US20190263888A1 - Therapeutic peptides and methods for treating autoimmune related disease - Google Patents
Therapeutic peptides and methods for treating autoimmune related disease Download PDFInfo
- Publication number
- US20190263888A1 US20190263888A1 US16/240,630 US201916240630A US2019263888A1 US 20190263888 A1 US20190263888 A1 US 20190263888A1 US 201916240630 A US201916240630 A US 201916240630A US 2019263888 A1 US2019263888 A1 US 2019263888A1
- Authority
- US
- United States
- Prior art keywords
- canceled
- peptide
- seq
- cells
- protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 509
- 238000000034 method Methods 0.000 title claims abstract description 163
- 102000004196 processed proteins & peptides Human genes 0.000 title abstract description 108
- 230000001363 autoimmune Effects 0.000 title abstract description 24
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 title description 38
- 201000010099 disease Diseases 0.000 title description 34
- 230000001225 therapeutic effect Effects 0.000 title description 3
- 101150013553 CD40 gene Proteins 0.000 claims abstract description 321
- 102100040245 Tumor necrosis factor receptor superfamily member 5 Human genes 0.000 claims abstract description 321
- 210000004027 cell Anatomy 0.000 claims abstract description 303
- 102100032937 CD40 ligand Human genes 0.000 claims abstract description 164
- 208000001072 type 2 diabetes mellitus Diseases 0.000 claims abstract description 124
- 102000018711 Facilitative Glucose Transport Proteins Human genes 0.000 claims abstract description 7
- 108010027279 Facilitative Glucose Transport Proteins Proteins 0.000 claims abstract description 7
- 230000003993 interaction Effects 0.000 claims description 130
- 230000027455 binding Effects 0.000 claims description 73
- 102000004127 Cytokines Human genes 0.000 claims description 17
- 108090000695 Cytokines Proteins 0.000 claims description 17
- 230000011664 signaling Effects 0.000 claims description 14
- 102000013691 Interleukin-17 Human genes 0.000 claims description 12
- 108050003558 Interleukin-17 Proteins 0.000 claims description 12
- 108010029697 CD40 Ligand Proteins 0.000 claims description 11
- 108010002350 Interleukin-2 Proteins 0.000 claims description 9
- 102000000588 Interleukin-2 Human genes 0.000 claims description 9
- 230000002401 inhibitory effect Effects 0.000 claims description 8
- 102000058061 Glucose Transporter Type 4 Human genes 0.000 claims description 2
- 108091006300 SLC2A4 Proteins 0.000 claims description 2
- 210000000612 antigen-presenting cell Anatomy 0.000 claims description 2
- 230000003393 splenic effect Effects 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 3
- 210000001185 bone marrow Anatomy 0.000 claims 1
- 206010061218 Inflammation Diseases 0.000 abstract description 90
- 230000004054 inflammatory process Effects 0.000 abstract description 90
- 201000001320 Atherosclerosis Diseases 0.000 abstract description 86
- 210000001744 T-lymphocyte Anatomy 0.000 abstract description 69
- 230000002452 interceptive effect Effects 0.000 abstract description 11
- 230000028709 inflammatory response Effects 0.000 abstract description 8
- 239000000463 material Substances 0.000 abstract description 7
- 238000008214 LDL Cholesterol Methods 0.000 abstract description 5
- 208000011594 Autoinflammatory disease Diseases 0.000 abstract description 2
- 238000011161 development Methods 0.000 description 114
- 230000018109 developmental process Effects 0.000 description 114
- 150000001413 amino acids Chemical class 0.000 description 85
- 241000699670 Mus sp. Species 0.000 description 81
- 238000003556 assay Methods 0.000 description 68
- 206010012601 diabetes mellitus Diseases 0.000 description 64
- 230000002757 inflammatory effect Effects 0.000 description 48
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 44
- 208000023275 Autoimmune disease Diseases 0.000 description 41
- 241001465754 Metazoa Species 0.000 description 36
- 108090000623 proteins and genes Proteins 0.000 description 33
- 102000004169 proteins and genes Human genes 0.000 description 32
- 239000003550 marker Substances 0.000 description 31
- 230000000694 effects Effects 0.000 description 29
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 27
- 238000011282 treatment Methods 0.000 description 27
- 230000001965 increasing effect Effects 0.000 description 25
- 208000024172 Cardiovascular disease Diseases 0.000 description 24
- 102100036011 T-cell surface glycoprotein CD4 Human genes 0.000 description 24
- 230000002441 reversible effect Effects 0.000 description 23
- 102000004877 Insulin Human genes 0.000 description 22
- 108090001061 Insulin Proteins 0.000 description 22
- 229940125396 insulin Drugs 0.000 description 22
- 230000004957 immunoregulator effect Effects 0.000 description 20
- 230000004913 activation Effects 0.000 description 19
- 210000004369 blood Anatomy 0.000 description 19
- 239000008280 blood Substances 0.000 description 19
- 238000012360 testing method Methods 0.000 description 19
- 241000699666 Mus <mouse, genus> Species 0.000 description 18
- 230000001419 dependent effect Effects 0.000 description 18
- 238000001514 detection method Methods 0.000 description 18
- 210000002865 immune cell Anatomy 0.000 description 18
- 230000009467 reduction Effects 0.000 description 18
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 17
- 239000008103 glucose Substances 0.000 description 17
- 102100029470 Apolipoprotein E Human genes 0.000 description 16
- 101710095339 Apolipoprotein E Proteins 0.000 description 16
- 102000017420 CD3 protein, epsilon/gamma/delta subunit Human genes 0.000 description 15
- 206010022489 Insulin Resistance Diseases 0.000 description 15
- 210000002376 aorta thoracic Anatomy 0.000 description 15
- 210000000056 organ Anatomy 0.000 description 15
- 230000002265 prevention Effects 0.000 description 15
- 238000004458 analytical method Methods 0.000 description 14
- 201000001421 hyperglycemia Diseases 0.000 description 14
- 230000008569 process Effects 0.000 description 14
- 238000005406 washing Methods 0.000 description 14
- 238000001990 intravenous administration Methods 0.000 description 13
- 210000002540 macrophage Anatomy 0.000 description 13
- 238000010172 mouse model Methods 0.000 description 13
- 208000037976 chronic inflammation Diseases 0.000 description 12
- 238000002347 injection Methods 0.000 description 12
- 239000007924 injection Substances 0.000 description 12
- 230000003902 lesion Effects 0.000 description 12
- 210000004698 lymphocyte Anatomy 0.000 description 12
- 210000000496 pancreas Anatomy 0.000 description 12
- 206010039073 rheumatoid arthritis Diseases 0.000 description 12
- 230000003143 atherosclerotic effect Effects 0.000 description 11
- 210000003719 b-lymphocyte Anatomy 0.000 description 11
- 230000008859 change Effects 0.000 description 11
- 230000006020 chronic inflammation Effects 0.000 description 11
- 230000007423 decrease Effects 0.000 description 11
- 201000006417 multiple sclerosis Diseases 0.000 description 11
- 230000002829 reductive effect Effects 0.000 description 11
- 108010074328 Interferon-gamma Proteins 0.000 description 10
- 238000000684 flow cytometry Methods 0.000 description 10
- 239000008194 pharmaceutical composition Substances 0.000 description 10
- 238000010186 staining Methods 0.000 description 10
- 210000001519 tissue Anatomy 0.000 description 10
- 208000038016 acute inflammation Diseases 0.000 description 9
- 230000006022 acute inflammation Effects 0.000 description 9
- 230000000903 blocking effect Effects 0.000 description 9
- 230000006870 function Effects 0.000 description 9
- 210000000440 neutrophil Anatomy 0.000 description 9
- 208000026872 Addison Disease Diseases 0.000 description 8
- 208000022559 Inflammatory bowel disease Diseases 0.000 description 8
- 108090001005 Interleukin-6 Proteins 0.000 description 8
- 102000004889 Interleukin-6 Human genes 0.000 description 8
- 210000000227 basophil cell of anterior lobe of hypophysis Anatomy 0.000 description 8
- 238000004113 cell culture Methods 0.000 description 8
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 8
- 208000025302 chronic primary adrenal insufficiency Diseases 0.000 description 8
- 230000034994 death Effects 0.000 description 8
- 231100000517 death Toxicity 0.000 description 8
- 230000003247 decreasing effect Effects 0.000 description 8
- 230000002950 deficient Effects 0.000 description 8
- 235000005911 diet Nutrition 0.000 description 8
- 230000037213 diet Effects 0.000 description 8
- 210000000987 immune system Anatomy 0.000 description 8
- 229960003130 interferon gamma Drugs 0.000 description 8
- 229940100601 interleukin-6 Drugs 0.000 description 8
- 230000001404 mediated effect Effects 0.000 description 8
- 241000282412 Homo Species 0.000 description 7
- 206010020772 Hypertension Diseases 0.000 description 7
- 102000008070 Interferon-gamma Human genes 0.000 description 7
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 7
- 230000005784 autoimmunity Effects 0.000 description 7
- 239000003795 chemical substances by application Substances 0.000 description 7
- 150000001875 compounds Chemical class 0.000 description 7
- 230000005764 inhibitory process Effects 0.000 description 7
- 238000005259 measurement Methods 0.000 description 7
- 230000004048 modification Effects 0.000 description 7
- 238000012986 modification Methods 0.000 description 7
- 210000001616 monocyte Anatomy 0.000 description 7
- 230000035755 proliferation Effects 0.000 description 7
- 210000000952 spleen Anatomy 0.000 description 7
- 208000024891 symptom Diseases 0.000 description 7
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 6
- 206010001052 Acute respiratory distress syndrome Diseases 0.000 description 6
- 208000008439 Biliary Liver Cirrhosis Diseases 0.000 description 6
- 208000033222 Biliary cirrhosis primary Diseases 0.000 description 6
- 208000006545 Chronic Obstructive Pulmonary Disease Diseases 0.000 description 6
- 208000011231 Crohn disease Diseases 0.000 description 6
- 238000002965 ELISA Methods 0.000 description 6
- 102000003814 Interleukin-10 Human genes 0.000 description 6
- 108090000174 Interleukin-10 Proteins 0.000 description 6
- 102100030704 Interleukin-21 Human genes 0.000 description 6
- 102100030703 Interleukin-22 Human genes 0.000 description 6
- 102000004388 Interleukin-4 Human genes 0.000 description 6
- 108090000978 Interleukin-4 Proteins 0.000 description 6
- 108060001084 Luciferase Proteins 0.000 description 6
- 239000005089 Luciferase Substances 0.000 description 6
- 208000010191 Osteitis Deformans Diseases 0.000 description 6
- 208000027868 Paget disease Diseases 0.000 description 6
- 208000012654 Primary biliary cholangitis Diseases 0.000 description 6
- 108091008874 T cell receptors Proteins 0.000 description 6
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 6
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 6
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 6
- 206010047115 Vasculitis Diseases 0.000 description 6
- 206010069351 acute lung injury Diseases 0.000 description 6
- 210000000709 aorta Anatomy 0.000 description 6
- 208000006673 asthma Diseases 0.000 description 6
- 210000003651 basophil Anatomy 0.000 description 6
- 210000001772 blood platelet Anatomy 0.000 description 6
- 230000001684 chronic effect Effects 0.000 description 6
- 210000002889 endothelial cell Anatomy 0.000 description 6
- 210000003979 eosinophil Anatomy 0.000 description 6
- 239000000499 gel Substances 0.000 description 6
- 210000003630 histaminocyte Anatomy 0.000 description 6
- 210000004969 inflammatory cell Anatomy 0.000 description 6
- 229940076144 interleukin-10 Drugs 0.000 description 6
- 108010074108 interleukin-21 Proteins 0.000 description 6
- 108010074109 interleukin-22 Proteins 0.000 description 6
- 229940028885 interleukin-4 Drugs 0.000 description 6
- 238000007918 intramuscular administration Methods 0.000 description 6
- 230000000670 limiting effect Effects 0.000 description 6
- 208000027202 mammary Paget disease Diseases 0.000 description 6
- 244000005700 microbiome Species 0.000 description 6
- 210000003205 muscle Anatomy 0.000 description 6
- 210000005259 peripheral blood Anatomy 0.000 description 6
- 239000011886 peripheral blood Substances 0.000 description 6
- 238000007920 subcutaneous administration Methods 0.000 description 6
- 206010043778 thyroiditis Diseases 0.000 description 6
- 238000001262 western blot Methods 0.000 description 6
- -1 HIS-tags Substances 0.000 description 5
- 208000008589 Obesity Diseases 0.000 description 5
- 108010058846 Ovalbumin Proteins 0.000 description 5
- 208000013616 Respiratory Distress Syndrome Diseases 0.000 description 5
- 230000006044 T cell activation Effects 0.000 description 5
- 208000007536 Thrombosis Diseases 0.000 description 5
- 201000000028 adult respiratory distress syndrome Diseases 0.000 description 5
- 230000009286 beneficial effect Effects 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 208000029078 coronary artery disease Diseases 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- 235000009200 high fat diet Nutrition 0.000 description 5
- 230000028993 immune response Effects 0.000 description 5
- 230000008595 infiltration Effects 0.000 description 5
- 238000001764 infiltration Methods 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 239000013642 negative control Substances 0.000 description 5
- 230000010807 negative regulation of binding Effects 0.000 description 5
- 235000020824 obesity Nutrition 0.000 description 5
- 229940092253 ovalbumin Drugs 0.000 description 5
- 150000003384 small molecules Chemical class 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 230000003442 weekly effect Effects 0.000 description 5
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 4
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 4
- 238000011740 C57BL/6 mouse Methods 0.000 description 4
- 206010018429 Glucose tolerance impaired Diseases 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- 208000015023 Graves' disease Diseases 0.000 description 4
- 208000030836 Hashimoto thyroiditis Diseases 0.000 description 4
- 241000976416 Isatis tinctoria subsp. canescens Species 0.000 description 4
- 241000124008 Mammalia Species 0.000 description 4
- 229930040373 Paraformaldehyde Natural products 0.000 description 4
- 102100034922 T-cell surface glycoprotein CD8 alpha chain Human genes 0.000 description 4
- 239000000427 antigen Substances 0.000 description 4
- 108091007433 antigens Proteins 0.000 description 4
- 102000036639 antigens Human genes 0.000 description 4
- 238000013459 approach Methods 0.000 description 4
- 238000012754 cardiac puncture Methods 0.000 description 4
- 230000001413 cellular effect Effects 0.000 description 4
- 230000002860 competitive effect Effects 0.000 description 4
- 230000001276 controlling effect Effects 0.000 description 4
- 230000002596 correlated effect Effects 0.000 description 4
- 230000000139 costimulatory effect Effects 0.000 description 4
- 238000002716 delivery method Methods 0.000 description 4
- 230000036541 health Effects 0.000 description 4
- 239000007943 implant Substances 0.000 description 4
- 208000015181 infectious disease Diseases 0.000 description 4
- 208000014674 injury Diseases 0.000 description 4
- 206010025135 lupus erythematosus Diseases 0.000 description 4
- 208000030159 metabolic disease Diseases 0.000 description 4
- 230000002503 metabolic effect Effects 0.000 description 4
- 208000010125 myocardial infarction Diseases 0.000 description 4
- 229920002866 paraformaldehyde Polymers 0.000 description 4
- 230000002093 peripheral effect Effects 0.000 description 4
- 230000007505 plaque formation Effects 0.000 description 4
- 108020003175 receptors Proteins 0.000 description 4
- 102000005962 receptors Human genes 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 230000004044 response Effects 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 238000006467 substitution reaction Methods 0.000 description 4
- 230000009885 systemic effect Effects 0.000 description 4
- 201000000596 systemic lupus erythematosus Diseases 0.000 description 4
- 238000002560 therapeutic procedure Methods 0.000 description 4
- 230000008733 trauma Effects 0.000 description 4
- 102000003390 tumor necrosis factor Human genes 0.000 description 4
- 210000003462 vein Anatomy 0.000 description 4
- IYMAXBFPHPZYIK-BQBZGAKWSA-N Arg-Gly-Asp Chemical compound NC(N)=NCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O IYMAXBFPHPZYIK-BQBZGAKWSA-N 0.000 description 3
- 101100015199 Caenorhabditis elegans gly-11 gene Proteins 0.000 description 3
- 101100505076 Caenorhabditis elegans gly-2 gene Proteins 0.000 description 3
- 101100067721 Caenorhabditis elegans gly-3 gene Proteins 0.000 description 3
- 101100228196 Caenorhabditis elegans gly-4 gene Proteins 0.000 description 3
- 101100228200 Caenorhabditis elegans gly-5 gene Proteins 0.000 description 3
- 101100228206 Caenorhabditis elegans gly-6 gene Proteins 0.000 description 3
- 101100228210 Caenorhabditis elegans gly-7 gene Proteins 0.000 description 3
- 101100335897 Caenorhabditis elegans gly-9 gene Proteins 0.000 description 3
- 101100505161 Caenorhabditis elegans mel-32 gene Proteins 0.000 description 3
- 102000008186 Collagen Human genes 0.000 description 3
- 108010035532 Collagen Proteins 0.000 description 3
- 101001057504 Homo sapiens Interferon-stimulated gene 20 kDa protein Proteins 0.000 description 3
- 101001055144 Homo sapiens Interleukin-2 receptor subunit alpha Proteins 0.000 description 3
- 108010034143 Inflammasomes Proteins 0.000 description 3
- 102100037850 Interferon gamma Human genes 0.000 description 3
- 102100026878 Interleukin-2 receptor subunit alpha Human genes 0.000 description 3
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 3
- 102000001183 RAG-1 Human genes 0.000 description 3
- 108060006897 RAG1 Proteins 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 208000006011 Stroke Diseases 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 3
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 3
- 239000000090 biomarker Substances 0.000 description 3
- 229960002685 biotin Drugs 0.000 description 3
- 235000020958 biotin Nutrition 0.000 description 3
- 239000011616 biotin Substances 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 229920001436 collagen Polymers 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 208000035475 disorder Diseases 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 230000002526 effect on cardiovascular system Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000007613 environmental effect Effects 0.000 description 3
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 101150089730 gly-10 gene Proteins 0.000 description 3
- 239000008187 granular material Substances 0.000 description 3
- 238000005469 granulation Methods 0.000 description 3
- 230000003179 granulation Effects 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 210000004185 liver Anatomy 0.000 description 3
- 229960000485 methotrexate Drugs 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 201000009104 prediabetes syndrome Diseases 0.000 description 3
