the incubation time, an oil red working solution was prepared by mixing 3.6 ml of a stock solution with 20 ml of isopropanol and 2.4 ml of distilled water. Once the incubation time is over, formalin is discarded, and then the cells are washed twice with water. Then the cells are permeable by incubating the cells with 60% isopropanol for 5 minutes. Then 60% of the isopropanol is discarded, and then the cells are stained with an oil red working solution. It was incubated for 15 minutes under gentle stirring. After that, discard the oil red o solution, and wash the cells with water 3 times, then add hematoxylin to dye the nucleus blue, and incubate for 1 minute to enable it to be visualized. Then discard hematoxylin, and wash the cells with water 3 times. Then cover the cells with water and observe under a microscope to visualize and check the accumulation of intracellular lipids. Then quantitatively dye by adding 100 μl of 100% isopropanol and using an ELISA plate reader to accurately read the plate at a wavelength of 572nm. Data are analyzed using a student's t test.

[0332] Glucose uptake in cells overexpressing LRP::FLAG was assessed.

[0333] Colorimetric ELISA

[0334] This plate-based assay was performed to determine whether overexpression of LRP::FLAG resulted in improved glucose uptake in cells cultured in high glucose. Therefore, we used a colorimetric ELISA to assess glucose uptake between untreated pCIneo-moLRP::FLAG transfected, untreated non-transfected, glucose-treated non-transfected, and glucose-treated pCIneo-moLRP::FLAG transfected THP-1 cells. The colorimetric ELISA was chosen because it is rapid, highly sensitive, and specific. The glucose assay kit of the invention evaluates the uptake of glucose by THP-1 cells. This kit consists of a buffer, a glucose probe, a glucose standard, and a glucose enzyme mixture that oxidizes the glucose present and produces a product that reacts with the probe to produce a color change. The intensity of the color is proportional to the amount of glucose present in the sample.

[0335] Then about 20 μ l of the extracted lysate was loaded in quadruplicate, then 30 μ l of glucose assay buffer was added to make a total volume of 50 μ l, while chaps and glucose buffer were used as negative controls. Thereafter, 50 μ l of glucose reagent mixture (which was prepared by mixing 46 μ l of glucose assay buffer with 2 μ l of glucose probe and 2 μ l of glucose enzyme mixture) was added to all plates containing extracted lysate and controls. It was then incubated at 37 ° C for 30 minutes and covered with foil because the reagent is light sensitive. The plate was then read using an enzyme-linked immunosorbent assay (ELISA) plate reader at an absorbance of 570 nm. The data obtained from the ELISA plate reader was analyzed using a student's t test.

[0336] result

[0337] Detection of LRP::FLAG overexpression in THP-1 cells using Western blotting

[0338] Cell culture model THP- cells were stably transfected with the pCIneo-moLRP::FLAG plasmid. This was done to induce overexpression of LRP::FLAG, which was confirmed using western blotting. β-actin was determined as a loading control to confirm that protein loading was the same throughout the gel and to ensure that the results were not due to loading or protein transfer errors. Figure 6 As shown, β-actin was detected in pCIneo-moLRP::FLAG transfected and non-transfected cells. LRP::FLAG was only detected in cells transfected with pCIneo-moLRP::FLAG, indicating that the transfection procedure was successful (lanes 1-4). Figure 6 Shown is the use of Western blotting to detect LRP::FLAG overexpression in THP-1 cells. Western blot analysis of β-actin and LRP::FLAG overexpression in THP-1 transfected cells (lanes 1-4) and THP-1 non-transfected cells (lanes 5-8). Detection of LRP::FLAG protein is achieved using mouse anti-FLAG primary antibody and anti-mouse IgG secondary antibody coupled to HRP. In addition, loading control (β-actin) is also determined in pCIneo-moLRP::FLAG transfected and non-transfected cells. β-actin is detected using monoclonal anti-β-actin peroxidase conjugated antibodies. LRP::FLAG is determined to be expressed only in THP-1pCIneo-moLRP::FLAG transfected cells.

[0339] Overexpression of LRP::FLAG significantly increased telomerase activity in THP-1 cells, independent of glucose treatment.