- 210000000329 smooth muscle myocyte Anatomy 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 230000003827 upregulation Effects 0.000 description 3
- 239000003981 vehicle Substances 0.000 description 3
- 108010088751 Albumins Proteins 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 2
- 108010032595 Antibody Binding Sites Proteins 0.000 description 2
- 206010003178 Arterial thrombosis Diseases 0.000 description 2
- 206010003211 Arteriosclerosis coronary artery Diseases 0.000 description 2
- 210000002237 B-cell of pancreatic islet Anatomy 0.000 description 2
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 description 2
- 208000023328 Basedow disease Diseases 0.000 description 2
- 108010074051 C-Reactive Protein Proteins 0.000 description 2
- 210000004366 CD4-positive T-lymphocyte Anatomy 0.000 description 2
- 101100335894 Caenorhabditis elegans gly-8 gene Proteins 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- 206010008909 Chronic Hepatitis Diseases 0.000 description 2
- 206010053567 Coagulopathies Diseases 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 2
- 102100032768 Complement receptor type 2 Human genes 0.000 description 2
- 206010061818 Disease progression Diseases 0.000 description 2
- 102100025137 Early activation antigen CD69 Human genes 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 208000002705 Glucose Intolerance Diseases 0.000 description 2
- 102000005720 Glutathione transferase Human genes 0.000 description 2
- 108010070675 Glutathione transferase Proteins 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 208000003807 Graves Disease Diseases 0.000 description 2
- 208000001204 Hashimoto Disease Diseases 0.000 description 2
- 206010019755 Hepatitis chronic active Diseases 0.000 description 2
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 2
- 101000941929 Homo sapiens Complement receptor type 2 Proteins 0.000 description 2
- 101000934374 Homo sapiens Early activation antigen CD69 Proteins 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- 102000003777 Interleukin-1 beta Human genes 0.000 description 2
- 108090000193 Interleukin-1 beta Proteins 0.000 description 2
- 102000003810 Interleukin-18 Human genes 0.000 description 2
- 108090000171 Interleukin-18 Proteins 0.000 description 2
- 241001529936 Murinae Species 0.000 description 2
- 208000031845 Pernicious anaemia Diseases 0.000 description 2
- 239000004793 Polystyrene Substances 0.000 description 2
- 208000001280 Prediabetic State Diseases 0.000 description 2
- 206010040047 Sepsis Diseases 0.000 description 2
- 210000000662 T-lymphocyte subset Anatomy 0.000 description 2
- 102000002933 Thioredoxin Human genes 0.000 description 2
- 108060008683 Tumor Necrosis Factor Receptor Proteins 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 210000004100 adrenal gland Anatomy 0.000 description 2
- 238000007818 agglutination assay Methods 0.000 description 2
- 239000005557 antagonist Substances 0.000 description 2
- 108010072041 arginyl-glycyl-aspartic acid Proteins 0.000 description 2
- 210000001367 artery Anatomy 0.000 description 2
- 230000007214 atherothrombosis Effects 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 210000004204 blood vessel Anatomy 0.000 description 2
- 210000002168 brachiocephalic trunk Anatomy 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 229960001838 canakinumab Drugs 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 230000005754 cellular signaling Effects 0.000 description 2
- 238000007385 chemical modification Methods 0.000 description 2
- 235000012000 cholesterol Nutrition 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 230000012085 chronic inflammatory response Effects 0.000 description 2
- 230000035602 clotting Effects 0.000 description 2
- 230000009918 complex formation Effects 0.000 description 2
- 208000026758 coronary atherosclerosis Diseases 0.000 description 2
- 238000007822 cytometric assay Methods 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 230000007850 degeneration Effects 0.000 description 2
- 210000004443 dendritic cell Anatomy 0.000 description 2
- 230000001627 detrimental effect Effects 0.000 description 2
- 208000016097 disease of metabolism Diseases 0.000 description 2
- 230000005750 disease progression Effects 0.000 description 2
- 239000006185 dispersion Substances 0.000 description 2
- 238000010494 dissociation reaction Methods 0.000 description 2
- 230000005593 dissociations Effects 0.000 description 2
- 230000003828 downregulation Effects 0.000 description 2
- 239000003974 emollient agent Substances 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 230000001747 exhibiting effect Effects 0.000 description 2
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 238000003304 gavage Methods 0.000 description 2
- 238000007429 general method Methods 0.000 description 2
- 230000013595 glycosylation Effects 0.000 description 2
- 238000006206 glycosylation reaction Methods 0.000 description 2
- 210000003714 granulocyte Anatomy 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 229920001477 hydrophilic polymer Polymers 0.000 description 2
- 230000036737 immune function Effects 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 238000003119 immunoblot Methods 0.000 description 2
- 238000001114 immunoprecipitation Methods 0.000 description 2
- 239000003547 immunosorbent Substances 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000000099 in vitro assay Methods 0.000 description 2
- 238000005462 in vivo assay Methods 0.000 description 2
- 238000011065 in-situ storage Methods 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 208000027866 inflammatory disease Diseases 0.000 description 2
- 238000007912 intraperitoneal administration Methods 0.000 description 2
- 238000007913 intrathecal administration Methods 0.000 description 2
- 239000002085 irritant Substances 0.000 description 2
- 231100000021 irritant Toxicity 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 239000004816 latex Substances 0.000 description 2
- 229920000126 latex Polymers 0.000 description 2
- 230000006372 lipid accumulation Effects 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 210000001165 lymph node Anatomy 0.000 description 2
- 239000006249 magnetic particle Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000011859 microparticle Substances 0.000 description 2
- 239000004005 microsphere Substances 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 206010028417 myasthenia gravis Diseases 0.000 description 2
- 239000002105 nanoparticle Substances 0.000 description 2
- 239000002077 nanosphere Substances 0.000 description 2
- 210000000653 nervous system Anatomy 0.000 description 2
- 229940021182 non-steroidal anti-inflammatory drug Drugs 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 230000006320 pegylation Effects 0.000 description 2
- 108010009779 peptide 32 Proteins 0.000 description 2
- 230000002085 persistent effect Effects 0.000 description 2
- 239000008177 pharmaceutical agent Substances 0.000 description 2
- 239000006187 pill Substances 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 229920003023 plastic Polymers 0.000 description 2
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 229920002223 polystyrene Polymers 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000000770 proinflammatory effect Effects 0.000 description 2
- 230000002685 pulmonary effect Effects 0.000 description 2
- 238000003127 radioimmunoassay Methods 0.000 description 2
- 238000012827 research and development Methods 0.000 description 2
- 230000000630 rising effect Effects 0.000 description 2
- 230000001953 sensory effect Effects 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 208000002491 severe combined immunodeficiency Diseases 0.000 description 2
- 210000003491 skin Anatomy 0.000 description 2
- 230000000391 smoking effect Effects 0.000 description 2
- 210000002460 smooth muscle Anatomy 0.000 description 2
- 238000000638 solvent extraction Methods 0.000 description 2
- 238000012289 standard assay Methods 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 239000006188 syrup Substances 0.000 description 2
- 235000020357 syrup Nutrition 0.000 description 2
- 239000003826 tablet Substances 0.000 description 2
- 239000010409 thin film Substances 0.000 description 2
- 108060008226 thioredoxin Proteins 0.000 description 2
- 229940094937 thioredoxin Drugs 0.000 description 2
- 210000001685 thyroid gland Anatomy 0.000 description 2
- 238000011200 topical administration Methods 0.000 description 2
- 238000002054 transplantation Methods 0.000 description 2
- 102000003298 tumor necrosis factor receptor Human genes 0.000 description 2
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- 102100029647 Apoptosis-associated speck-like protein containing a CARD Human genes 0.000 description 1
- 208000037260 Atherosclerotic Plaque Diseases 0.000 description 1
- 206010003694 Atrophy Diseases 0.000 description 1
- 208000030767 Autoimmune encephalitis Diseases 0.000 description 1
- 210000001266 CD8-positive T-lymphocyte Anatomy 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 102100035904 Caspase-1 Human genes 0.000 description 1
- 108090000426 Caspase-1 Proteins 0.000 description 1
- 102100038916 Caspase-5 Human genes 0.000 description 1
- 101710090333 Caspase-5 Proteins 0.000 description 1
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 description 1
- 101100346152 Drosophila melanogaster modSP gene Proteins 0.000 description 1
- 208000005189 Embolism Diseases 0.000 description 1
- 206010048554 Endothelial dysfunction Diseases 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 208000010201 Exanthema Diseases 0.000 description 1
- 208000009386 Experimental Arthritis Diseases 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 108010073385 Fibrin Proteins 0.000 description 1
- 102000009123 Fibrin Human genes 0.000 description 1
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- 206010018852 Haematoma Diseases 0.000 description 1
- 208000032843 Hemorrhage Diseases 0.000 description 1
- 101000728679 Homo sapiens Apoptosis-associated speck-like protein containing a CARD Proteins 0.000 description 1
- 208000013038 Hypocalcemia Diseases 0.000 description 1
- 208000032382 Ischaemic stroke Diseases 0.000 description 1
- 101150013552 LDLR gene Proteins 0.000 description 1
- 206010024264 Lethargy Diseases 0.000 description 1
- 108010006444 Leucine-Rich Repeat Proteins Proteins 0.000 description 1
- 208000005777 Lupus Nephritis Diseases 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 102000012064 NLR Proteins Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 208000005764 Peripheral Arterial Disease Diseases 0.000 description 1
- 208000030831 Peripheral arterial occlusive disease Diseases 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 210000001789 adipocyte Anatomy 0.000 description 1
- 210000000593 adipose tissue white Anatomy 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 208000007502 anemia Diseases 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000001064 anti-interferon Effects 0.000 description 1
- 239000003472 antidiabetic agent Substances 0.000 description 1
- 229940125708 antidiabetic agent Drugs 0.000 description 1
- 210000001765 aortic valve Anatomy 0.000 description 1
- 230000000923 atherogenic effect Effects 0.000 description 1
- 230000037444 atrophy Effects 0.000 description 1
- 201000004983 autoimmune atherosclerosis Diseases 0.000 description 1
- 229960002170 azathioprine Drugs 0.000 description 1
- LMEKQMALGUDUQG-UHFFFAOYSA-N azathioprine Chemical compound CN1C=NC([N+]([O-])=O)=C1SC1=NC=NC2=C1NC=N2 LMEKQMALGUDUQG-UHFFFAOYSA-N 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- KQNZDYYTLMIZCT-KQPMLPITSA-N brefeldin A Chemical compound O[C@@H]1\C=C\C(=O)O[C@@H](C)CCC\C=C\[C@@H]2C[C@H](O)C[C@H]21 KQNZDYYTLMIZCT-KQPMLPITSA-N 0.000 description 1
- JUMGSHROWPPKFX-UHFFFAOYSA-N brefeldin-A Natural products CC1CCCC=CC2(C)CC(O)CC2(C)C(O)C=CC(=O)O1 JUMGSHROWPPKFX-UHFFFAOYSA-N 0.000 description 1
- 230000002308 calcification Effects 0.000 description 1
- 230000008614 cellular interaction Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 208000037893 chronic inflammatory disorder Diseases 0.000 description 1
- 235000019504 cigarettes Nutrition 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 230000003750 conditioning effect Effects 0.000 description 1
- 238000004624 confocal microscopy Methods 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 229960004397 cyclophosphamide Drugs 0.000 description 1
- 230000016396 cytokine production Effects 0.000 description 1
- 230000006735 deficit Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000001904 diabetogenic effect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000011038 discontinuous diafiltration by volume reduction Methods 0.000 description 1
- 230000000225 effect on diabetes Effects 0.000 description 1
- 230000003073 embolic effect Effects 0.000 description 1
- 230000008694 endothelial dysfunction Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 201000005884 exanthem Diseases 0.000 description 1
- 206010016256 fatigue Diseases 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 229950003499 fibrin Drugs 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 230000004761 fibrosis Effects 0.000 description 1
- 210000000497 foam cell Anatomy 0.000 description 1
- 230000001434 glomerular Effects 0.000 description 1
- 230000004190 glucose uptake Effects 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 230000003345 hyperglycaemic effect Effects 0.000 description 1
- 230000000705 hypocalcaemia Effects 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 208000026278 immune system disease Diseases 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 229960003444 immunosuppressant agent Drugs 0.000 description 1
- 230000001861 immunosuppressant effect Effects 0.000 description 1
- 239000003018 immunosuppressive agent Substances 0.000 description 1
- 230000004968 inflammatory condition Effects 0.000 description 1
- 230000003914 insulin secretion Effects 0.000 description 1
- 102000006495 integrins Human genes 0.000 description 1
- 108010044426 integrins Proteins 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 210000004153 islets of langerhan Anatomy 0.000 description 1
- 210000004901 leucine-rich repeat Anatomy 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 150000002617 leukotrienes Chemical class 0.000 description 1
- 210000000191 macrophage derived foam cell Anatomy 0.000 description 1
- 238000007726 management method Methods 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 235000012054 meals Nutrition 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 210000000663 muscle cell Anatomy 0.000 description 1
- 208000031225 myocardial ischemia Diseases 0.000 description 1
- 210000000822 natural killer cell Anatomy 0.000 description 1
- 230000001338 necrotic effect Effects 0.000 description 1
- 230000008692 neointimal formation Effects 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 208000004235 neutropenia Diseases 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 230000005937 nuclear translocation Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 238000006384 oligomerization reaction Methods 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 229940094443 oxytocics prostaglandins Drugs 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 230000001991 pathophysiological effect Effects 0.000 description 1
- 230000007310 pathophysiology Effects 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 150000003180 prostaglandins Chemical class 0.000 description 1
- 201000007094 prostatitis Diseases 0.000 description 1
- 229940024999 proteolytic enzymes for treatment of wounds and ulcers Drugs 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 206010037844 rash Diseases 0.000 description 1
- 230000000306 recurrent effect Effects 0.000 description 1
- 210000003289 regulatory T cell Anatomy 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000009256 replacement therapy Methods 0.000 description 1
- 208000037803 restenosis Diseases 0.000 description 1
- 239000007320 rich medium Substances 0.000 description 1
- 238000012502 risk assessment Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 208000023516 stroke disease Diseases 0.000 description 1
- FIAFUQMPZJWCLV-UHFFFAOYSA-N suramin Chemical compound OS(=O)(=O)C1=CC(S(O)(=O)=O)=C2C(NC(=O)C3=CC=C(C(=C3)NC(=O)C=3C=C(NC(=O)NC=4C=C(C=CC=4)C(=O)NC=4C(=CC=C(C=4)C(=O)NC=4C5=C(C=C(C=C5C(=CC=4)S(O)(=O)=O)S(O)(=O)=O)S(O)(=O)=O)C)C=CC=3)C)=CC=C(S(O)(=O)=O)C2=C1 FIAFUQMPZJWCLV-UHFFFAOYSA-N 0.000 description 1
- 229960005314 suramin Drugs 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000011285 therapeutic regimen Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000011830 transgenic mouse model Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- 239000013638 trimer Substances 0.000 description 1
- 230000006441 vascular event Effects 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/191—Tumor necrosis factors [TNF], e.g. lymphotoxin [LT], i.e. TNF-beta
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/08—Peptides having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/10—Peptides having 12 to 20 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
- A61K38/1774—Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/4643—Vertebrate antigens
- A61K39/4644—Cancer antigens
- A61K39/464402—Receptors, cell surface antigens or cell surface determinants
- A61K39/464416—Receptors for cytokines
- A61K39/464417—Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR], CD30
Definitions
- the present disclosure relates to methods or uses for prevention, modulation, and reduction of cardiovascular disease and/or atherosclerosis in a subject in need of a therapeutically effective amount of a peptide that inhibits the interaction of CD40 and CD154, and the use of such compounds in modulating T-cell activity and in treating disease. Furthermore, the present disclosure relates to methods of preventing, modulating, reducing, treating and/or reversing of type 2 diabetes mellitus and/or auto-inflammatory disease, via administration of a therapeutically effective amount of a CD40-binding peptide that inhibits, influences, disrupts, blocks, and/or changes the interaction of CD40 and CD154 are disclosed.
- Autoimmune diseases are conditions arising from an abnormal immune response to a normal body part. More than 80 diseases occur because of the immune system attacking the body's own organs, tissues, and cells. Type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosus, and inflammatory bowel disease are common autoimmune diseases that affect a wide range of people across entire populations. Significantly, the autoimmune disorders mentioned above afflict substantial number of people affecting their daily lives and routines and require significant monetary and healthcare resources, time and care from healthcare providers.
- CVDs cardiovascular diseases
- AHRQ Healthcare Cost and Utilization Project
- Atherosclerosis is defined by arterial plaque formation that may lead to heart attack and stroke.
- Arterial plaque formation is caused by the deposition of cells, substances, waste products, and cellular debris including, but not limited to: cholesterol, dead cells, dendritic cells, foam cells, macrophages, mast cells, monocytes, smooth muscle cells, T-cells, collagen, calcium, and fibrin.
- Inflammatory changes within the arterial wall and plaque may play a crucial and causative role in atherosclerotic disease development. Consequently, the concept of atherosclerosis as an autoimmune and inflammatory disease has been investigated; however, a therapeutic control has not been established. The importance of controlling inflammation is highlighted by current clinical trials targeting other aspects of autoimmune, inflammation, and cardiovascular disease and death.
- CIRT Cardiovascular Inflammation Reduction Trial
- IL-6 interleukin-6
- Another example includes, CANTOS (Canakinumab Anti-inflammatory Thrombosis Outcomes Study) which is studying whether canakinumab can block the pro-inflammatory cytokine interleukin-1 ⁇ (IL-1 ⁇ ) to reduce rates of recurrent myocardial infarction, stroke, and cardiovascular death rates in heart attack patients who remain at a high risk.
- CANTOS Canakinumab Anti-inflammatory Thrombosis Outcomes Study
- hsCRP high sensitivity C-reactive protein
- Mammalian and human atherosclerotic lesions are characterized as a chronic inflammatory-fibroproliferative disease of the blood vessel wall containing monocytes, macrophages, endothelial cells, smooth muscle cells, platelets, and T-cells. Each of these cell types can express either or both of the CD40/CD154 costimulatory pair.
- This dyad is responsible for enhancing the immune response and may contribute to many chronic inflammatory diseases including rheumatoid arthritis, multiple sclerosis, and type 1 diabetes (T1D).
- T1D type 1 diabetes
- Inflammation may occur when inflammatory cells, such as neutrophils, eosinophils, basophils, mast cells, macrophages, platelets, and endothelial cells, respond to inflammatory events or harmful stimuli, such as, invading microorganisms, damages cells, or other irritants.
- the body's inflammatory response is beneficial because for example, in the case of invading microorganisms, the inflammatory response is an important step in localizing the infecting agent for removal by the immune system.
- ACI aseptic chronic inflammation
- the inflammation is detrimental since it destroys normal tissues.
- the results of this aseptic inflammation are life-altering and in some cases life-threatening.
- this process is mediated by immune cells, including T-cells.
- ACI is not limited to a single disease, but is instrumental in numerous autoimmune diseases, including, but not limited to: type 1 diabetes (T1D), multiple sclerosis (MS), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Crohn's disease, inflammatory bowel disease (IBS), chronic obstructive pulmonary disease (COPD) including types of autoimmune asthma, atherosclerosis, vasculitis, hypertension, thyroiditis including Hashimoto's and Graves diseases, primary biliary cirrhosis, Paget's disease, Addison's disease, acute respiratory distress syndrome (ARDS), acute lung injury, and aseptic chronic inflammation (ACI) associated with organ transplantation.
- T1D type 1 diabetes
- MS multiple sclerosis
- SLE systemic lupus erythematosus
- RA rheumatoid arthritis
- IBS inflammatory bowel disease
- COPD chronic obstructive pulmonary disease
- organ-specific autoimmune disorders are classified into two types: organ-specific (directed mainly at one organ) and non-organ-specific (widely spread throughout the body).
- organ-specific autoimmune disorders are insulin-dependent Type 1 diabetes (T1D) which affects the pancreas; Hashimoto's thyroiditis and Graves' disease, which affect the thyroid gland; pernicious anemia, which affects the blood; Addison's disease, which affects the adrenal glands; chronic active hepatitis, which affects the liver; myasthenia gravis which affects the muscle; and multiple sclerosis (MS), which affects tissue of the nervous system.
- T1D insulin-dependent Type 1 diabetes
- Hashimoto's thyroiditis and Graves' disease which affect the thyroid gland
- pernicious anemia which affects the blood
- Addison's disease which affects the adrenal glands
- chronic active hepatitis which affects the liver
- myasthenia gravis which affects the muscle
- MS multiple sclerosis
- Acute inflammation is also immune cell mediated. While a comprehensive, complete, and exhaustive list of the molecular mediators in this process have not yet been identified, a prominent role for T-cells, lymphocytes, neutrophils, macrophages, monocytes, neutrophils, eosinophils, basophils, mast cells, and other inflammatory cells is strongly implicated. Therefore, a process to modulate these cell types may control the inflammatory response.
- Type 2 diabetes T2D has historically and scientifically been primarily categorized as a metabolic disorder; however, type 2 diabetes is in the process of being redefined as an autoimmune disease rather than just a metabolic disorder (Winer, D, et al. Nature Medicine (2011) 17:610-617).
- type 2 diabetes most often develops in people over the age of 45, type 2 diabetes increased 21% in American youth from 2001 to 2009 and a large study called SEARCH for Diabetes in Youth found that newly diagnosed cases of Type 2 diabetes in children and teens increased by about 4.8 percent in each year of the study's period between 2002 and 2012 (“Rates of new diagnosed cases of type 1 and type 2 diabetes on the rise among children, teens” National Institutes of Health, Apr. 13, 2017, available at https://www.nih.gov/news-events/news-releases/rates-new-diagnosed-cases-type-1-type-2-diabetes-rise-among-children-teens).
- An ideal anti-diabetic agent may be an agent which corrects hyperglycemia, prevents macrovascular complications, and corrects the pathophysiological disturbances responsible for Type 2 Diabetes (“T2D”).
- T2D Type 2 Diabetes
- Insulin resistance is basic to T2D, but ⁇ -cell failure eventually occurs with imbalance between insulin resistance and insulin secretion being a further complication. Therefore, therapeutically beneficial treatment approaches may aim to reverse insulin resistance and improve ⁇ -cell function.
- T2D often conincides with obesity however genetic and environmental factors recently have been described as disease contributors (Comuzzie A G, Best Pract. Res. Clin. Endocrinol. Metab. (2002) 16(4):611-21. PubMed PMID: 12468410; van Tilberg J., et al. J. Med. Genet . (2001) 38(9):569-78. Pub Med PMID: 11546824; PMCID: PMC 1734947). Moreover, additional research has emerged that indicates that T2D, like T1D, has prominent inflammation component that is a contributing and/or driving factor of the T2D disease commencement, development and progression.
- the CD40-CD154 inflammatory dyad may act as a molecular driver to propel auto-immune inflammation and influence excessive levels of the dyad may be an unappreciated but contributing factor to T2D.
- inflammation may occur when inflammatory cells, such as neutrophils, eosinophils, basophils, mast cells, macrophages, platelets, endothelial cells, and lymphocytes, including but not limited to T cells and B cells respond to inflammatory events or harmful stimuli, such as, invading microorganisms, damaged cells, or other irritants.
- inflammatory cells such as neutrophils, eosinophils, basophils, mast cells, macrophages, platelets, endothelial cells, and lymphocytes, including but not limited to T cells and B cells respond to inflammatory events or harmful stimuli, such as, invading microorganisms, damaged cells, or other irritants.
- the body's inflammatory response is beneficial because, for example, in the case of invading microorganisms, the inflammatory response is an important step in localizing the infecting agent for removal by the immune system.
- autoimmunity there is no infection, yet severe inflammation is present or persistent.
- aseptic chronic inflammation is detrimental since it destroys normal tissues.
- ACI aseptic chronic inflammation
- the results of this aseptic inflammation are life-altering and in some cases life-threatening.
- this process is mediated by immune cells, including T-cells.
- ACI is not limited to a single disease, but is instrumental in numerous autoimmune diseases, including, but not limited to: type 1 diabetes (T1D), multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, Crohn's disease, inflammatory bowel disease, chronic obstructive pulmonary disease including types of autoimmune asthma, atherosclerosis, vasculitis, hypertension, thyroiditis including Hashimoto's and Graves diseases, primary biliary cirrhosis, Paget's disease, Addison's disease, acute respiratory distress syndrome, acute lung injury, and ACI associated with organ transplantation.
- T1D type 1 diabetes
- T1D type 1 diabetes
- multiple sclerosis systemic lupus erythematosus
- rheumatoid arthritis Crohn's disease
- inflammatory bowel disease chronic obstructive pulmonary disease including types of autoimmune asthma, atherosclerosis, vasculitis, hypertension, thyroiditis including Ha
- organ-specific autoimmune disorders are classified into two types: organ-specific (directed mainly at one organ) and non-organ-specific (widely spread throughout the body).
- organ-specific autoimmune disorders are insulin-dependent Type 1 diabetes (T1D) which affects the pancreas; Hashimoto's thyroiditis and Graves' disease, which affect the thyroid gland; pernicious anemia, which affects the blood; Addison's disease, which affects the adrenal glands; chronic active hepatitis, which affects the liver; myasthenia gravis which affects the receptors at the junction between nerves and muscles; and multiple sclerosis, which affects tissue of the nervous system.
- T1D insulin-dependent Type 1 diabetes
- Hashimoto's thyroiditis and Graves' disease which affect the thyroid gland
- pernicious anemia which affects the blood
- Addison's disease which affects the adrenal glands
- chronic active hepatitis which affects the liver
- myasthenia gravis which affects the receptors at
- Acute inflammation is also immune cell mediated. While a comprehensive, complete, and exhaustive list of the molecular mediators in this process have not yet been identified, a prominent role for T-cells, lymphocytes, neutrophils, macrophages, monocytes, neutrophils, eosinophils, basophils, mast cells, and other inflammatory cells is strongly implicated. Therefore, a process to modulate these cell types may control the inflammatory response.
- T cell subset has been shown to be instrumental in the development of autoimmune disease.
- These cells are phenotypically characterized as CD4loCD40+ (Waid, D. M., et al., Eur. J. of Immunol., 34:1488, 2004; Vaitaitis, G. M., et al., Cutting Edge, J. Immunol., 170:3455, 2003; Wagner, D. H., Jr., et al., Proc. Nat'l. Acad. Sci. USA, 99:3782, 2002; Wagner, D. H., Jr., et al., Int'l J. of Mol. Med. 4:231, 1999), and are referred to as Th40 cells. (Waid, D. M., et al., Eur. J. of Immunol., 34:1488, 2004; Vaitaitis, G. M., et al., Cutting Edge, J. Immunol., 170:3455, 2003; Wagner
- CD40 expression typically is associated with antigen presenting cells and the majority of prior art describes CD40 as being expressed on B cells, macrophages, monocytes, and other cells; however, CD40 proteins are also expressed on T cells (Waid, D.
- Th40 cells comprise a proportion of the peripheral CD4+ compartment in na ⁇ ve, non-autoimmune mice (Waid, D. M., et al., Eur. J. of Immunol., 34:1488, 2004; Wagner, D.
- T cells do not express early activation markers and occur in the na ⁇ ve phenotype of non-challenged mice.
- Th40 cells occur at exaggerated levels in spleen, lymph nodes and the pancreas, even prior to diabetes onset (Waid, D. M., et al., Eur. J. of Immunol. 34:1488, 2004; Wagner, D. H., Jr., et al., Proc. Nat'l Acad. Sci. USA 99:3782, 2002).
- An elevated number and percentage of these T cells are seen in peripheral blood of type 1 diabetic (T1D) patients when compared to non-autoimmune controls and type 2 diabetic patients (Waid. D. M., et al., Clin. Immunol., 124:138, 2007).
- Th40 cells were antigen responsive or that CD40 expression is activation induced.
- CD40+ several diabetogenic T cell clones are CD40+ (Wagner, D. H., Jr., et al., Proc. Nat'l Acad. Sci. USA 99:3782, 2002).
- Th40 cells infiltrate islet beta cells destroying insulin production thus suggesting islet antigen specificity (Waid, D. M., et al., Eur. J. of Immunol. 34:1488, 2004; Wagner, D. H., Jr., et al., Proc. Nat'l Acad. Sci. USA 99:3782, 2002). It has also been shown that Th40 cells are required for diabetes transfer.
- CD154 is temporally induced on activated T-cells in response to CD3/TCR stimulation (Lederman, S. et al., J. of Exp. Med., 175:1091, 1992). CD154 expression has also been demonstrated on platelets, monocytes, basophils, eosinophils, dendritic cells, fibroblasts, smooth muscle, and endothelial cells (Russo, S. et al., J. Immunol. 171:5489, 2003; Stumpf, C., et al., Eur. J.
- CD154 is a member of the tumor necrosis factor (TNF) super-family and a soluble form of CD154 (sCD154) has been described (Russo, S., et al., J. Immunol. 171:5489 2003; Stumpf, C., et al., Eur. J. Heart Fail 5:629, 2003; Toubi, E., et al., Autoimmunity 37:457, 2004). Therefore, sCD154 may act like a cytokine (Stumpf, C., et al., Eur. J. Heart Fail.
- CD154 has not been genetically linked in T1D studies, sCD154 is significantly elevated in T1D and may play a role in the disease process (Varo, N. et al., Circulation 107:2664, 2003; Cipollone, F., et al., Diabetologia 48:1216, 2005; Devaraj, S., et al., Diabetes 55:774, 2006).
- the importance of CD40-CD154 interaction in autoimmunity has been established (Wagner, D. H., Jr., et al., Proc. Nat'l Acad. Sci. USA 99:3782, 2002; Kobata, T., et al., Rev. Immunogenet.
- Blocking CD40-CD154 interaction may prevent collagen induced arthritis, (Durie, F. H., et al., Science 281:1328, 1993) experimental autoimmune encephalitis (Howard, L. M., et al., Autoimmunity 37:411, 2004), prostatitis (Grossman, M. E., et al., J. Immunother.
- Th40 cell subset induces RAG1 and RAG2 (Recombination-Activating Genes) transcription, translation and nuclear translocation (Vaitaitis, G. M., et al., Cutting Edge, J. Immunol. 170:3455, 2003) when CD40 is engaged (Vaitaitis, G. M. et al., Cutting Edge, J. Immunol. 170:3455, 2003).
- CD3 engagement does not induce RAG1 or RAG2 in T-cells (Vaitaitis, G. M., et al., Cutting Edge, J. Immunol. 170:3455, 2003).
- TCR T-cell receptor
- CD40 is a 50-kDa integral membrane protein of the tumor necrosis factor receptor (TNF-R) family. It is constitutively expressed as a homotrimer (Foy T M, et al., Ann. Rev. of Immunol., 14:591, 1996). In general, stimulation of all CD40-expressing cell types induces operations which contribute to inflammation, such as enhancement of costimulatory and adhesion molecules, and up-regulation of proteolytic enzymes (Mach, F. et al., Atherosclerosis. 137 Suppl:S89-95, 1998).
- TNF-R tumor necrosis factor receptor
- the main source of sCD154 is platelets.
- mice are utilized for research and development concerning atherosclerosis and cardiovascular disease because wild type mice are generally highly resistant to development and progression of atherosclerosis.
- Prior studies have attempted to block the CD40/CD154 interaction by using monoclonal antibodies and this approach has proven efficacious in several mouse model studies utilizing the Apoe ⁇ / ⁇ or LDLr deficient atherosclerotic models.
- these same mouse models built with a deletion of CD154 saw significant reductions in overall plaque formation and may have also contributed to production of a more stable plaque phenotype.
- stable plaques are identifiable and denoted by increased collagen and smooth muscle content, a thick fibrous cap, and an observable decrease in T cell, macrophage, and lipid accumulation.
- mice models are utilized for research and development concerning T2D because mouse models can portray insulin resistance and the inability of the beta cell to sufficiently compensate, which are characteristic of T2D in humans.
- Many animal models, including mouse models for T2D are obese, reflecting the human condition where obesity is closely linked to T2D development.
- NSAIDS non-steroidal anti-inflammatory drugs
- Other approaches use more powerful immunosuppressant drugs such as cyclophosphamide, methotrexate and azathioprine that suppress the immune response and stop the progression of the disease.
- Still other treatments involve the use of monoclonal antibodies (mAb) designed to alter the immune responses to self-tissues, as occurs during autoimmune diseases.
- mAb monoclonal antibodies
- type 2 diabetes in humans typically develops through a progressive series of increasingly disruptive phases or stages.
- pre-diabetes is characterized by impaired glucose tolerance, wherein the body has difficulty clearing glucose after a meal (postprandial hyperglycemia) and/or the body may have decreased sensitivity to insulin.
- postprandial hyperglycemia and basal hyperglycemia occur while insulin producing beta cells of the pancreas become dysfunctional at an increasing rate.
- hyperglycemia occurs even after fasting and at a cellular level significant beta cell atrophy takes place.
- beta cells can no longer produce and/or release insulin and the patient requires insulin replacement therapy.
- T2D is clinically characterized by hyperglycemia and pathologically by insulin resistance with relative insulin secretory impairment. Individuals who are genetically prone to developing T2D may experience insulin resistance (the earliest detectable metabolic defect) between 15 and 25 years or more before the clinical onset of overt diabetes. (Kahn, C R, Diabetes . (1994) 43:1066-1084). T2D classically has been associated with age and obesity; however, the increased diagnosis of youth with T2D has demonstrated that these two factors alone are not the sole reliable predictors of the disease. Body mass index (BMI) is also clearly associated with T2D, but both genetic and environmental factors are now identified as contributory as well (Wu Y, et al. Int. J. Med. Sci .
- BMI Body mass index
- CVDs cardiovascular diseases
- ACI aseptic chronic inflammation
- the present developments may provide novel methods for preventing, modulating, and/or reducing atherosclerosis that arises in a corporeal body.