[0340] Telomere activity was determined between untreated pCIneo-moLRP::FLAG transfected, untreated non-transfected, glucose-treated pCIneo-moLRP::FLAG transfected, and glucose-treated non-transfected THP-1 cells. It was found that overexpression of LRP::FLAG resulted in increased telomerase activity in untreated THP-1pCIneo-moLRP::FLAG transfected, glucose-treated pCIneo-moLRP::FLAG, and glucose-treated non-transfected THP-1 cells relative to untreated non-transfected THP-1 cells ( Figure 7 ). Increase in telomerase activity between glucose-treated pCIneo-moLRP::FLAG-transfected cells and glucose-treated non-transfected cells. Figure 7 Overexpression of LRP::FLAG significantly increased telomerase activity in THP-1 cells that mimic a diabetic state. The results showed that when compared to untreated non-transfected cells, untreated pCIneo-moLRP::FLAG transfected cells (p = 1 × 10 -4 ) and glucose-treated non-transfected THP (p = 8 × 10 -4 ) increased their telomerase activity by 4.7-fold and 1.7-fold, respectively. Applicants also observed that glucose-treated pCIneo-moLRP::FLAG-transfected and untreated non-transfected cells (p = 1 × 10 -4 ) significantly increased by 4.7-fold between glucose-treated non-transfected cells and glucose-treated pCIneo-moLRP::FLAG-transfected cells (p = 4 × 10 -5 ) increased by 2.8-fold between 2 and 1.5 fold. Data plotted are relative to untreated, non-transfected THP-1 cells set as 100%.

[0341] Overexpression of LRP::FLAG significantly reduced lipid content in THP-1 pCIneo-moLRP::FLAG-transfected cells. quantity.

[0342] Lipid content is determined in untreated pCIneo-moLRP::FLAG transfected, untreated non-transfected, glucose-treated pCIneo-moLRP::FLAG and glucose-treated non-transfected THP-1 cells. In order to assess lipid content, lipids were stained using oil red staining, which was quantified on an ELISA plate reader. Oil red staining shows that, as shown in Figure 8, compared with glucose-treated non-transfected cells, the overexpression of LRP::FLAG significantly reduces the lipid content shown as pink staining in glucose-treated pCIneo-moLRP::FLAG transfected cells.

[0343] Figure 8 shows that overexpression of LRP::FLAG significantly reduces lipid content in THP-1 cells that simulate a type II diabetic state. (a) Images obtained after 10-fold magnification show that the lipid content in glucose-treated non-transfected cells is higher (shown as pink staining) compared to glucose-treated pCIneo-moLRP::FLAG-transfected cells, untreated non-transfected cells, and untreated pCIneo-moLRP::FLAG-transfected cells. The lipid content shown in the pink staining of glucose-treated pCIneo-moLRP::FLAG-transfected THP-1 cells was reduced to approximately the same level as that of untreated non-transfected THP-1 cells. (b) When compared to glucose-treated non-transfected cells (p=4×10 -5 ) were compared to glucose-treated pCIneo-moLRP::FLAG transfected cells. We also observed a 1.1-fold decrease in lipid content between untreated pCIneo-moLRP::FLAG transfected cells and untreated non-transfected cells, while a 1.04-fold increase in lipid content between untreated non-transfected cells and glucose-treated pCIneo-moLRP::FLAG transfected cells (p = 0.008). Untreated non-transfected cells and glucose-treated non-transfected cells (p = 3.7 × 10 -6 ) showed a significant 1.3-fold increase in lipid content between 1 and 2 . Data plotted are relative to untreated, non-transfected THP-1 cells set as 100%.

[0344] LRP::FLAG overexpression induces increased glucose uptake in THP-1 cells that mimic a diabetic state

[0345] It is found that the overexpression of LRP::FLAG has an impact on the ability of cell uptake of glucose, and is confirmed by glucose determination based on ELISA.The glucose uptake of THP-1 cells is determined between pCIneo-moLRP::FLAG transfected, untreated non-transfected, glucose-treated pCIneo-moLRP::FLAG and glucose-treated non-transfected cells. Glucose determination based on ELISA shows that, relative to glucose-treated non-transfected cells, the glucose uptake of glucose-treated pCIneo-moLRP::FLAG transfected cells significantly increases, and between untreated pCIneo-moLRP::FLAG and untreated non-transfected cells is also the case. In addition, we observe that, relative to the culture medium of the untreated pCIneo-moLRP::FLAG transfected of THP-1, the glucose in the culture medium of the untreated non-transfected cells of THP-1 increases.