- Atherosclerosis may arise as a result of chronic inflammatory response of white blood cells in the walls of arteries. It is postulated that the chronic inflammatory response and the subsequent buildups of plaque in arteries may be caused by elevated levels of cholesterol and triglycerides in the blood, high blood pressure, and cigarette smoking.
- the present developments are based on the knowledge that interaction of CD40-ligand (CD154 protein) with CD40 protein expressed on T-cells (Th40 cells), may be important in the development of atherosclerosis and autoimmune disease.
- the present developments may be based on the elucidation of the critical residues in CD40 and CD154 that may be important for this interaction.
- the present developments relate to blocking the interaction between a CD40 protein and a CD154 protein through the use of small peptides that interact with the CD40 protein at a site where the CD154 protein would normally bind.
- the present developments also relate to using such peptides to reduce the level of Th40 cells, thereby reducing the severity of disease.
- One embodiment of the present developments is a method for preventing atherosclerosis comprising contacting the CD40 protein with a peptide that interacts with the CD40 protein.
- Preferred peptides may be those that are less than 25 amino acids in length, and that bind to a CD40 protein, thereby inhibiting its interaction with a CD154 protein; however, the length of the peptide should not be considered a limitation on the developments herein as there are numerous other factors that may affect the ability of the peptide to perform its intended and desired result.
- One embodiment of the present developments is a method for preventing, modulating, and/or reducing atherosclerosis, the method comprising inhibiting interaction between a CD40 protein and a CD154 protein with a peptide that interacts with the CD40 protein.
- Preferred peptides interact with the CD40 protein at the CD154-binding site.
- Preferably such peptides are less than 25 amino acids in length.
- Even more preferred peptides are those amino acid sequences selected from SEQ ID NOs 3-9 and 25-30.
- One embodiment of the present developments is a method for preventing, modulating, and/or reducing atherosclerosis, the method comprising administering to a subject in need thereof, a therapeutically effective amount of a peptide that affects the interaction of CD40 with CD154/CD40-ligand.
- a peptide that affects the interaction of CD40 with CD154/CD40-ligand may be that the peptide binds to CD40.
- the peptide may bind to a CD40 protein with a Kd of greater than 10 ⁇ 6 .
- the peptide may affect the interaction between CD40 and CD154.
- a preferred embodiment may inhibit the binding of CD40 to CD154.
- the peptide binds to CD40 at the site where CD40 interacts with CD154.
- the peptide affects the interaction of CD40 with CD154 in such a manner as to prevent the expansion of Th40 cells. In this embodiment, the peptide affects the interaction of CD40 with CD154 in such a manner as to reduce the number of Th40 cells. In this embodiment, the peptide affects the interaction of CD40 with CD154 in such a manner as to alter the cytokine expression profile of a cell population, treated with said peptide.
- One embodiment of the present developments is a method to modulate and/or reduce atherosclerosis in an animal, the method comprising administering to the animal, a peptide that interacts with a CD40 protein in such a manner as to modulate IFN ⁇ (interferon gamma).
- Preferred peptides are those that interact with the CD40 protein at the CD154-binding site, thereby modulating IFN ⁇ .
- Preferred peptides modulate inflammation by reducing the level of Th40 cells to no more than 25% of the total T-cell population.
- Such methods can be used to prevent and/or reduce atherosclerosis and symptoms that may accompany cardiovascular diseases, more generally.
- One embodiment of the present developments is a method to identify a patient at risk for developing cardiovascular disease and/or atherosclerosis, the method comprising obtaining a sample containing T-cells from a patient to be tested, contacting the sample with a peptide that binds the CD40 protein, detecting the CD-40 bound peptide, and determining the level of Th40 cells from the amount of CD40 bound, wherein a level of Th40 cells greater than 25% of the total T-cell population indicates the patient is at risk for developing cardiovascular disease and/or developing atherosclerosis.
- Another embodiment of the present developments is a method to prevent, modulate, or reduce calcium buildup, or calcification of vessel walls, the method comprising administering to the subject in need thereof, a therapeutically effective amount of a peptide which specifically binds to a CD40 presenting cells at the CD154 binding site.
- Another embodiment of the present developments is a method to administer a CD40-binding peptide to prevent, modulate, and/or reduce atherosclerosis, comprising selecting a peptide that interacts with a CD40 protein and CD154 binding site, selecting a delivery method selected from the group comprising intramuscular (IM) delivery, intravenous (IV) delivery, subcutaneous (SC) delivery, oral delivery, gavage delivery, emollient/skin delivery, or transdermal patch.
- IM intramuscular
- IV intravenous
- SC subcutaneous
- oral delivery gavage delivery
- emollient/skin delivery or transdermal patch.
- Another embodiment of the present developments is a method to administer a CD40-binding peptide to prevent, modulate, and/or reduce atherosclerosis in an animal, comprising selecting a peptide that interacts with a CD40 protein and CD154 binding site, using an extended delivery method selected from the group comprising an implantable device, a hydrophilic polymer formulation, a permeable polymeric membrane, injectable gel implants, solvent extraction system, phase inversion system, thermosensitive gels, pH dependent in situ gels, microparticles, microspheres, nanoparticles, nanospheres, bio-degradable implants, or photoactivated depot.
- Another embodiment of the present developments is a method to lower LDL cholesterol in a subject, the method comprising administering to the subject in need thereof, a therapeutically effective amount of a peptide which specifically binds to a CD40 presenting cells at the CD154 binding site.
- the present developments may provide novel methods for preventing, modulating, reducing, treating and/or reversing T2D that arises in a corporeal body.
- the developments disclosed herein are therapeutic methods which may additionally be used for the prevention, control, and treatment of diseases, disorders, and conditions, in particular immune and inflammatory diseases.
- the present developments are based on the knowledge that interaction of CD40-ligand (CD154 protein) with CD40 protein expressed on T-cells (Th40 cells), may be important in the development of type 2 diabetes and autoimmune diseases.
- the present developments may be based on the elucidation of the critical residues in CD40 and CD154 that may be important for this interaction.
- the present developments relate to blocking and/or disrupting the interaction between a CD40 protein and a CD154 protein through the use of small synthesized peptides that interact and/or associate with the CD40 protein at a site where the CD154 protein would normally bind.
- the present developments also relate to using such peptides to reduce the level of Th40 cells, thereby reducing the severity of disease.
- the peptides of the current developments may bind directly and/or alternatively associate with the CD40 molecule in such a way as to alter CD40 function.
- the peptide may alter CD40 signals to no longer be inflammatory.
- the peptides of the current development may block, disrupt, interfere, and/or inhibit CD40 function at sites including but not limited to Th40 cells, pancreas beta cells, endothelial cells, B cells, monocytes, and/or macrophages.
- SIPs small interfering peptides
- One embodiment of the present developments is a method for preventing, modulating, reducing, and/or reversing type 2 diabetes comprising contacting the CD40 protein with a peptide that interacts with the CD40 protein.
- Preferred peptides are those that are less than 25 amino acids in length, and that bind to a CD40 protein, thereby inhibiting its interaction with a CD154 protein.
- One embodiment of the present developments is a method for preventing, modulating, reducing and/or reversing type 2 diabetes, the method comprising inhibiting interaction between a CD40 protein and a CD154 protein with a peptide that interacts with the CD40 protein.
- Preferred peptides interact with the CD40 protein at the CD154-binding site.
- Preferably such peptides are less than 25 amino acids in length.
- Even more preferred peptides are those amino acid sequences selected from SEQ ID NOs 3-9 and 25-30.
- the present development provides a method for modulating and/or increasing glucose transport protein 4 (GLUT4) the method comprising administering to a patient a therapeutically sufficient dose of a peptide selected from SEQ ID NOs 3-9 and 25-30.
- GLUT4 glucose transport protein 4
- One embodiment of the present developments is a method to prevent, modulate, reduce and/or reverse type 2 diabetes in an animal, the method comprising administering to the animal, a peptide that interacts with a CD40 protein in such a manner as to modulate glucose transport protein 4 (“GLUT4”).
- Preferred peptides are those that interact with the CD40 protein at the CD154-binding site, thereby modulating GLUT4.
- Preferred peptides may modulate, upregulate, or increase GLUT4 in both adipose and muscle tissue compared to untreated populations.
- Such methods can be used to prevent and/or reduce T2D and symptoms that may accompany T2D and autoimmune related inflammation, more generally.
- Another embodiment of the present developments is a method to administer a CD40-binding peptide to prevent, modulate, reduce and/or reverse type 2 diabetes, comprising selecting a peptide that interacts with a CD40 protein and CD154 binding site, selecting a delivery method selected from the group comprising intramuscular (IM) delivery, intravenous (IV) delivery, subcutaneous (SC) delivery, oral delivery, gavage delivery, emollient/skin delivery, transdermal patch, or nasal administration.
- IM intramuscular
- IV intravenous
- SC subcutaneous
- oral delivery gavage delivery
- emollient/skin delivery emollient/skin delivery
- transdermal patch emollient/skin delivery
- Another embodiment of the present developments is a method to administer a CD40-binding peptide to prevent, modulate, reduce and/or reverse type 2 diabetes in an animal, comprising selecting a peptide that interacts with a CD40 protein and CD154 binding site, using an extended delivery method selected from the group comprising an implantable device, a hydrophilic polymer formulation, a permeable polymeric membrane, injectable gel implants, solvent extraction system, phase inversion system, thermosensitive gels, pH dependent in situ gels, microparticles, microspheres, nanoparticles, nanospheres, bio-degradable implants, or photoactivated depot.
- Another embodiment of the present developments is a method of modulating, controlling, and/or increasing GLUT4 in a subject, the method comprising administering to the subject in need thereof, a therapeutically effective amount of a peptide selected from SEQ ID NOs 3-9 and 25-30.
- Another embodiment of the present developments is a method to modulate, affect and/or reduce interleukin-2 signaling in a cell or a subject comprising administering a peptide selected from SEQ ID NOs 3-9 and 25-30, in an amount sufficient to reduce or inhibit interleukin-2 signaling, wherein, the interleukin-2 signaling is associated with a condition selected from the group comprising type I diabetes, multiple sclerosis, systemic lupus erythematosa, rheumatoid arthritis, Crohn's disease, inflammatory bowel disease, chronic obstructive pulmonary disease, asthma, atherosclerosis, vasculitis, hypertension, thyroiditis, primary biliary cirrhosis, Paget's disease, Addison's disease, acute respiratory distress syndrome, acute lung injury, and/or aseptic chronic inflammation, more generally.
- Another embodiment of the present developments is a method to modulate and/or reduce interleukin-2 signaling in a cell or a subject comprising administering a peptide selected from SEQ ID NOs 3-9 and 25-30 in an amount sufficient to reduce or inhibit interleukin-2 signaling, wherein, the interleukin-2 signaling is associated with type 2 diabetes.
- Another embodiment of the present developments is a method to modulate and/or reduce IFN- ⁇ in a cell or a subject comprising administering a peptide selected from SEQ ID NOs 3-9 and 25-30, in an amount sufficient to reduce or inhibit IFN- ⁇ signaling, wherein, the IFN- ⁇ signaling is associated with type 2 diabetes.
- Another embodiment of the present developments is a method to modulate, affect, and/or induce changes of interleukin-21 (IL-21), interleukin-22 (IL-22), IFN ⁇ , TNF ⁇ , interleukin-6 (IL-6), granulocyte-macrophage colongy-stimulating factor (GM-CSF), interleukin-4 (IL-4), interleukin-10 (IL-10) and transforming growth factor beta (TGF ⁇ ) in a cell or subject comprising administering a peptide selected from SEQ ID NOs 3-9 and 25-30, in an amount sufficient to change said interleukin-21 (IL-21), interleukin-22 (IL-22), IFN ⁇ , TNF ⁇ , interleukin-6 (IL-6), granulocyte-macrophage colongy-stimulating factor (GM-CSF), interleukin-4 (IL-4), interleukin-10 (IL-10) and transforming growth factor beta (TGF ⁇ ), wherein the said interleukin-21 (IL-21), interleukin-22 (IL-22
- Another embodiment of the present developments includes a method to modulate and/or reduce interleukin 17 (IL-17) in a cell or a subject comprising administering a peptide selected from SEQ ID NOs 3-9 and 25-30 in an amount sufficient to reduce or inhibit interleukin 17 (IL-17) signaling, wherein, the IL-17 signaling is associated with a condition selected from the group comprising type I diabetes, multiple sclerosis, systemic lupus erythematosa, rheumatoid arthritis, Crohn's disease, inflammatory bowel disease, chronic obstructive pulmonary disease, asthma, atherosclerosis, vasculitis, hypertension, thyroiditis, primary biliary cirrhosis, Paget's disease, Addison's disease, acute respiratory distress syndrome, acute lung injury, and/or aseptic chronic inflammation, more generally.
- IL-17 signaling is associated with a condition selected from the group comprising type I diabetes, multiple sclerosis, systemic lupus erythematos
- Another embodiment of the present developments includes a method to modulate and/or reduce interleukin 17 (IL-17) in a cell or a subject comprising administering a peptide selected from SEQ ID NOs 3-9 and 25-30 in an amount sufficient to reduce or inhibit interleukin 17 (IL-17) signaling, wherein, the IL-17 is associated with type 2 diabetes.
- IL-17 interleukin 17
- One embodiment of the present developments is a method to identify a patient at risk for developing type 2 diabetes, the method comprising obtaining a sample containing T-cells from a patient to be tested, contacting the sample with a peptide that binds the CD40 protein, detecting the CD-40 bound peptide, and determining the level of Th40 cells from the amount of CD40 bound, wherein a level of Th40 cells greater than 25% of the total T-cell population indicates the patient is at risk for developing type 2 diabetes.
- One embodiment of the present developments is the composition of matter of small interfering peptides of those in SEQ ID NOs: 4, 27, 28, 29, and 30.
- These embodiments of the current developments may be used for the for the treatment of disease selected from the group comprising type I diabetes, multiple sclerosis, systemic lupus erythematosa, rheumatoid arthritis, Crohn's disease, inflammatory bowel disease, chronic obstructive pulmonary disease, asthma, atherosclerosis, vasculitis, hypertension, thyroiditis, primary biliary cirrhosis, Paget's disease, Addison's disease, acute respiratory distress syndrome, acute lung injury, type 2 diabetes, and/or aseptic chronic inflammation, more generally.
- disease selected from the group comprising type I diabetes, multiple sclerosis, systemic lupus erythematosa, rheumatoid arthritis, Crohn's disease, inflammatory bowel disease, chronic obstructive pulmonary disease, asthma, atherosclerosis,
- FIG. 1 is a chart of the effect of various peptides of CD154 on the development of diabetes in NOD mice.
- the 8-mer (SEQ ID NO: 5), 10-mer (SEQ NO: 24), 13-mer (SEQ ID NO:25), 15-mer (SEQ ID NO: 7), and 24-mer (SEQ ID NO:26) were tested.
- FIG. 2A is a chart of the effect of a 15-mer peptide from CD154 on the CD4/CD8 ratio in NOD mice.
- FIG. 2B is a chart of the effect of 15-mer peptide on beta-islet infiltration in treated versus control pancreata excised, examined, and scored.
- FIG. 3 is a graph of reversal of diabetes in NOD mice using a 15-mer peptide from CD154.
- FIG. 4 is a dot-plot of the detection of Th40 cells using a SIP-15-mer peptide from CD154.
- FIG. 5 is a dot-plot of a screening of B cells using a SIP-15-mer peptide from CD154.
- FIG. 6 is a chart demonstrating a comparison of Th40 cell levels in diabetic and non-diabetic mice.
- FIG. 7 is a chart demonstrating the effect of treatment with the 15-mer peptide on insulin granulation of the pancreas.
- FIG. 8 is a graph that shows the effect of mutations in the 15-mer peptide on the ability of the 15-mer peptide to inhibit development of diabetes in NOD mice.
- FIG. 9 is a chart showing the number of cells ( ⁇ 10 6 ) of CD3+CD4+CD40+ in different mice models.
- FIG. 10 is a chart showing the percentage of Th40 cells in the peripheral blood in human subjects in control and diabetic subjects.
- FIG. 11 is a dot-plot comparing CD4+ and CD40 cell data obtained through flow cytometry. Th40 cells (CD4+CD40+) are in the upper right quadrant.
- FIG. 12 is a chart showing the percentage of Th40 cells of CD3+CD4+ population of mice models.
- FIG. 13 is a chart that shows Th40 Cell percentage of CD3+CD4+ population within the murine aorta of C57B-6, young non-diabetic NOD mice, and diabetic NOD mice.
- FIG. 14 is an image at 200 ⁇ magnification showing Th40 cells in the shoulder region of plaque in the ApoE ⁇ / ⁇ mouse model.
- FIG. 15 is a graph of interferon gamma control of Th40 proliferation.
- FIG. 16 is a plot of CD40 stimulated proliferation of Th40 cells in the absence/presence of anti-interferon gamma antibody ( ⁇ IFN ⁇ ) demonstrating that interferon gamma mediates CD40 induced proliferation.
- ⁇ IFN ⁇ anti-interferon gamma antibody
- FIG. 17 is a sample of stains of aortic arch of the lesser curvature of the aortic arch.
- FIG. 18 is a stain of lesser curvature of aortic arch in control and treated subjects.
- FIG. 19 is a chart of area measurements of the lesser curvature of aortic arches and plaques.
- FIG. 20-23 are charts of treated and control ApoE mice subjects.
- FIG. 24 is a table of blood clot data from human blood in the presence of 15-mer peptide compared to normal clotting.
- FIG. 25A is a table providing the relative peptide stability assessed by ExPASy analysis.
- FIG. 25B is a table providing the relative peptides stability assessed by ExPASy analysis.
- FIG. 26 is a western blot comparing control and treated samples from subject mice.
- FIG. 27 is a graph of LDL cholesterol measured in treated and untreated subjects.
- FIG. 28A is an image of KGYY6 treated aortic en-face Sudan IV staining.
- FIG. 28B is an image of control aortic en-face Sudan IV staining.
- FIG. 29 is a graph demonstrating the reduction of lesion areas of Sudan IV staining.
- FIG. 30 is a graph of plaque volume reduction for area under the curve.
- FIG. 31 is a graph of plaque composition for KGYY6 (SEQ ID NO:29) treated and control subject mice.
- FIG. 32A is an image of trichrome stained sections of KGYY6 (SEQ ID NO:29) treated subject.
- FIG. 32B is an image of trichrome stained sections of control subjects.
- FIG. 33( a )-( b ) are graphs that show a statistically significant improvement in glucose tolerance (GTT) and insulin sensitivity in response to SEQ ID NO: 29.
- FIG. 34 is a western blot analysis of GLUT4 (insulin regulated glucose transport protein) comparing adipose and muscle tissue of treated (with SEQ ID NO: 29) and untreated mice.
- GLUT4 insulin regulated glucose transport protein
- FIG. 35 is a graph of the percentage change of in-vitro lymphocyte cytokines measured in spleen cells from ApoE ⁇ / ⁇ and C57BL/6 mice as measured by flow cytometry.
- Th40 cells may be instrumental in autoimmune inflammation.
- involvement of Th40 cells in the autoimmune process may be dependent on the interaction between CD40 protein expressed on the surface of the T-cell, and CD154 protein.
- Interaction of CD40 and CD154 results in activation signals being delivered between the cells, and subsequent activation of the Th40 cell.
- Such activation results in propagation of the Th40 cell and an increase in inflammation (e.g., an increase in the number of immune cells and immunoregulatory molecules, present in the system). Accordingly, inhibition of the CD40/CD154 interaction can modulate Th40 cell activity, and thereby affect inflammation.
- the present subject matter relates to the peptides, and administration thereof, that may affect the interaction between a CD40 protein and a CD154 protein, thereby modulating inflammation.
- the present subject matter relates to peptides that affect the interaction between CD40 protein expressed on the surface of a T-cell, and a CD154 protein, thereby affecting T-cell activity, controlling inflammation, and consequently preventing, modulating, and reducing atherosclerosis.
- the present subject matter also encompasses the use of such peptides to detect Th40 cells.
- a entity or an entity refers to one or more of that entity.
- a nucleic acid molecule refers to one or more nucleic acid molecules.
- the terms “a”, “an”, “one or more” and “at least one” can be used interchangeably.
- the terms “comprising”, “including” and “having” can be used interchangeably.
- animal refers to a vertebrate, preferably a mammal, more preferably a human.
- Suitable mammals on which to use the methods of the present invention include but are not limited farm animals, sports animals, pets, primates, mice, rats, horses, dogs, cats, and humans.
- the term animal can be used interchangeably with the terms subject or patient.
- One embodiment of the present subject matter is a peptide that interacts with a CD40 protein in such a manner as to prevent atherosclerosis.
- the terms interact, interaction, and the like mean that two molecules come into sufficient physical proximity such that they cause a modulation of inflammation.
- One such type of interaction is a binding interaction.
- the peptide associates with CD40 to form a complex.
- An example of complex formation is the association of an antigen with an antibody.
- binding of a peptide hereof to a CD40 protein can be reversible (e.g., non-covalent binding interactions) or non-reversible (e.g., covalent binding interactions).
- a reversible interaction can be strong or weak, the strength of the interaction being determined by the forces (e.g., ionic charges, hydrogen binding, van der Walls interactions, etc.) exerted by each protein on the other protein in the complex.
- forces e.g., ionic charges, hydrogen binding, van der Walls interactions, etc.
- Kd dissociation constant
- Preferred peptides of the present invention are those that bind to a CD40 protein with a Kd of no more than about 1 ⁇ 10 ⁇ 6 M, about 1 ⁇ 10 ⁇ 7 M, or about 1 ⁇ 10 ⁇ 8 M.
- Particularly preferred peptides are those having a Kd of less than about 1 ⁇ 10 ⁇ 9 M.
- a peptide hereof binds to a CD40 protein with a Kd of less than 100 nM, less than 50 nM, less than 25 nM, less than 10 nM, less than 5 nM, less than 3 nM, less than 2 nM, or less than 1 nM.
- Methods of measuring and analyzing binding interactions between a peptide and a CD40 protein are known by those of skill in the art.
- the change the level of Th40 cells present in an animal, or in a culture of T cells may be indicative of modulation of inflammation.
- level, number, count and concentration can be used interchangeably.
- Modulation of inflammation may mean an increase or decrease in the number of Th40 cells present in the inflammatory environment; however, the modulation of inflammation should not be limited to cell numbers or counts.
- modulation can be referred to as positive or negative.
- Positive modulation also referred to as up-regulation
- Negative modulation also referred to as down-regulation
- the level, count, or concentration of Th40 cells may not be indicative of inflammation in the inflammatory environment.
- a preferred peptide may be one that down-regulates inflammation, thereby reducing the number of Th40 cells present in the inflammatory environment.
- Positive and negative modulation of inflammation may or may not result in a change in the type and amount of immunoregulatory molecules present in the inflammatory environment.
- modulating inflammation may refer to changes in the Th40 levels, numbers, or concentration in an corporeal body or sample, and may also refer to changes in inflammation more generally that may be associated with disease, inflammatory cytokines, and/or cell derived inflammatory mediator molecules.
- both a cell culture system and the immune system of an animal comprise basal levels of immune cells and immunoregulatory molecules.
- basal level and normal level can be used interchangeably.
- the basal level of a type of immune cell e.g., Th40 cell
- a immunoregulatory molecule refers to the average number of that cell type, or immunoregulatory molecule, present in a population of individuals considered healthy (i.e., free of metabolic, autoimmune, or infectious disease).
- the basal level of a type of immune cell, or an immunoregulatory molecule refers to the average level of that cell type, or immunoregulatory molecule, present in a population of cells that is non-activated.
- Those skilled in the art are capable of determining if a T-cell, or a population of such cells, is activated. For example, the expression of CD69, CD25 and/or CD154 proteins by a cell indicates that the cell has been activated.
- the basal level of a cell or molecule can be a specific amount (e.g., a specific concentration) or it can encompass a range of amounts.
- Basal levels, or ranges, of immune cells and immunoregulatory molecules are known to those in the art.
- the normal level of CD4+ T-cells present in human blood is 500-1500 cells/ml. Variability in this measurement can result from differences in the method used to determine the cell count.
- normal levels of cells can also be reported as a percentage of a total cell population. For example, in a healthy individual, Th40 cells make up less than 25% of the total T cell population.
- the term inflammation refers to an inflammatory environment in which Th40 cells make up greater than about 25%, greater than about 30%, greater than about 35%, greater than about 40%, greater than about 45%, greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, or greater than about 80% of the total T-cell population.
- a preferred peptide herein is one that reduces the level of Th40 cells to less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 27%, or equal to about 25% of the total T-cell population.
- the phrase inflammatory environment refers to the overall population of immune cells, and related immunoregulatory molecules, that are present in a culture of cells, or in the body of an animal.
- the phrase inflammatory environment encompasses the types, and/or the relative amounts of immune cells and immunoregulatory molecules (e.g., cytokines) present in a culture of cells, or in an animal, which are involved in affecting an inflammatory reaction.
- immunoregulatory molecules e.g., cytokines
- Examples of cells encompassed by the term inflammatory environment include, but are not limited to, T cells, neutrophils, macrophages, granulocytes, and the like.
- the inflammatory environment relates to cells and molecules that mediate both acute and chronic inflammation. It will be appreciated by those skilled in the art that the inflammatory environment refers to the system to which peptides hereof are administered.
- the system is a cell culture system.
- the system is a whole animal.
- a preferred peptide hereof is one that selectively interacts with a CD40 protein in solution, as determined using an assay such as an immunosorbent assay, or on the surface of a T-cell.
- an assay such as an immunosorbent assay
- the terms selectively, selective, specific, and the like indicate the peptide has a greater affinity for a CD40 protein than it does for proteins unrelated to the CD40 protein. More specifically, the terms selectively, selective, specific, and the like indicate that the affinity of the peptide for CD40 is statistically significantly higher than its affinity for a negative control (e.g., an unrelated protein such as albumin) as measured using a standard assay (e.g., ELISA).
- a negative control e.g., an unrelated protein such as albumin
- Suitable techniques for assaying the ability of a peptide to selectively interact with a CD40 protein are known to those skilled in the art.
- Such assays can be in vitro or in vivo assays.
- useful assays include, but are not limited to, an enzyme-linked immunoassay, a competitive enzyme-linked immunoassay, a radioimmunoassay, a fluorescence immunoassay, a chemiluminescent assay, a lateral flow assay, a flow-through assay, an agglutination assay, a particulate-based assay (e.g., using particulates such as, but not limited to, magnetic particles or plastic polymers, such as latex or polystyrene beads), an immunoprecipitation assay, an immunoblot assay (e.g., a western blot), a phosphorescence assay, a flow-through assay, a chromatography assay, a polyacrylamide gel electrophor
- an assay can be performed using cells in culture, or it can be performed in a whole animal. Assays can be designed to give qualitative, quantitative or semi-quantitative results, depending on how they are used and the type of result that is desired.
- CVD cardiovascular Disease
- Atherosclerosis Atherosclerosis
- One embodiment hereof is a peptide that interacts with a CD40 protein in such a manner as to affect the interaction of the CD40 protein with a CD154 protein, thereby modulating inflammation.
- the effect of the peptide on the CD40/CD154 interaction can be positive or it can be negative.
- the peptide can interact with the CD40 protein in such a manner that the strength of the interaction between the CD40 protein and a CD154 protein is increased.
- the peptide can interact with the CD40 protein such that the strength of the interaction between the CD40 protein and a CD154 protein is decreased. Methods of measuring the strength of binding between the peptide and a CD40 protein are known to those skilled in the art.
- a preferred peptide hereof is one that reduces the strength of the interaction between a CD40 protein and a CD154 protein.
- Preferred peptides hereof reduce the strength of binding between a CD40 protein and a CD154 protein by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%.
- a particularly preferred peptide is one that completely inhibits binding of CD40 to CD154.
- Complete inhibition of binding between CD40 and CD154 means that when a peptide hereof is brought into proximity with a CD40 protein and a CD154 protein under conditions that would normally allow the interaction of CD40 and CD154, no such interaction occurs and activation signals are not stimulated in the CD40-expressing cell.
- the peptide interacts with the CD40 protein in such a manner as to reduce the level of inflammation in the system. In one embodiment, the peptide interacts with the CD40 protein in such a manner as to inhibit the development of inflammation in the system.
- peptides hereof can interact with any site on the CD40 protein, preferred peptides interact with the CD40 protein at a location that overlaps with the CD154 binding site.
- a peptide hereof interacts with the CD40 protein at the CD154 binding site.
- An example of such a peptide is a CD40 ligand competitive antagonist.
- peptides that interfere with, or inhibit, the binding of a CD154 protein to a CD40 protein are referred to as small interfering peptides (SIPs).
- a small interfering peptide is a peptide that, through physio-chemical properties, interferes with the interaction of a CD40 protein with a CD154 protein, thereby preventing activation signals from being delivered to the CD40-bearing cell, thus limiting the activation of the CD40-bearing cell, and consequently, inflammation.
- the consequences of such interference are prevention of T-cell activation and propagation, and a prevention or reduction of inflammation.
- the results of such inhibition or prevention of interaction between CD40 and CD154 may include observable data that demonstrates that atherosclerosis, and characteristics of diseases associated therewith, are prevented, modulated, and/or reduced.
- a small interfering peptide may, through its physio-chemical properties, interfere with the interaction of a CD40 protein with a CD154 protein, thereby preventing activation signals from being delivered to the CD40-bearing cell, thus limiting the activation of the CD40-bearing cell, and consequently, modulating, inhibiting, and preventing atherosclerosis.
- the consequences of such interference are prevention of T cell activation and propagation, and a prevention, reduction, or modulation of atherosclerotic developments.
- a peptide useful for practicing methods of the present developments should be of a size sufficient to interact with CD40 protein in such a manner as to modulate atherosclerosis. It is understood by those skilled in the art that preferred peptides are relatively short since they are easier and less expensive to produce. Preferred peptides may be those that are less than 25 amino acids in length; however, the length of the peptide may be longer than 25 amino acids in some instances. A preferred peptide may be one that is 4, 6, 8, 10, 13, 15, or 24 amino acids in length.
- the peptide is an amino acid selected from the group of SEQ ID NO:3 (Core-sequence see Table 1), SEQ ID NO:4 (6-mer see Table 1), SEQ ID NO:5 (8-mer mouse see Table 1), SEQ ID NO:6 (8-mer human see Table 1), SEQ IN NO:7 (15-mer see Table 1), SEQ ID NO:8 (15-mer human see Table 1), SEQ ID NO:9 (24-mer see Table 1), SEQ ID NO: 24 (10-mer see Table 1), SEQ ID NO: 25 (13-mer see Table 1), SEQ ID NO: 26 (24-mer see Table 1), SEQ ID NO: 27 (6-mer (Form 2) see Table 1), SEQ ID NO: 28 (6-mer (Form 3) see Table 1), SEQ ID NO: 29 (6-mer (Form 4) see Table 1), SEQ ID NO: 30 (6-mer (Form 4) see Table 1) and SEQ ID NO:32 (24-mer-mouse (Form 2)).
- SEQ ID NO:3 Core
- a peptide comprising only a short portion of the CD154 region that interacts with CD40 is capable of binding to a CD40 protein, thereby modulating atherosclerosis.
- a peptide that comprises at least a portion of the amino acid sequence of a CD154 protein such that the peptide interacts with CD40 protein in such a manner as to modulate atherosclerosis.
- interaction of the peptide with CD40 protein results in negative modulation of atherosclerosis.
- the peptide comprises at least a portion of SEQ ID NO:1 or SEQ ID NO:2.
- the peptide is as short as possible yet comprises enough of the CD154 protein to allow interaction with a CD40 protein in such a manner as to modulate atherosclerosis.
- a peptide hereof comprises 6, 13 or 15 contiguous amino acids from SEQ ID NO:1 or SEQ ID NO:2, and interacts with CD40 in such a manner as to modulate atherosclerosis.
- a preferred peptide comprises a core sequence of lysine-glycine-tyrosine-tyrosine (KGYY; SEQ ID NO:3), which corresponds to amino acids 142-145 of SEQ ID NO:1 and amino acids 143-146 of SEQ ID NO:2.
- Useful peptides can comprise additional regions of sequence from SEQ ID NO:1 or SEQ ID NO:2 that are adjacent to the core sequence, so long as the peptide is capable of modulating atherosclerosis.
- a peptide comprises at least one sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:32, so long as the peptide interacts with CD40 protein in such a manner as to modulate atherosclerosis.
- a peptide hereof is a sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:32.
- a peptide hereof is a sequence selected from SEQ ID NOs 3-9, 25-30, and 32.
- peptides of the present subject matter may be selected entirely of or from sequences that are responsible for the interaction of the peptide with a CD40 protein, they may additionally contain amino acid sequences that do not interact with a CD40 protein, but which have other useful functions. Any useful, additional amino acid sequence can be added to the CD40-interacting sequence, so long as the additional sequences do not have an unwanted effect on the ability of the CD40 interacting sequence to interact with a CD40 protein.
- a peptide hereof can contain amino acid sequences that are useful for visualizing or purifying the peptide. Such sequences act as labels (e.g., enzymes) or tags (antibody binding sites).
- labels and tags include, but are not limited to, B-galactosidase, luciferase, glutathione-s-transferase, thioredoxin, HIS-tags, biotin tags, and fluorescent tags.
- Other useful sequences for labeling and tagging proteins are known to those of skill in the art.
- peptides hereof can be modified, so long as such modification does not significantly affect the ability of the peptide to modulate atherosclerosis.
- modifications can be made, for example, to increase the stability, solubility or absorbability of the protein. Examples of such modifications include, but are not limited to pegylation, glycosylation and chemical modification of the peptide.
- Peptides hereof may be obtained from nature (e.g., obtained from plants, animals or microorganisms) or they may be produced in a laboratory (e.g., recombinantly or synthetically). Preferred peptides are those that are synthesized. Also encompassed are peptides that are combinations of natural and synthetic molecules. General methods for producing and isolating recombinant or synthetic peptides are known to those skilled in the art. It should be noted that, as used herein, an isolated, or biologically pure, molecule, is one that has been removed from its natural milieu. As such the terms isolated, biologically pure, and the like, do not necessarily reflect the extent to which the protein has been purified.
- one embodiment is a method to reduce the interaction between a CD40 protein and a CD154 protein comprising introducing into an environment containing a CD40 protein and a CD154 protein, a peptide, that interacts with the CD40 protein in such a manner as to reduce the interaction between the CD40 protein and the CD154 protein.
- the peptide reduces the interaction between the CD40 protein and the CD154 protein by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In one embodiment, the peptide reduces the interaction between the CD40 protein and the CD154 protein by a factor of at least 10, at least 100, at least 1,000, at least 10,000. Methods of measuring the strength of the interaction between the CD40 protein and the CD154 protein have been discussed previously, and are also know to those of skill in the art.