[0346] Fig. 9 LRP::FLAG overexpression induces increased glucose uptake in THP-1 cells that mimic a diabetic state. Untreated pCIneo-moLRP::FLAG transfected cells were determined to have a 1.18-fold increase in glucose uptake relative to untreated non-transfected cells (p=0.03). The results also showed that glucose-treated pCIneo-moLRP::FLAG transfected cells and glucose-treated non-transfected cells (p=7×10 -4 ) increased by 1.1-fold, whereas glucose uptake decreased by 1.12-fold between the culture medium of untreated pCIneo-moLRP::FLAG-transfected cells and untreated non-transfected cells.

[0347] Discussion and Conclusion:

[0348] Western blotting reveals that LRP::FLAG in pCIneo-moLRP::FLAG THP-1 transfected cells is overexpressed. This shows that the transfection procedure is successful. The results show that the telomerase activity in untreated pCIneo-moLRP::FLAG transfected, glucose-treated non-transfected and glucose-treated pCIneo-moLRP::FLAG transfected THP-1 increases. This shows that telomerase activity is enhanced, thereby extending telomeres. Along with the increase in telomerase activity, it is further found that overexpression of LRP::FLAG reduces the lipid content in THP-1 cells.

[0349] It was further shown that LRP::FLAG-dependent telomerase activation resulted in a significant reduction in lipid content of glucose-treated pCIneo-moLRP::FLAG to approximately the same level as controls (untreated non-transfected and untreated pCIneo-moLRP::FLAG-transfected THP-1 cells). Diabetic cells have been found to have increased lipid content, which is also associated with insulin resistance (Gavin et al., 2017). Studies have shown that increased lipid content leads to insulin resistance (Palmer et al., 2015).

[0350] Thus, by overexpressing LRP::FLAG in THP-1 cells, telomerase activity is increased. Without being limited to theory, this may lead to a reduction in ROS species, which also play a role in the regulation of lipid metabolism by activating fatty acid synthase (FAS) by increasing the expression of sterol regulatory element binding protein (SREBP) cleavage activator protein (Ryu et al., 2015). FAS is an adipogenic gene that stimulates adipogenesis, and this contributes to insulin resistance. It is known that increased FAS levels reduce mitochondrial β-oxidation in skeletal muscle cells and hepatocytes. This leads to an increase in (DAG) and (LCCoA), inducing serine / threonine phosphorylation at the IRS-1 site, thereby inhibiting IRS-1 phosphorylation and activation of phosphatidylinositol 3-phosphate (PI3P) signaling. (Savage et al., 2007). Inhibition of PI3P activation in bone leads to reduced GLUT4 synthesis, thereby reducing glucose uptake by skeletal muscle. Inhibition of PI3P activation in hepatocytes leads to a decrease in forkhead box protein O (FOXO) phosphorylation, which in turn leads to an increase in hepatic gluconeogenesis (Savage et al., 2007). Therefore, without being limited by theory, overexpression of LRP::FLAG can lead to a decrease in ROS species, which can lead to a decrease in lipid metabolism and lipid content, as shown in the figure. The decrease in lipid content may inhibit insulin resistance by improving insulin sensitivity.

[0351] Improved insulin sensitivity and reduced insulin resistance lead to increased cellular glucose uptake and reduced glucose production. Insulin activates the MAPK, PI3K-Akt and CAP / Cbl and small G protein TC10 pathways to increase glucose uptake. MAPK inhibits gluconeogenesis, PI3K-Akt increases GLUT-4 and CAP / Cbl, and small G protein TC10 increases the inhibition of lipogenesis (Hajiaghaalipour et al., 2015). This is consistent with our results, in which we observed increased glucose uptake in THP-1pCIneo-moLRP-FLAG transfected cells that mimic type II diabetes. This increase in cellular glucose uptake means that the hyperglycemic state, a major hallmark of diabetes, is reduced.

[0352] Therefore, by using LRP::FLAG, increasing hTERT levels and telomerase activity, it may lead to a decrease in ROS and thereby a reduction in the hyperglycemic state, such as seen in type II diabetes, such as Figure 7 shown.

[0353] Reducing lipid concentrations in diabetic cells will improve impaired insulin signaling therein, thereby treating and / or preventing obesity, insulin resistance and / or diabetes.

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[0407] Van Deursen,M.(2014).“The role of senescent cells in ageing”.Nature.509(7501):439–446.

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[0410] Weinrich,S.L.,Pruzan,R.,Ma,L.,Ouellette,M.,Tesmer,V.M.,Holt,S.E.,Bodnar,A.G.,Lichtsteiner,S.,Kim,N.W.,Trager,J.B.,Taylor,R.D.,Carlos,R.,Andrews,W.H.,Wright,W.E.,Shay,J.W.,Harley,C.B.and Morin,G.B.(1997).Reconstitution of human telomerase with the template RNA component hTR andthe catalytic protein subunit hTRT.Nat Genet.17:498–502.