- One embodiment hereof is a method to modulate atherosclerosis comprising contacting a CD40 protein with a peptide that interacts to the CD40 protein in such a manner as to modulate inflammation.
- interaction of the peptide with the CD40 protein decreases the number of Th40 cells by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%.
- interaction of the peptide with the CD40 protein decreases the number of Th40 cells by a factor of at least 10, at least 100, at least 1,000, at least 10,000.
- One aspect of the current developments hereof is a method to modulate atherosclerosis, cardiovascular disease, and/or cholesterol levels comprising contacting a CD40 protein with a peptide that interacts with the CD40 protein in such a manner as to ameliorate inflammation.
- One aspect of this alternative embodiment is that the Th40 levels do not change in response to contacting said CD40 protein with a peptide that interacts with the CD40 protein.
- Th40 cells treated with peptide may stop, slow, reduce, or retard production of inflammatory cytokines. In this aspect the number of Th40 levels may remain relatively unchanged.
- One aspect is a method to reduce atherosclerosis in a patient, the method comprising administering a peptide hereof to the patient.
- the peptide comprises an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:32.
- the peptide is an amino acid sequence selected from SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30 and SEQ ID NO:32.
- interaction of the peptide with the CD40 protein decreases the number of Th40 cells by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%.
- interaction of the peptide with the CD40 protein decreases the number of Th40 cells by a factor of at least 10, at least 100, at least 1,000, at least 10,000.
- the level of Th40 cells is reduced so that Th40 cells comprise no more than about 20%, about 25%, about 30%, about 35%, or about 40% of the total T-cell population.
- Peptides and methods hereof are suitable for use in cell culture as well as for treating a patient.
- patient refers to any animal in need of such treatment.
- the animal can be a human or a non-human animal.
- a preferred animal to treat is a mammal.
- a peptide can be administered or applied per se, or as pharmaceutical compositions.
- a peptide hereof, or a pharmaceutical composition thereof can be administered to a patient by a variety of routes, including, but limited to, by injection (e.g., intravenous, intramuscular, subcutaneous, intrathecal, intraperitoneal), by inhalation, by oral (e.g., in a pill, tablet, capsule, powder, syrup, solution, suspension, thin film, dispersion or emulsion.), transdermal, transmucosal, pulmonary, buccal, intranasal, sublingual, intracerebral, intravaginal rectal or topical administration or by any other convenient method known to those of skill in the art.
- routes including, but limited to, by injection (e.g., intravenous, intramuscular, subcutaneous, intrathecal, intraperitoneal), by inhalation, by oral (e.g., in a pill, tablet, capsule, powder, syrup, solution, suspension, thin film, dispersion or emulsion.), transdermal, transmucosal, pulmonary
- the amount of a peptide hereof and/or a pharmaceutical composition thereof that will be effective can be determined by standard clinical techniques known in the art. Such an amount is dependent on, among other factors, the patient being treated, including, but not limited to the weight, age, and condition of the patient, the intended effect of the compound, the manner of administration and the judgment of the prescribing physician. Also, in this context, it should be noted that in treating a patient exhibiting a disorder of interest, a therapeutically effective amount of an agent or agents such as these is administered.
- a therapeutically effective dose refers to that amount of the compound that results in amelioration of one or more symptoms or a prolongation of survival in a patient.
- a peptide hereof, or a pharmaceutical composition thereof can be administered alone or in combination with one or more other pharmaceutical agents, including other compounds of the present disclosure.
- the specific pharmaceutical composition depends on the desired mode of administration, as is well known to the skilled artisan.
- one embodiment hereof is a method to treat atherosclerotic disease in a patient in need of such treatment, the method comprising administering to a patient a peptide that interacts with the CD40 protein, thereby reducing atherosclerosis.
- the peptide interacts with the CD40 protein in such a manner as to affect the interaction of CD40 and CD154, thereby reducing atherosclerosis.
- interaction of the peptide with the CD40 protein reduces the number of Th40 cells in a patient to a level equal to that observed in subjects that do not have cardiovascular disease.
- the present developments are suitable for treating any patient having an autoimmune disease and/or cardiovascular disease, the development of which is dependent on Th40 cells. More specifically, peptides hereof are suitable for reducing the level of Th40 cells in such patients.
- a peptide hereof reduces the level of Th40 cells in a patient suffering from a cardiovascular disease to no more than about 25% of the total T-cell population.
- a peptide hereof reduces inflammatory cytokine levels while having no effect on the Th40 levels in a patient.
- Atherosclerosis inflammatory changes of the arterial wall occur resulting in the formation and buildup of arterial plaque. Consequently, control of inflammatory cells and cell signaling via CD40-CD154 interaction may be able to be used to control, modulate, and/or reduce atherosclerotic lesions that are characterized as chronic inflammatory-fibroproliferative disease of the vessel wall.
- Several murine models of T2D and/or atherosclerosis have been developed.
- AHA type 1 may be characterized by early or initial lesions, may be comprised of histologically “normal” cells, macrophage infiltration, and isolated foam cells.
- AHA type V may be characterized by advanced or complicated legions, including but not limited to increased endothelial dysfunction characterized by surface defects, hematoma, hemorrhage, and/or thrombosis.
- one embodiment of the present developments is a method to prevent atherosclerosis in an individual at risk for developing atherosclerosis, the method comprising administering to the individual a peptide to selectively bind to a CD40 expressing cell.
- Atherosclerosis may be particularly amenable to treatment using a peptide of the current development.
- the risk for atherosclerosis may result from familial factors (e.g., inheritance) or from other factors, such as the physical condition of the individual.
- the level of atherosclerotic activity and disease may vary from individual to individual depending on numerous factors such as level of activity, diet, smoking status, and other variable factors that are dynamic such as levels of inflammation.
- the method of treatment comprises administering to a patient in need thereof, a peptide that comprises an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 7, SEQ ID NO:8, and SEQ ID NO:9, so long as the peptide can down-regulate inflammation.
- the peptide is an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30 and SEQ ID NO:32.
- one aspect of the present subject matter is a method to reverse atherosclerosis comprising administering to a patient diagnosed as having atherosclerosis, a peptide hereof.
- the peptide comprises an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 7, SEQ ID NO:8, and SEQ ID NO:9, so long as the peptide can down-regulate inflammation.
- the peptide is an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9.
- the phrase to reverse atherosclerosis means to reduce the aortic-arch infiltration, plaque, and lesions of an individual with atherosclerosis to a level comparable to that observably lower levels or in some instances to levels that may be more common in a non-atherosclerotic individual.
- peptides of the present invention selectively bind to a CD40 expressing cell. Consequently, peptides of the present subject matter can be used to identify Th40 cells.
- a method to detect Th40-dependent atherosclerosis comprising contacting a T-cell population with a peptide hereof.
- the peptide is labeled with a detectable marker, such as, for example, luciferase or alkaline phosphatase. Such detection can be performed using assay techniques known to those skilled in the art.
- an assay for detecting Th40 cells using a peptide hereof comprises (a) obtaining a sample of cells; (b) contacting a peptide hereof with said cells under condition suitable to allow binding of the peptide to Th40 cells, if present; (c) washing said cells using conditions that disrupt non-specific interactions, and that remove unbound peptide; and (d) detecting peptide bound to cells.
- Detection of bound peptide can be achieved directly or indirectly. For example, direct detection can be achieved using a peptide labeled using a detectable marker, as disclosed herein. Following the wash step listed above, the cells are then simply screened for the presence of detectable marker.
- detectable marker in the cell sample indicates the presence of Th40 cells, and thus Th40-dependent atherosclerosis.
- indirect detection involves the use of a second molecule, such as an antibody, that binds to the peptide.
- a detection molecule that binds the peptide is added to the cell sample.
- the detection molecule is labeled with a detectable marker. After washing away unbound detection molecule, the cells are screened for the presence of detectable marker.
- the presence of detectable marker in the cell sample indicates the presence of Th40 cells.
- Suitable assay techniques are known to those skilled in the art, and are also disclosed in, for example, Molecular Cloning: A Laboratory Manual, Sambrook, J., Fritsch, E. F., and Maniatis, T, Cold Spring Harbor Laboratory Press; 2nd Edition (December 1989). All references cited herein are incorporated herein in their entirety.
- the assay technology described above can also be used to identify other molecules that affect the interaction of a CD40 protein with a CD154 protein.
- examples of such molecules include, but are not limited to, proteins, peptides and small molecules.
- assays can be designed that test the ability of molecules to compete with a peptide of the present developments for binding to a Th40 cell.
- a peptide labeled with a detectable marker can be mixed with a test molecule and a population of cells known to contain Th40 cells, under conditions that allow binding of the peptide to the Th40 cells. Following an appropriate incubation period, the cells are washed to remove unbound peptide, and the cells screened for the presence of detectable marker.
- the labeled peptide could be bound to Th40 cells first, and after a wash step to remove unbound peptide, the test molecule could be added to the cells containing bound peptide. Following an incubating period and a wash step to remove unbound molecule, or released peptide, the cells are screened for the presence of detectable marker. In either case, absence of the detectable marker in the cell sample indicates the test molecule is able to compete with the peptide for binding to the Th40 cells, while presence of the detectable marker would indicate the test molecule does not inhibit binding of the peptide to Th40 cells. Inhibition of binding need not be 100%, as such assay would also be useful for identifying molecules that partially inhibit binding of the peptide to Th40 cells. It is understood by those skilled in the art that such assays would involve the use of positive controls (e.g., unlabeled peptide) and negative controls (e.g., a protein/molecule that is known not to bind to Th40 cells).
- positive controls e.g.
- one embodiment of the present developments is a method to identify a patient at risk for developing autoimmune related atherosclerosis.
- patients at risk for developing atherosclerosis are identified by obtaining a sample from a patient to be tested, contacting the T-cell portion of said sample with a peptide hereof, and determining the level of Th40 cells present in the sample, wherein a level of Th40 cells greater than about 25% of the total T-cell population indicates the patient is at risk for developing atherosclerotic disease.
- the peptide comprises an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:7, SEQ ID NO:9 SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:32 so long as the peptide binds to the CD40 protein.
- the peptide is an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:32.
- the peptide is labeled with a suitable detectable marker such as, for example, luciferase or alkaline phosphatase.
- kits useful for practicing the methods disclosed herein comprising a peptide that interacts with a CD40 protein in such a manner as to modulate atherosclerosis.
- the peptide comprises an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, so long as the peptide can down-regulate atherosclerosis.
- the peptide is an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9.
- kits for determining the level of Th40 cells comprising a peptide that interacts with a CD40 protein, and methods for detecting CD40-bound peptide.
- Kits can also contain associated reagents and components, such as, but not limited to, buffers, labels, containers, inserts, tubing, vials, syringes, and the like.
- Th40 cells that may be instrumental in autoimmune inflammation, including conditions such as type 2 diabetes.
- involvement of Th40 cells in the autoimmune process may be dependent on the interaction between CD40 protein expressed on the surface of the T-cell, and CD154 protein.
- Interaction of CD40 and CD154 results in activation signals being delivered between the cells, and subsequent activation of the Th40 cell.
- Such activation results in propagation of the Th40 cell and an increase in inflammation (e.g., an increase in the number of immune cells and immunoregulatory molecules, present in the system). Accordingly, inhibition of the CD40/CD154 interaction can modulate Th40 cell activity, and thereby affect inflammation.
- the present subject matter relates to the peptides, and administration thereof, that may affect the interaction between a CD40 protein and a CD154 protein, thereby modulating inflammation.
- the present subject matter relates to peptides that affect the interaction between CD40 protein expressed on the surface of a T-cell, and a CD154 protein, thereby affecting T-cell activity, controlling inflammation, and consequently preventing, modulating, reducing and/or reversing type 2 diabetes.
- the present subject matter also encompasses the use of such peptides to detect Th40 cells.
- One embodiment of the present subject matter is a peptide that interacts with a CD40 protein in such a manner as to prevent type 2 diabetes.
- the terms interact, interaction, and the like mean that two molecules come into sufficient physical proximity such that they cause a modulation of inflammation.
- One such type of interaction is a binding interaction.
- the peptide associates with CD40 to form a complex.
- An example of complex formation is the association of an antigen with an antibody.
- binding of a peptide hereof to a CD40 protein can be reversible (e.g., non-covalent binding interactions) or non-reversible (e.g., covalent binding interactions).
- a reversible interaction can be strong or weak, the strength of the interaction being determined by the forces (e.g., ionic charges, hydrogen binding, van der Walls interactions, etc.) exerted by each protein on the other protein in the complex.
- forces e.g., ionic charges, hydrogen binding, van der Walls interactions, etc.
- Kd dissociation constant
- Preferred peptides of the present invention are those that bind to a CD40 protein with a Kd of no more than about 1 ⁇ 10 ⁇ 6 M, about 1 ⁇ 10 ⁇ 7 M, or about 1 ⁇ 10 ⁇ 8 M.
- Particularly preferred peptides are those having a Kd of less than about 1 ⁇ 10 ⁇ 9 M.
- a peptide hereof binds to a CD40 protein with a Kd of less than 100 nM, less than 50 nM, less than 25 nM, less than 10 nM, less than 5 nM, less than 3 nM, less than 2 nM, or less than 1 nM.
- Methods of measuring and analyzing binding interactions between a peptide and a CD40 protein are known by those of skill in the art.
- the change the level of Th40 cells present in an animal, or in a culture of T cells may be indicative of modulation of inflammation.
- level, number, count and concentration can be used interchangeably.
- Modulation of inflammation may mean an increase or decrease in the number of Th40 cells present in the inflammatory environment; however, the modulation of inflammation should not be limited to cell numbers or counts. Consequently, modulation can be referred to as positive or negative.
- Positive modulation (also referred to as up-regulation) of inflammation may result in an increase in the number of Th40 cells in the inflammatory environment.
- Negative modulation also referred to as down-regulation
- of inflammation may result in a reduction in the number of Th40 cells present in the inflammatory environment.
- the level, count, or concentration of Th40 cells may not be indicative of inflammation in the inflammatory environment.
- a preferred peptide may be one that down-regulates inflammation, thereby reducing the number of Th40 cells present in the inflammatory environment.
- Positive and negative modulation of inflammation may or may not result in a change in the type and amount of immunoregulatory molecules present in the inflammatory environment.
- the Th40 levels will not change but the activity of those cells is altered such that they are no longer exerting or increasing inflammatory cytokines and other biomarkers of inflammation.
- modulating inflammation may refer to changes in the Th40 levels, numbers, or concentration in a corporeal body or sample, and may also refer to changes in inflammation more generally that may be associated with disease, inflammatory cytokines, and/or cell derived inflammatory mediator molecules.
- both a cell culture system and the immune system of an animal comprise basal levels of immune cells and immunoregulatory molecules.
- basal level and normal level can be used interchangeably.
- the basal level of a type of immune cell e.g., Th40 cell
- a immunoregulatory molecule refers to the average number of that cell type, or immunoregulatory molecule, present in a population of individuals considered healthy (i.e., free of metabolic, autoimmune, or infectious disease).
- the basal level of a type of immune cell, or an immunoregulatory molecule refers to the average level of that cell type, or immunoregulatory molecule, present in a population of cells that is non-activated.
- Those skilled in the art are capable of determining if a T-cell, or a population of such cells, is activated. For example, the expression of CD69, CD25 and/or CD154 proteins by a cell indicates that the cell has been activated.
- the basal level of a cell or molecule can be a specific amount (e.g., a specific concentration) or it can encompass a range of amounts.
- Basal levels, or ranges, of immune cells and immunoregulatory molecules are known to those in the art.
- the normal level of CD4+ T-cells present in human blood is 500-1500 cells/ml. Variability in this measurement can result from differences in the method used to determine the cell count.
- normal levels of cells can also be reported as a percentage of a total cell population. For example, in a healthy individual, Th40 cells make up less than 25% of the total T cell population.
- the term inflammation refers to an inflammatory environment in which Th40 cells make up greater than about 25%, greater than about 30%, greater than about 35%, greater than about 40%, greater than about 45%, greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, or greater than about 80% of the total T-cell population.
- a preferred peptide herein is one that reduces the level of Th40 cells to less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 27%, or equal to about 25% of the total T-cell population.
- the phrase inflammatory environment refers to the overall population of immune cells, and related immunoregulatory molecules, that are present in a culture of cells, or in the body of an animal.
- the phrase inflammatory environment encompasses the types, and/or the relative amounts of immune cells and immunoregulatory molecules (e.g., cytokines) present in a culture of cells, or in an animal, which are involved in affecting an inflammatory reaction.
- immunoregulatory molecules e.g., cytokines
- Examples of cells encompassed by the term inflammatory environment include, but are not limited to, T cells, neutrophils, macrophages, granulocytes, and the like.
- the inflammatory environment relates to cells and molecules that mediate both acute and chronic inflammation. It will be appreciated by those skilled in the art that the inflammatory environment refers to the system to which peptides hereof are administered.
- the system is a cell culture system.
- the system is a whole animal.
- a preferred peptide hereof is one that selectively interacts with a CD40 protein in solution, as determined using an assay such as an immunosorbent assay, or on the surface of a T-cell.
- an assay such as an immunosorbent assay
- the terms selectively, selective, specific, and the like indicate the peptide has a greater affinity for a CD40 protein than it does for proteins unrelated to the CD40 protein. More specifically, the terms selectively, selective, specific, and the like indicate that the affinity of the peptide for CD40 is statistically significantly higher than its affinity for a negative control (e.g., an unrelated protein such as albumin) as measured using a standard assay (e.g., ELISA).