[0411] Weiss,S.F.T.(2017).Bad Boy with a Twist:Targeting the 37kda / 67KDaLaminin Receptor for Treatment of Cancer and Neurodegenerative Diseases andfor Changing Telomere Dynamics.Cell Cellular Lif Sci J.2(2):000114

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[0414] Zhao,J.,Zhu,Y.,Lin J.,Matsuguchi,T.,Blackburn,E.,Zhang,Y.,Cole,SA,Best,LG,Lee,ETand Howard,BV(2013).Short leukocyte telomere lengthpredicts risk of diabetes in American Indians: the Strong Heart FamilyStudy.Diabetes.63:354–362.

[0415] Applicants believe that the subject matter disclosed herein improves upon at least one of the shortcomings known in the prior art.

[0416] Although the subject matter of the present disclosure has been described in detail with respect to specific embodiments and / or examples thereof, it should be understood that those skilled in the art, after understanding the foregoing, can easily conceive of changes, variations and equivalents of these embodiments. Therefore, the scope of the present disclosure should be assessed as the scope of the claims and any equivalents thereof, which are attached hereto. Sequence Listing <110> University of the Witwatersrand, Johannesburg <120> Biopharmaceuticals for reducing lipid content in cells <130> P1181PC <140> <141> <150> 2018 / 04509 <151> 2018-07-06 <160> 5 <170> PatentIn Version 3.5 <210> 1 <211> 295 <212> PRT <213> Homo sapiens <400> 1 Met Ser Gly Ala Leu Asp Val Leu Gln Met Lys Glu Glu Asp Val Leu 1 5 10 15 Lys Phe Leu Ala Ala Gly Thr His Leu Gly Gly Thr Asn Leu Asp Phe 20 25 30 Gln Met Glu Gln Tyr Ile Tyr Lys Arg Lys Ser Asp Gly Ile Tyr Ile 35 40 45 Ile Asn Leu Lys Arg Thr Trp Glu Lys Leu Leu Leu Ala Ala Arg Ala 50 55 60 Ile Val Ala Ile Glu Asn Pro Ala Asp Val Ser Val Ile Ser Ser Arg 65 70 75 80 Asn Thr Gly Gln Arg Ala Val Leu Lys Phe Ala Ala Ala Thr Gly Ala 85 90 95 Thr Pro Ile Ala Gly Arg Phe Thr Pro Gly Thr Phe Thr Asn Gln Ile 100 105 110 Gln Ala Ala Phe Arg Glu Pro Arg Leu Leu Val Val Thr Asp Pro Arg 115 120 125 Ala Asp His Gln Pro Leu Thr Glu Ala Ser Tyr Val Asn Leu Pro Thr 130 135 140 Ile Ala Leu Cys Asn Thr Asp Ser Pro Leu Arg Tyr Val Asp Ile Ala 145 150 155 160 Ile Pro Cys Asn Asn Lys Gly Ala His Ser Val Gly Leu Met Trp Trp 165 170 175 Met Leu Ala Arg Glu Val Leu Arg Met Arg Gly Thr Ile Ser Arg Glu 180 185 190 His Pro Trp Glu Val Met Pro Asp Leu Tyr Phe Tyr Arg Asp Pro Glu 195 200 205 Glu Ile Glu Lys Glu Glu Gln Ala Ala Ala Glu Lys Ala Val Thr Lys 210 215 220 Glu Glu Phe Gln Gly Glu Trp Thr Ala Pro Ala Pro Glu Phe Thr Ala 225 230 235 240 Thr Gln Pro Glu Val Ala Asp Trp Ser Glu Gly Val Gln Val Pro Ser 245 250 255 Val Pro Ile Gln Gln Phe Pro Thr Glu Asp Trp Ser Ala Gln Pro Ala 260 265 270 Thr Glu Asp Trp Ser Ala Ala Pro Thr Ala Gln Ala Thr Glu Trp Val 275 280 285 Gly Ala Thr Thr Asp Trp Ser 290 295 <210> 2 <211> 295 <212> PRT <213> Mus musculus <400> 2 Met Ser Gly Ala Leu Asp Val Leu Gln Met Lys Glu Glu Asp Val Leu 1 5 10 15 Lys Phe Leu Ala Ala Gly Thr His Leu Gly Gly Thr Asn Leu Asp Phe 20 25 30 Gln Met Glu Gln Tyr Ile Tyr Lys Arg Lys Ser Asp Gly Ile Tyr Ile 35 40 45 Ile Asn Leu Lys Arg Thr Trp Glu Lys Leu Leu Leu Ala Ala Arg Ala 50 55 60 