- a negative control e.g., an unrelated protein such as albumin
- Suitable techniques for assaying the ability of a peptide to selectively interact with a CD40 protein are known to those skilled in the art.
- Such assays can be in vitro or in vivo assays.
- useful assays include, but are not limited to, an enzyme-linked immunoassay, a competitive enzyme-linked immunoassay, a radioimmunoassay, a fluorescence immunoassay, a chemiluminescent assay, a lateral flow assay, a flow-through assay, an agglutination assay, a particulate-based assay (e.g., using particulates such as, but not limited to, magnetic particles or plastic polymers, such as latex or polystyrene beads), an immunoprecipitation assay, an immunoblot assay (e.g., a western blot), a phosphorescence assay, a flow-through assay, a chromatography assay, a polyacrylamide gel electrophor
- an assay can be performed using cells in culture, or it can be performed in a whole animal. Assays can be designed to give qualitative, quantitative or semi-quantitative results, depending on how they are used and the type of result that is desired.
- One embodiment hereof is a peptide that interacts with a CD40 protein in such a manner as to affect the interaction of the CD40 protein with a CD154 protein, thereby modulating inflammation.
- the effect of the peptide on the CD40/CD154 interaction can be positive or it can be negative.
- the peptide can interact with the CD40 protein in such a manner that the strength of the interaction between the CD40 protein and a CD154 protein is increased.
- the peptide can interact with the CD40 protein such that the strength of the interaction between the CD40 protein and a CD154 protein is decreased. Methods of measuring the strength of binding between the peptide and a CD40 protein are known to those skilled in the art.
- a preferred peptide hereof is one that reduces the strength of the interaction between a CD40 protein and a CD154 protein.
- Preferred peptides hereof reduce the strength of binding between a CD40 protein and a CD154 protein by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%.
- a particularly preferred peptide is one that completely inhibits binding of CD40 to CD154.
- Complete inhibition of binding between CD40 and CD154 means that when a peptide hereof is brought into proximity with a CD40 protein and a CD154 protein under conditions that would normally allow the interaction of CD40 and CD154, no such interaction occurs and activation signals are not stimulated in the CD40-expressing cell.
- the peptide interacts with the CD40 protein in such a manner as to reduce the level of inflammation in the system. In one embodiment, the peptide interacts with the CD40 protein in such a manner as to inhibit the development of inflammation in the system. In one aspect, the peptide may alter the way the cells may interact with other molecules that normally occur during cell-to-cell interactions. Such cell-to-cell interactions may be those that result in inflammation.
- the peptides of the developments hereof, may disrupt the inflammasome complex, which may promote the maturation of pro-inflammatory cytokines interleukin-1 ⁇ (IL-1 ⁇ ) and interleukin-18 (IL-18).
- peptides hereof may disrupt the inflammasome complex of caspase 1, PYCARD, NALP, caspase 5, nucleotide-binding oligomerization domain and leucine-rich repeat-containing receptors (NLRs) and ALRs (AIM2-like receptors).
- One aspect of the peptides described herein is that administration of the peptide may alter the way the cell may interact with other molecules that normally occur during cell to cell interactions which may be indicative of or result in inflammation.
- One aspect of the peptides described herein is that these peptide(s) may disrupt the inflammasome and thus alter inflammatory outcomes.
- peptides hereof can interact with any site on the CD40 protein, preferred peptides interact with the CD40 protein at a location that overlaps with the CD154 binding site.
- a peptide hereof interacts with the CD40 protein at the CD154 binding site.
- An example of such a peptide is a CD40 ligand competitive antagonist.
- peptides that interfere with, or inhibit, the binding of a CD154 protein to a CD40 protein are referred to as small interfering peptides (SIPs).
- a small interfering peptide is a peptide that, through physio-chemical properties, interferes with the interaction of a CD40 protein with a CD154 protein, thereby preventing activation signals from being delivered to the CD40-bearing cell, thus limiting the activation of the CD40-bearing cell, and consequently, inflammation.
- the consequences of such interference are prevention of T-cell activation and propagation, and a prevention or reduction of inflammation.
- a small interfering peptide may, through its physio-chemical properties, interfere with the interaction of a CD40 protein with a CD154 protein, thereby preventing activation signals from being delivered to the CD40-bearing cell, thus limiting the activation of the CD40-bearing cell, and consequently, modulating, inhibiting, preventing, and/or reversing type 2 diabetes.
- the consequences of such interference are prevention of T cell activation and propagation, and a prevention, reduction, modulation, and reversal of type 2 diabetic developments.
- a peptide useful for practicing methods of the present developments should be of a size sufficient to interact with CD40 protein in such a manner as to modulate type 2 diabetes. It is understood by those skilled in the art that preferred peptides are relatively short since they are easier and less expensive to produce. Preferred peptides are those that are less than 20 amino acids in length. A preferred peptide is one that is 4, 6, 8, 10, 13, 15, or 24 amino acids in length.
- the peptide is an amino acid selected from the group of SEQ ID NO:3 (Core-sequence see Table 1), SEQ ID NO:4 (6-mer see Table 1), SEQ ID NO:5 (8-mer mouse see Table 1), SEQ ID NO:6 (8-mer human see Table 1), SEQ ID NO:7 (15-mer mouse see Table 1), SEQ ID NO:8 (15-mer human see Table 1), SEQ ID NO:9 (24-mer see Table 1), SEQ ID NO: 24 (10-mer see Table 1), SEQ ID NO: 25 (13-mer see Table 1), SEQ ID NO: 26 (24-mer see Table 1), SEQ ID NO: 27 (6-mer (Form 2) see Table 1), SEQ ID NO: 28 (6-mer (Form 3) see Table 1), SEQ ID NO: 29 (6-mer (Form 4) see Table 1), SEQ ID NO: 30 (6-mer (Form 4) see Table 1) and SEQ ID NO:32 (24-mer-mouse (Form 2)).
- SEQ ID NO:3 (
- a peptide comprising only a short portion of the CD154 region that interacts with CD40 is capable of binding to a CD40 protein, thereby modulating type 2 diabetes.
- a peptide that comprises at least a portion of the amino acid sequence of a CD154 protein such that the peptide interacts with CD40 protein in such a manner as to modulate type 2 diabetes.
- interaction of the peptide with CD40 protein results in negative modulation, reduction, or inhibition of type 2 diabetes.
- the peptide comprises at least a portion of SEQ ID NO:1 or SEQ ID NO:2.
- the peptide is as short as possible yet comprises enough of the CD154 protein to allow interaction with a CD 40 protein in such a manner as to modulate type 2 diabetes.
- a peptide hereof comprises 6, 13 or 15 contiguous amino acids from SEQ ID NO:1 or SEQ ID NO:2, and interacts with CD40 in such a manner as to modulate type 2 diabetes.
- a preferred peptide comprises a core sequence of lysine-glycine-tyrosine-tyrosine (KGYY; SEQ ID NO:3), which corresponds to amino acids 142-145 of SEQ ID NO:1 and amino acids 143-146 of SEQ ID NO:2.
- another preferred peptide comprises a core sequence of lysine-glycine-tyrosine-tyrosine-threonine-methionine (KGYYTM; SEQ ID NO:27), which corresponds to amino acids 142-147 of SEQ ID NO:1 and amino acids 143-148 of SEQ ID NO:2.
- Useful peptides can comprise additional regions of sequence from SEQ ID NO:1 or SEQ ID NO:2 that are adjacent to the core sequence, so long as the peptide is capable of modulating type 2 diabetes.
- a peptide comprises at least one sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30 and SEQ ID NO:32 so long as the peptide interacts with CD40 protein in such a manner as to modulate type 2 diabetes.
- a peptide hereof is a sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30 and SEQ ID NO:32.
- peptides of the present subject matter can be selected entirely of or from sequences that are responsible for the interaction of the peptide with a CD40 protein, they may additionally contain amino acid sequences that do not interact with a CD40 protein, but which have other useful functions. Any useful, additional amino acid sequence can be added to the CD40-interacting sequence, so long as the additional sequences do not have an unwanted effect on the ability of the CD40 interacting sequence to interact with a CD40 protein.
- a peptide hereof can contain amino acid sequences that are useful for visualizing or purifying the peptide. Such sequences act as labels (e.g., enzymes) or tags (antibody binding sites).
- a peptide hereof can contain amino acid sequences that are useful for visualizing or purifying the peptide.
- labels and tags include, but are not limited to, B-galactosidase, luciferase, glutathione-s-transferase, thioredoxin, HIS-tags, biotin tags, and fluorescent tags.
- acetyl groups and amides may be appended on the N-terminus or C-terminus, and the developments hereof contemplate these and other variations that may enhance stability or other traits desired of such a peptide.
- Other useful sequences for labeling and tagging proteins are known to those of skill in the art.
- peptides hereof can be modified, so long as such modification does not significantly affect the ability of the peptide to modulate type 2 diabetes.
- modifications can be made, for example, to increase the stability, solubility or absorbability of the protein. Examples of such modifications include, but are not limited to pegylation, glycosylation and chemical modification of the peptide.
- Peptides hereof may possibly be derived from nature (e.g., obtained from plants, animals or microorganisms) or they can be produced in a laboratory (e.g., recombinantly or synthetically). Preferred peptides are those that are synthesized. Also encompassed are peptides that are combinations of natural and synthetic molecules. General methods for producing and isolating recombinant or synthetic peptides are known to those skilled in the art. It should be noted that, as used herein, an isolated, or biologically pure, molecule, is one that has been removed from its natural milieu. As such the terms isolated, biologically pure, and the like, do not necessarily reflect the extent to which the protein has been purified.
- the peptides hereof do not arise naturally in a corporeal body, but rather must be constructed and synthesized to obtain the small interfering peptides. Certain aspects of the design and synthesis may affect the peptides stability and ability to perform its intended use.
- the peptides hereof may vary in length from four amino acids in length as in SEQ ID NO:3, five amino acids in length, six amino acids in length as in SEQ ID NOs: 4, 27, 28, 29, and 30, seven amino acids in length, eight amino acids in length as in SEQ ID NOs: 5 and 6, nine amino acids in length, ten amino acids in length as in SEQ ID NOs: 24, eleven amino acids in length, twelve amino acids in length, thirteen amino acids in length as in SEQ ID NO: 25, fourteen amino acids in length, fifteen amino acids in length as in SEQ ID NOs: 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, and 20, sixteen amino acids in length, seventeen amino acids in length, eighteen amino acids in length, nineteen amino acids in length, twenty amino acids in length, twenty-one amino acids in length, twenty-two amino acids in length, twenty-three amino acids in length, twenty-four amino acids in length as in SEQ ID NO: 26 and 32, and twenty-five amino acids in length.
- the embodiments of this development may be up to fifty or more amino acids in length and in such instances, repeats of the core sequence of SEQ ID NO: 3, may occur one, two, three, four, five, six, seven, eight, nine, ten, eleven or more times in such a peptide.
- the embodiments of this development may be up to fifty or more amino acids in length, and in such instances repeats of the core sequence of SEQ ID NO:3, may be varied with other sequences known to provide the desired effect such as those of SEQ ID NOS: 4-9, 25-30, and 32.
- the repeats and sequences of said variations are countless; however, the developments herein contemplate a peptide that maintains its ability to perform its intended use of interacting with CD40 in such a manner as change, control, or affect inflammation in the subject.
- one embodiment is a method to reduce the interaction between a CD40 protein and a CD154 protein comprising introducing into an environment containing a CD40 protein and a CD154 protein, a peptide, that interacts with the CD40 protein in such a manner as to reduce the interaction between the CD40 protein and the CD154 protein.
- the peptide reduces the interaction between the CD40 protein and the CD154 protein by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In one embodiment, the peptide reduces the interaction between the CD40 protein and the CD154 protein by a factor of at least 10, at least 100, at least 1,000, at least 10,000. Methods of measuring the strength of the interaction between the CD40 protein and the CD154 protein have been discussed previously, and are also know to those of skill in the art.
- One embodiment hereof is a method to modulate type 2 diabetes comprising contacting a CD40 protein with a peptide that interacts to the CD40 protein in such a manner as to modulate inflammation.
- interaction of the peptide with the CD40 protein increases the number of Th40 cells by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%.
- interaction of the peptide with the CD40 protein increases the number of Th40 cells by a factor of at least 10, at least 100, at least 1,000, at least 10,000.
- One aspect is a method to reduce type 2 diabetes in a patient, the method comprising administering a peptide hereof to the patient.
- the peptide comprises an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO:30 and SEQ ID NO:32.
- the peptide is an amino acid sequence selected from SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:27, SEQ ID NO: 28, and SEQ ID NO: 29.
- interaction of the peptide with the CD40 protein decreases the number of Th40 cells by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%.
- interaction of the peptide with the CD40 protein decreases the number of Th40 cells by a factor of at least 10, at least 100, at least 1,000, at least 10,000.
- the level of Th40 cells is reduced so that Th40 cells comprise no more than about 20%, about 25%, about 30%, about 35%, or about 40% of the total T-cell population.
- Peptides and methods hereof are suitable for use in cell culture as well as for treating a patient.
- patient refers to any animal in need of such treatment.
- the animal can be a human or a non-human animal.
- a preferred animal to treat is a mammal.
- a peptide can be administered or applied per se, or as pharmaceutical compositions.
- a peptide hereof, or a pharmaceutical composition thereof can be administered to a patient by a variety of routes, including, but limited to, by injection (e.g., intravenous, intramuscular, subcutaneous, intrathecal, intraperitoneal), by inhalation, by oral (e.g., in a pill, tablet, capsule, powder, syrup, solution, suspension, thin film, dispersion or emulsion.), transdermal, transmucosal, pulmonary, buccal, intranasal, sublingual, intracerebral, intravaginal rectal or topical administration or by any other convenient method known to those of skill in the art.
- routes including, but limited to, by injection (e.g., intravenous, intramuscular, subcutaneous, intrathecal, intraperitoneal), by inhalation, by oral (e.g., in a pill, tablet, capsule, powder, syrup, solution, suspension, thin film, dispersion or emulsion.), transdermal, transmucosal, pulmonary
- the amount of a peptide hereof and/or a pharmaceutical composition thereof that will be effective can be determined by standard clinical techniques known in the art. Such an amount is dependent on, among other factors, the patient being treated, including, but not limited to the weight, age, and condition of the patient, the intended effect of the compound, the manner of administration and the judgment of the prescribing physician. Also, in this context, it should be noted that in treating a patient exhibiting a disorder of interest, a therapeutically effective amount of an agent or agents such as these is administered.
- a therapeutically effective dose refers to that amount of the compound that results in amelioration of one or more symptoms or a prolongation of survival in a patient.
- a peptide hereof, or a pharmaceutical composition thereof can be administered alone or in combination with one or more other pharmaceutical agents, including other compounds of the present disclosure.
- the specific pharmaceutical composition depends on the desired mode of administration, as is well known to the skilled artisan.
- one embodiment hereof is a method to treat type 2 diabetes in a patient in need of such treatment, the method comprising administering to a patient a peptide that interacts with the CD40 protein, thereby reducing type 2 diabetes.
- the peptide interacts with the CD40 protein in such a manner as to affect the interaction of CD40 and CD154, thereby reducing type 2 diabetes.
- interaction of the peptide with the CD40 protein reduces the number of Th40 cells in a patient to a level equal to that observed in subjects that do not have type 2 diabetes.
- interaction of the peptide with the CD40 protein reduces the inflammatory cytokine levels in a patient. In another embodiment, interaction of the peptide with the CD40 protein reduces the inflammatory cytokine levels in a patient to a level equal or similar to those observed in subjects that do not have type 2 diabetes.
- the present developments are suitable for treating any patient having an autoimmune disease and/or cardiovascular disease, the development of which may be related to, correlated with, or dependent on Th40 cells.
- the present developments may also be suitable for treating any patient having an autoimmune disease and/or cardiovascular disease, the development of which is not related to, correlated with, or dependent on Th40 cell counts, levels, and or concentrations.
- peptides hereof may be suitable for reducing the level of Th40 cells in such patients.
- a peptide hereof may reduce the level of Th40 cells in a patient suffering from an autoimmune disease to no more than about 25% of the total T-cell population.
- Type 2 diabetes glucose tolerance is reduced, insulin sensitivity is decreased, and plasma insulin levels are increased. Consequently, control of inflammatory cells and cell signaling via CD40-CD154 interaction may be able to be used to control, modulate, reduce and/or reverse type 2 diabetes symptoms that are characterized as by glucose intolerance, insulin resistance, and increased plasma insulin levels.
- T2D and/or atherosclerosis Several murine models of T2D and/or atherosclerosis have been developed. For initial studies, ApoE ⁇ / ⁇ mice were selected due to their ability to develop type 2 diabetes from a high fat diet. Glucose tolerance and insulin testing were performed on all of the mice.
- mice were administered the 6-mer peptide (SEQ ID NO:29) at a rate of 1 mg/kg weekly via I.V. injection and monitored.
- Peptide treated ApoE deficient mice demonstrated significantly increased glucose tolerance as well as significantly improved insulin sensitivity, improve insulin resistance, and lowered plasma insulin levels compared to controls.
- the peptide may be selected from SEQ ID NOS: 3-9 and 24-30.
- one aspect of the present subject matter is a method to reverse type 2 diabetes comprising administering to a patient diagnosed as having type 2 diabetes, a peptide hereof.
- the peptide comprises an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, so long as the peptide can down-regulate or reduce inflammation.
- the peptide is an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30.
- the phrase to reverse type 2 diabetes means to increase glucose tolerance, decrease insulin resistance, and decrease plasma insulin levels to levels closer to or more comparable to those observed in individuals who do not have type 2 diabetes.