Ile Val Ala Ile Glu Asn Pro Ala Asp Val Ser Val Ile Ser Ser Arg 65 70 75 80 Asn Thr Gly Gln Arg Ala Val Leu Lys Phe Ala Ala Ala Thr Gly Ala 85 90 95 Thr Pro Ile Ala Gly Arg Phe Thr Pro Gly Thr Phe Thr Asn Gln Ile 100 105 110 Gln Ala Ala Phe Arg Glu Pro Arg Leu Leu Val Val Thr Asp Pro Arg 115 120 125 Ala Asp His Gln Pro Leu Thr Glu Ala Ser Tyr Val Asn Leu Pro Thr 130 135 140 Ile Ala Leu Cys Asn Thr Asp Ser Pro Leu Arg Tyr Val Asp Ile Ala 145 150 155 160 Ile Pro Cys Asn Asn Lys Gly Ala His Ser Val Gly Leu Met Trp Trp 165 170 175 Met Leu Ala Arg Glu Val Leu Arg Met Arg Gly Thr Ile Ser Arg Glu 180 185 190 His Pro Trp Glu Val Met Pro Asp Leu Tyr Phe Tyr Arg Asp Pro Glu 195 200 205 Glu Ile Glu Lys Glu Glu Gln Ala Ala Ala Glu Lys Ala Val Thr Lys 210 215 220 Glu Glu Phe Gln Gly Glu Trp Thr Ala Pro Ala Pro Glu Phe Thr Ala 225 230 235 240 Ala Gln Pro Glu Val Ala Asp Trp Ser Glu Gly Val Gln Val Pro Ser 245 250 255 Val Pro Ile Gln Gln Phe Pro Thr Glu Asp Trp Ser Ala Gln Pro Ala 260 265 270 Thr Glu Asp Trp Ser Ala Ala Pro Thr Ala Gln Ala Thr Glu Trp Val 275 280 285 Gly Ala Thr Thr Glu Trp Ser 290 295 <210> 3 <211> 8 <212> PRT <213> Escherichia coli <400> 3 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 <210> 4 <211> 194 <212> PRT <213> Homo sapiens <400> 4 Arg Phe Thr Pro Gly Thr Phe Thr Asn Gln Ile Gln Ala Ala Phe Arg 1 5 10 15 Glu Pro Arg Leu Leu Val Val Thr Asp Pro Arg Ala Asp His Gln Pro 20 25 30 Leu Thr Glu Ala Ser Tyr Val Asn Leu Pro Thr Ile Ala Leu Cys Asn 35 40 45 Thr Asp Ser Pro Leu Arg Tyr Val Asp Ile Ala Ile Pro Cys Asn Asn 50 55 60 Lys Gly Ala His Ser Val Gly Leu Met Trp Trp Met Leu Ala Arg Glu 65 70 75 80 Val Leu Arg Met Arg Gly Thr Ile Ser Arg Glu His Pro Trp Glu Val 85 90 95 Met Pro Asp Leu Tyr Phe Tyr Arg Asp Pro Glu Glu Ile Glu Lys Glu 100 105 110 Glu Gln Ala Ala Ala Glu Lys Ala Val Thr Lys Glu Glu Phe Gln Gly 115 120 125 Glu Trp Thr Ala Pro Ala Pro Glu Phe Thr Ala Thr Gln Pro Glu Val 130 135 140 Ala Asp Trp Ser Glu Gly Val Gln Val Pro Ser Val Pro Ile Gln Gln 145 150 155 160 Phe Pro Thr Glu Asp Trp Ser Ala Gln Pro Ala Thr Glu Asp Trp Ser 165 170 175 Ala Ala Pro Thr Ala Gln Ala Thr Glu Trp Val Gly Ala Thr Thr Asp 180 185 190 Trp Ser <210> 5 <211> 194 <212> PRT <213> Mus musculus <400> 5 Arg Phe Thr Pro Gly Thr Phe Thr Asn Gln Ile Gln Ala Ala Phe Arg 1 5 10 15 Glu Pro Arg Leu Leu Val Val Thr Asp Pro Arg Ala Asp His Gln Pro 20 25 30 Leu Thr Glu Ala Ser Tyr Val Asn Leu Pro Thr Ile Ala Leu Cys Asn 35 40 45 Thr Asp Ser Pro Leu Arg Tyr Val Asp Ile Ala Ile Pro Cys Asn Asn 50 55 60 Lys Gly Ala His Ser Val Gly Leu Met Trp Trp Met Leu Ala Arg Glu 65 70 75 80 Val Leu Arg Met Arg Gly Thr Ile Ser Arg Glu His Pro Trp Glu Val 85 90 95 Met Pro Asp Leu Tyr Phe Tyr Arg Asp Pro Glu Glu Ile Glu Lys Glu 100 105 110 Glu Gln Ala Ala Ala Glu Lys Ala Val Thr Lys Glu Glu Phe Gln Gly 115 120 125 Glu Trp Thr Ala Pro Ala Pro Glu Phe Thr Ala Ala Gln Pro Glu Val 130 135 140 Ala Asp Trp Ser Glu Gly Val Gln Val Pro Ser Val Pro Ile Gln Gln 145 150 155 160 Phe Pro Thr Glu Asp Trp Ser Ala Gln Pro Ala Thr Glu Asp Trp Ser 165 170 175 Ala Ala Pro Thr Ala Gln Ala Thr Glu Trp Val Gly Ala Thr Thr Glu 180 185 190 Trp Ser

Claims

1. Use of a biopharmaceutical in the preparation of a medicament for treating and / or preventing obesity, the biopharmaceutical comprising 37 kDa / 67 kDa laminin receptor precursor / high affinity laminin receptor (LRP / LR) and / or fragments thereof, wherein LRP / LR and / or the fragments thereof are used for administration to target cells of a subject in need thereof, and wherein the target cells are at least one of the following groups: vascular endothelial cells, vascular smooth muscle cells, pancreatic cells including α cells, β cells, δ cells and γ cells, such that administration of the biopharmaceutical reduces the lipid content in the target cells when used, and wherein the protein sequence of the LRP / LR is shown in SEQ ID NO: 1 or SEQ ID NO: 2, and the protein sequence of the fragments thereof is shown in SEQ ID NO: 4 or SEQ ID NO:

5.

2. The use according to claim 1, wherein the LRP / LR is encapsulated into a delivery vehicle comprising nanoparticles.

3. The use according to claim 2, wherein the nanoparticle is functionalized with a chemical, biochemical or biological moiety to ensure site-specific delivery to the target cell, wherein the chemical, biochemical or biological moiety acts as a ligand to ensure site-specific delivery at the target cell.

4. The use according to claim 2, wherein the delivery means is formulated as a pharmaceutical composition comprising a pharmaceutical carrier for parenteral or non-parenteral administration to the subject.

5. The use according to claim 4, wherein the pharmaceutical composition further comprises an active pharmaceutical ingredient (API).

6. The use according to claim 5, wherein the pharmaceutical composition further comprises an antioxidant, such that the antioxidant scavenge reactive oxygen species when used at the target cell.

7. The use according to claim 1, wherein the LRP / LR forms part of a transfection agent for expressing 37 kDa / 67 kDa laminin receptor precursor / high affinity laminin receptor (LRP / LR) and / or fragments thereof.

8. Use according to any one of claims 2 to 6, wherein the LRP / LR forms part of a transfection agent for expressing 37 kDa / 67 kDa laminin receptor precursor / high affinity laminin receptor (LRP / LR) and / or fragments thereof.

9. The use according to claim 8, wherein the transfection agent is pCIneo-moLRP::FLAG plasmid.

10. The use according to any one of claims 2 to 6 and 9, wherein the nanoparticles are polymer nanoparticles and are biodegradable to provide a reduced risk of an immunogenic response to the polymer nanoparticles when used, and wherein the polymer nanoparticles are biocompatible to mitigate the risk of any immunogenic response to the polymer nanoparticles, and wherein the polymer nanoparticles are one or more selected from the following group: acrylic resin, gum arabic, carrageenan, cellulose, hydroxypropyl cellulose (HPC), methyl cellulose (MC), hydroxypropyl methyl cellulose (HPMC), lactic acid-glycolic acid copolymer (PLGA), chitin, pectin, pullulan, natural rubber, polyethylene olefins, polypropylene, polystyrene, polyamide, polyacrylonitrile, polyvinyl chloride, polyvinyl alcohol (PVA), polyethylene glycol (PEG), polyethylene oxide (PEO), poly (D-lactide) (PDLA), polylactic acid (PLLA), polygalacturonate, methylcellulose (polyacetal), poly (ε-caprolactone), phospholipids, polysaccharides, polyanionic polysaccharides, carboxymethyl cellulose, carboxymethyl amylose, chondroitin 6-sulfate, dermatan sulfate, heparin, heparin sulfate, poly (hydroxyethyl methacrylate), collagen, fibrinogen, albumin, fibrin, acrylamide, hydroxypropyl methacrylamide copolymer, polyacrylamide, poly (N-isopropyl acrylamide) (pNIPAAm), polyvinyl pyrrolidone, poly (Methacrylic acid-g-ethylene glycol), poly(N-2-hydroxypropyl methacrylamide), poly(glycolic acid) (PGA), poly(lactic acid) (PLA), chitosan, poly(2-hydroxyethyl methacrylate) (HEMA), polynitrile, phosphorylcholine, hyaluronic acid (HA), hydroxyethyl methacrylate (HEMA), methylene-bis-acrylamide (MBAAm), poly(acrylic acid) (PAAc), polyacrylamide (PAAm), polyacrylonitrile (PAN), polyoxybutylene (PBO), polycaprolactone (PCL), poly(ethyleneimine) (PEI), poly(ethyl methacrylate) (PEMA), propylene glycol fumarate (PF), poly(glucose ethyl methacrylate) (PGEMA) , poly(hydroxybutyrate) (PHB), poly(hydroxyethyl methacrylate) (PHEMA), poly(hydroxypropyl methacrylamide) (PHPMA), poly(methyl methacrylate) (PMMA), poly(N-vinyl pyrrolidone) (PNVP), poly(propylene oxide) (PPO), poly(vinyl acetate) (PVAc), poly(vinylamine), chondroitin sulfate, dextran sulfate, polylysine, gelatin, carboxymethyl chitin, dextran, agarose, pullulan, polyester, PEG-PLA-PEG, PEG-PLGA-PEG, PEG-PCL-PEG, PLA-PEG-PLA, poly(PF-co-EG), poly(PEG / PBO-terephthalate),PEG-bis-(PLA-acrylate), PEG6CD, PEG-g-poly(AAm-co-vinylamine), poly(NIPAAm-co-AAc), poly(NIPAAm-co-EMA), PNVP, poly(MMA-co-HEMA), poly(AN-co-allyl sulfonate), poly(biscarboxy-phenoxy-phosphazene), poly(GEMA-sulfate), poly(PEG-co-peptide), alginate-g-(PEO-PPO-PEO), poly(PLGA-co-serine), collagen-acrylate, alginate, alginate-acrylate, poly(HPMA-g-peptide), HA-g-NIPAAm and poly(vinyl methyl ether) (PVME) and / or derivatives of any one or more of the above.

11. The use according to claim 8, wherein the nanoparticles are polymer nanoparticles and are biodegradable to provide a reduced risk of immunogenic response to the polymer nanoparticles when used, and wherein the polymer nanoparticles are biocompatible to mitigate the risk of any immunogenic response to the polymer nanoparticles, and wherein the polymer nanoparticles are one or more selected from the following group: acrylic resin, gum arabic, carrageenan, cellulose, hydroxypropyl cellulose (HPC), methyl cellulose (MC), hydroxypropyl methyl cellulose (HPMC), lactic acid-glycolic acid copolymer (PLGA), chitin, pectin, pullulan, natural rubber, polyethylene, polypropylene, polystyrene , polyamide, polyacrylonitrile, polyvinyl chloride, polyvinyl alcohol (PVA), polyethylene glycol (PEG), polyethylene oxide (PEO), poly (D-lactide) (PDLA), polylactic acid (PLLA), polygalacturonate, methylcellulose (polyacetal), poly (ε-caprolactone), phospholipids, polysaccharides, polyanionic polysaccharides, carboxymethyl cellulose, carboxymethyl amylose, chondroitin 6-sulfate, dermatan sulfate, heparin, heparin sulfate, poly (hydroxyethyl methacrylate), collagen, fibrinogen, albumin, fibrin, acrylamide, hydroxypropyl methacrylamide copolymer, polyacrylamide, poly (N-isopropyl acrylamide) (pNIPAAm), polyvinyl pyrrolidone, poly (methacrylic acid-g-ethylene glycol) ), poly(N-2-hydroxypropyl methacrylamide), poly(glycolic acid) (PGA), poly(lactic acid) (PLA), chitosan, poly(2-hydroxyethyl methacrylate) (HEMA), polynitrile, phosphorylcholine, hyaluronic acid (HA), hydroxyethyl methacrylate (HEMA), methylene-bis-acrylamide (MBAAm), poly(acrylic acid) (PAAc), polyacrylamide (PAAm), polyacrylonitrile (PAN), polybutylene oxide (PBO), polycaprolactone (PCL), poly(ethyleneimine) (PEI), poly(ethyl methacrylate) (PEMA), propylene glycol fumarate (PF), poly(glucose ethyl methacrylate) (PGEMA), poly(hydroxybutyrate) (PHB) , poly(hydroxyethyl methacrylate) (PHEMA), poly(hydroxypropyl methacrylamide) (PHPMA), poly(methyl methacrylate) (PMMA), poly(N-vinyl pyrrolidone) (PNVP), poly(propylene oxide) (PPO), poly(vinyl acetate) (PVAc), poly(vinylamine), chondroitin sulfate, dextran sulfate, polylysine, gelatin, carboxymethyl chitin, dextran, agarose, pullulan, polyester, PEG-PLA-PEG, PEG-PLGA-PEG, PEG-PCL-PEG, PLA-PEG-PLA, poly(PF-co-EG), poly(PEG / PBO-terephthalate), PEG-bis-(PLA-acrylate),PEG6CD, PEG-g-poly(AAm-co-vinylamine), poly(NIPAAm-co-AAc), poly(NIPAAm-co-EMA), PNVP, poly(MMA-co-HEMA), poly(AN-co-allyl sulfonate), poly(dicarboxy-phenoxy-phosphazene), poly(GEMA-sulfate), poly(PEG-co-peptide), alginate-g-(PEO-PPO-PEO), poly(PLGA-co-serine), collagen-acrylate, alginate, alginate-acrylate, poly(HPMA-g-peptide), HA-g-NIPAAm and poly(vinyl methyl ether) (PVME) and / or derivatives of any one or more of the above.

12. The use according to claim 10, wherein the delivery vehicle is formulated for oral delivery and comprises a coating at least partially surrounding it, the coating comprising a cytochrome P450 3A4 (CYP3A4) inhibitor and a P-glycoprotein (P-gp) efflux pump inhibitor.

13. A method for reducing lipid concentration in target cells of a human or animal subject in vitro, the method comprising the steps of: transfecting the cells to produce 37 kDa / 67 kDa laminin receptor precursor / high affinity laminin receptor (LRP / LR) and / or fragments thereof; or providing LRP / LR and / or fragments thereof to the cell; in, The protein sequence of the LRP / LR is shown in SEQ ID NO: 1 or SEQ ID NO: 2, and the protein sequence of the fragment thereof is shown in SEQ ID NO: 4 or SEQ ID NO:

5.

14. Use of a biopharmaceutical in the preparation of a medicament for treating and / or preventing atherosclerosis, the biopharmaceutical comprising 37 kDa / 67 kDa laminin receptor precursor / high affinity laminin receptor (LRP / LR) and / or fragments thereof, wherein LRP / LR and / or the fragments thereof are used for administration to target cells of a subject in need thereof, and wherein the target cells are at least one of the following groups: vascular endothelial cells and vascular smooth muscle cells, and wherein the biopharmaceutical reduces the lipid content in the target cells when used, and wherein the protein sequence of the LRP / LR is shown in SEQ ID NO: 1 or SEQ ID NO: 2, and the protein sequence of the fragments thereof is shown in SEQ ID NO: 4 or SEQ ID NO:

5.

15. Use of a biopharmaceutical in the preparation of a medicament for treating and / or preventing insulin resistance and / or diabetes, the biopharmaceutical comprising 37 kDa / 67 kDa laminin receptor precursor / high affinity laminin receptor (LRP / LR) and / or fragments thereof, wherein LRP / LR and / or the fragments thereof are used for administration to target cells of a subject in need thereof, and wherein the target cells are at least one of the following groups: pancreatic cells comprising α cells, β cells, δ cells and γ cells, and when used, the biopharmaceutical reduces the lipid content in the target cells, and wherein the protein sequence of the LRP / LR is shown in SEQ ID NO: 1 or SEQ ID NO: 2, and the protein sequence of the fragments thereof is shown in SEQ ID NO: 4 or SEQ ID NO: 5.

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