- one aspect of the present subject matter is a method to reverse type 2 diabetes comprising administering to a patient diagnosed as having type 2 diabetes, a peptide hereof.
- the peptide comprises an amino acid sequence selected from SEQ ID NOS: 3-9 and 24-30, so long as the peptide can control, modulate, reduce and/or reverse inflammation.
- the peptide is an amino acid sequence selected from SEQ ID NOS: 3-9 and 24-30, so long as the peptide can control, modulate, reduce and/or reverse type 2 diabetes.
- a peptide hereof may reduce the level of Th40 cells in a patient suffering from an autoimmune disease to no more than about 25% of the total T-cell population.
- interaction of the peptide with the CD40 protein reduces the number of Th40 cells in a patient to a level equal to that observed in subjects that do not have type 2 diabetes.
- interaction of the peptide with the CD40 protein reduces the inflammatory cytokine levels in a patient.
- interaction of the peptide with the CD40 protein reduces the inflammatory cytokine levels in a patient to a level equal or similar to those observed in subjects that do not have type 2 diabetes.
- the present developments may be suitable for treating any patient having an autoimmune disease and/or cardiovascular disease, the development of which may be related to, correlated with, or dependent on Th40 cells.
- the present developments may also be suitable for treating any patient having an autoimmune disease and/or cardiovascular disease, the development of which is not related to, correlated with, or dependent on Th40 cell counts, levels, and or concentrations.
- peptides of the present invention selectively bind to a CD40 expressing cell. Consequently, peptides of the present subject matter can be used to identify Th40 cells.
- a method to detect Th40-dependent type 2 diabetes comprising contacting a T-cell population with a peptide hereof.
- the peptide is labeled with a detectable marker, such as, for example, fluorescein, luciferase or alkaline phosphatase.
- a detectable marker such as, for example, fluorescein, luciferase or alkaline phosphatase.
- an assay for detecting Th40 cells using a peptide hereof comprises (a) obtaining a sample of cells; (b) contacting a peptide hereof with said cells under condition suitable to allow binding of the peptide to Th40 cells, if present; (c) washing said cells using conditions that disrupt non-specific interactions, and that remove unbound peptide; and (d) detecting peptide bound to cells.
- Detection of bound peptide can be achieved directly or indirectly. For example, direct detection can be achieved using a peptide labeled using a detectable marker, as disclosed herein. Following the wash step listed above, the cells are then simply screened for the presence of detectable marker.
- detectable marker in the cell sample indicates the presence of Th40 cells, and thus Th40-dependent type 2 diabetes.
- indirect detection involves the use of a second molecule, such as an antibody, that binds to the peptide.
- a detection molecule that binds the peptide is added to the cell sample.
- the detection molecule is labeled with a detectable marker. After washing away unbound detection molecule, the cells are screened for the presence of detectable marker.
- the presence of detectable marker in the cell sample indicates the presence of Th40 cells.
- Suitable assay techniques are known to those skilled in the art, and are also disclosed in, for example, Molecular Cloning: A Laboratory Manual, Sambrook, J., Fritsch, E. F., and Maniatis, T, Cold Spring Harbor Laboratory Press; 2nd Edition (December 1989). All references cited herein are incorporated herein in their entirety.
- the assay technology described above can also be used to identify other molecules that affect the interaction of a CD40 protein with a CD514 protein.
- examples of such molecules include, but are not limited to, proteins, peptides and small molecules.
- assays can be designed that test the ability of molecules to compete with a peptide of the present developments for binding to a Th40 cell.
- a peptide labeled with a detectable marker can be mixed with a test molecule and a population of cells known to contain Th40 cells, under conditions that allow binding of the peptide to the Th40 cells. Following an appropriate incubation period, the cells are washed to remove unbound peptide, and the cells screened for the presence of detectable marker.
- the labeled peptide could be bound to Th40 cells first, and after a wash step to remove unbound peptide, the test molecule could be added to the cells containing bound peptide. Following an incubating period and a wash step to remove unbound molecule, or released peptide, the cells are screened for the presence of detectable marker. In either case, absence of the detectable marker in the cell sample indicates the test molecule is able to compete with the peptide for binding to the Th40 cells, while presence of the detectable marker would indicate the test molecule does not inhibit binding of the peptide to Th40 cells. Inhibition of binding need not be 100%, as such assay would also be useful for identifying molecules that partially inhibit binding of the peptide to Th40 cells. It is understood by those skilled in the art that such assays would involve the use of positive controls (e.g., unlabeled peptide) and negative controls (e.g., a protein/molecule that is known not to bind to Th40 cells).
- positive controls e.g.
- the assay technology above can also be used to identify other molecules that affect the interaction of a CD40 protein with a CD154 proteins.
- examples of such molecules include, but are not limited to, proteins, peptides and small molecules.
- assays can be designed that test the ability of molecules to compete with a peptide of the present developments for binding to a CD40 protein of cells other than T cells, such as neutrophils, eosinophils, basophils, mast cells, macrophages, platelets, endothelial cells, and lymphocytes, including natural killer cells and B cells.
- a peptide labeled with a detectable marker can be mixed with a test molecule and a population of cells known to contain CD40 containing cells, under conditions that allow binding of the peptide to the CD40 bearing cells. Following an appropriate incubation period, the cells are washed to remove unbound peptide, and the cells screened for the presence of detectable marker.
- the labeled peptide could be bound to CD40 bearing cells first, and after a wash step to remove unbound peptide, the test molecule could be added to the cells containing bound peptide. Following an incubating period and a wash step to remove unbound molecule, or released peptide, the cells are screened for the presence of detectable marker.
- absence of the detectable marker in the cell sample indicates the test molecule is able to compete with the peptide for binding to the CD40 bearing cells, while presence of the detectable marker would indicate the test molecule does not inhibit binding of the peptide to CD40 bearing cells. Inhibition of binding need not be 100%, as such assay would also be useful for identifying molecules that partially inhibit binding of the peptide to CD40 bearing cells. It is understood by those skilled in the art that such assays would involve the use of positive controls (e.g., unlabeled peptide) and negative controls (e.g., a protein/molecule that is known not to bind to CD40 bearing cells).
- positive controls e.g., unlabeled peptide
- negative controls e.g., a protein/molecule that is known not to bind to CD40 bearing cells.
- one embodiment of the present developments is a method to identify a patient at risk for developing autoimmune related type 2 diabetes.
- patients at risk for developing type 2 diabetes are identified by obtaining a sample from a patient to be tested, contacting the T-cell portion of said sample with a peptide hereof, and determining the level of Th40 cells present in the sample, wherein a level of Th40 cells greater than about 25% of the total T-cell population indicates the patient is at risk for developing type 2 diabetes.
- the peptide comprises an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, so long as the peptide binds to the CD40 protein.
- the peptide is an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30.
- the peptide is labeled with a suitable detectable marker such as, for example, fluorescein, luciferase or alkaline phosphatase.
- a suitable detectable marker such as, for example, fluorescein, luciferase or alkaline phosphatase.
- the peptide comprises an amino acid sequence selected from SEQ ID NOs: 4-9, 24-30, and 32, so long as the peptide binds to the CD40 protein.
- kits useful for practicing the methods disclosed herein comprising a peptide that interacts with a CD40 protein in such a manner as to modulate, reduce, prevent, treat, or otherwise improve symptoms of type 2 diabetes.
- the peptide comprises an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, so long as the peptide can modulate type 2 diabetes.
- the peptide is an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30.
- Kits can also contain associated reagents and components, such as, but not limited to, buffers, labels, containers, inserts, tubing, vials, syringes, and the like.
- kits useful for practicing the methods disclosed herein comprising a peptide that interacts with a CD40 protein in such a manner as to modulate, reduce, prevent, treat, or otherwise improve symptoms of type 2 diabetes.
- the peptide comprises an amino acid sequence selected from SEQ ID NOs: 3-9 and 24-30, so long as the peptide can modulate type 2 diabetes.
- the peptide is an amino acid sequence selected from SEQ ID NOs: 3-9 and 24-30.
- kits for determining the level of Th40 cells the kit comprising a peptide that interacts with a CD40 protein, and methods for detecting CD40-bound peptide. Kits can also contain associated reagents and components, such as, but not limited to, buffers, labels, containers, inserts, tubing, vials, syringes, and the like.
- This Example demonstrates the effect of various peptide fragments of CD154 on CD4/CD8 ratios and the development of diabetes in NOD mice.
- the peptides were designed based on the amino acid sequence of mouse CD154 protein (SEQ ID NO:1) in the SwissPro database.
- the peptides (8-mer (SEQ ID NO: 5; SEQ ID NO: 6), 10-mer (SEQ ID NO:24), 13-mer (SEQ ID NO:25), 15-mer (SEQ ID NO: 7), 24-mer (SEQ ID NO:26), scrambled (SEQ ID NO: 23), and RGD (arginylglycylaspartic acid) were then ordered from New England Peptide.
- the RGD peptide is a 15-amino acid sequence from the CD154 sequence that does not include the CD40 binding motif.
- the lyophilized peptides were suspended in sterile saline at 1 mg/ml.
- mice 25 ug in 100 ul (1 mg/kg) of a particular peptide was then injected into the tail vein of 6-week old NOD mice.
- Control mice received 100 ul of sterile saline. This is well before the onset of diabetes (and atherosclerosis), but after damage to pancreatic islets has begun.
- Weekly after the initial injection another 25 ug of peptide (or 100 ul of saline in the case of the Control mice) was injected into the tail vein.
- mice were monitored for diabetes, as indicated by a blood glucose level greater than 250 mg/dL for three consecutive days. The results of this study are shown in FIG. 1 .
- treatment of mice with a 15-mer peptide derived from the CD154 protein prevented the onset of diabetes.
- the 13-mer peptides derived from the CD154 protein had significant effects on the development of diabetes.
- the data demonstrates that the 15-mer peptide did not result in compromise of the immune system, as determined by the CD4/CD8 ratio.
- This Example demonstrates the effect of the 15-mer peptide on hyperglycemia in newly diabetic NOD mice.
- mice from Example 1 Six mice from Example 1 that were not treated were allowed to subsequently develop diabetes. These mice were injected intravenously with 100 ug of the 15-mer peptide. These mice were then given weekly injections of the 15-mer peptide into their tail veins, and their blood glucose levels monitored twice-weekly. The 15-mer peptide was administered for a total of ten weeks, after which the treatment was stopped. The results of this study are shown in FIG. 3 .
- Total lymphocytes were isolated from 9 week old NOD mice. The lymphocytes were incubated with anti-CD4, anti-CD8, anti-CD40 and an FITC-labeled 15-mer peptide, and then analyzed by flow cytometry. Cells were gated for CD4 (both CD4hi and CD4lo populations were included) and CD40 versus the 15-mer peptide. The results of this analysis are shown in FIG. 4 .
- B cells were isolated from the spleens of NOD mice. Sorted MHC-II+ cells were purified from total lymphocytes. Cells were stained with FITC-labeled 15 mer peptide, anti-CD40, and B cell markers CD19 and CD21. MHC-II+ cells were gated for CD19+ and CD21+ and then 15-mer peptide versus CD40 antibody was measured. The results of this study are shown in FIG. 5 .
- This example demonstrates the level of CD40 positive cells in the blood of type-I diabetic subjects and non-diabetic (control) subjects.
- This example demonstrates the level of insulin granulation observed in the pancreas of NOD mice treated with either the 15-mer peptide or a peptide from ovalbumin.
- mice At the onset of diabetes, six NOD mice were injected with 100 ug/ml of the 15-mer peptide, resulting in the reversal of hyperglycemia in 80% of the recipients. Six weeks after reversal of hyperglycemia, mice were sacrificed, and the pancreas removed for analysis. The pancreas was fixed, sectioned and then stained using an aldehyde/fuschsin stain that allows detection of insulin granules.
- FIG. 8 provides results related to this Example 6.
- Peptide were designed and produced as described in Example 1. Variant peptides were produced so that in each variant, a glycine was substituted for an amino acid corresponding to an amino acid in positions 1-7 or 9-12 of SEQ ID NO:7, as follows:
- Gly-1 (SEQ ID NO: 11) G-L-Q-W-A-K-K-G-Y-Y-T-M-K-S-N Gly-2 (SEQ ID NO: 12) V-G-Q-W-A-K-K-G-Y-Y-T-M-K-S-N Gly-3 (SEQ ID NO: 13) V-L-G-W-A-K-K-G-Y-Y-T-M-K-S-N Gly-4 (SEQ ID NO: 14) V-L-Q-G-A-K-K-G-Y-Y-T-M-K-S-N Gly-5 (SEQ ID NO: 15) V-L-Q-W-G-K-K-G-Y-Y-T-M-K-S-N Gly-6 (SEQ ID NO: 16) V-L-Q-W-A-G-K-G-Y-Y-T-M-K-S-N Gly-7 (SEQ ID NO: 17) V-L
- NOD mice were placed in groups of 10, and the mice in each group injected IV weekly with 25 ug of either wild-type (WT; Legend) peptide or a variant peptide (in PBS, ph 7.2) listed above.
- WT wild-type
- PBS PBS, ph 7.2
- T1D human Type 1 Diabetes
- ApoE deficient mice on a normal chow diet were selected to receive a dose of 1 mg/kg of the 15-mer peptide (SEQ ID NO: 7) by IV tail injection, three times a week over a period of 26 weeks, beginning at 9 weeks of age and also utilized a control.
- the animals were euthanized, weighed, and then had blood, spleen, and pancreas removed for analysis.
- the subjects were then perfused through cardiac puncture with 4% paraformaldehyde.
- Aortic arches were dissected, dehydrated in sucrose gradient and then flash frozen. Approximately thirty-five 8 um longitudinal sections were obtained per mouse for various staining procedures.
- Flow cytometry was performed utilizing a MACSQuant® Analyzer 10 (Miltenyi Biotec Inc.). Additional analysis was performed using FlowJo® (FlowJo, LLC wholly owned by BectonDickinson, Inc.) single-cell flow cytometry software.
- C57BL/6 and ApoE ⁇ / ⁇ mice demonstrated increased levels of Th40 cells relative to all CD3+CD4+ cells prior to hyperglycemia as demonstrated in FIG. 12 .
- NOD mice tested in this study demonstrated significant Th40 infiltration in the aorta compared with control and young non-diabetic NOD mice populations, as shown in FIG. 13 .
- FIG. 14 An exemplar aortic plaque of one of the longitudinal sections was observed at 200 ⁇ magnification using oil-red-0, trichrome stain and immune-fluorescence, and is shown in FIG. 14 .
- This microscopy and stain of the aorta showed that not only are the Th40 cells increased in the aorta similarly to the peripheral blood as demonstrated in FIGS. 10-14 , but also the Th40 cells are found within the shoulder region of plaque in the ApoE ⁇ / ⁇ model (the growth region of plaque/atherosclerosis).
- 10 and 20 identify cells that represent CD3+, CD4+, and CD40+ (Th40 cells) that have significant intracellular CD40.
- 30 demonstrates Th40 cell with extracellular expression and no demonstrative CD40 intracellularly.
- 40 identifies CD3+, CD4+, CD40neg cell.
- CD3+CD4+CD40+ cells appear to produce interferon gamma (INF ⁇ ) in abundance. Additionally, interferon gamma controls Th40 proliferation.
- ApoE deficient mice on a normal chow diet were selected to receive a dose of 1 mg/kg of the 15-mer peptide (SEQ ID NO: 7) by IV tail injection, three times a week over a period of 26 weeks, beginning at 9 weeks of age and also utilized a control.
- the animals were euthanized, weighed, and then had blood, spleen, and pancreas removed for analysis.
- the subjects were then perfused through cardiac puncture with 4% paraformaldehyde.
- Aortic arches were dissected, dehydrated in sucrose gradient and then flash frozen. Approximately thirty-five 8 m longitudinal sections were obtained per mouse for various staining procedures.
- Flow cytometry was performed utilizing a MACSQuant® Analyzer 10 (Miltenyi Biotec Inc.). Additional analysis was performed using FlowJo® (FlowJo, LLC wholly owned by BectonDickinson, Inc.) single-cell flow cytometry software.
- CD40 can be internal or external to the CD3+CD4+ cell.
- Flow cytometry was further performed and demonstrated that while most CD3+ cells appear to have ability to produce CD40, the CD3+CD4+CD40+ cells appear to produce interferon gamma (IFN ⁇ ) in abundance.
- IFN ⁇ interferon gamma
- FIG. 16 demonstrates that interferon gamma controls Th40 proliferation.
- Isolated Th40 cells were cross-linked by antibody to CD40.
- the graph in FIG. 16 denotes proliferation of CD40 stimulated (CD40XL: activated) Th40 cells in the absence/presence of antibody to INF ⁇ ( ⁇ IFN ⁇ ) and non-stimulated controls (UN). Additionally, by blocking IFN ⁇ , activated Th40 cells do not proliferate.
- ApoE deficient mice on a normal chow diet were selected to receive a dose of 1 mg/kg of the 15-mer peptide (SEQ ID NO: 7) by IV tail injection, three times a week over a period of 26 weeks, beginning at 9 weeks of age and also utilized a control.
- the animals were euthanized, weighed, and then had blood, spleen, and pancreas removed for analysis.
- the subjects were then perfused through cardiac puncture with 4% paraformaldehyde. Aortic arches were dissected, dehydrated in sucrose gradient and then flash frozen.
- FIG. 17 provides an example of the lesser curvature of the aortic arch, defined proximally from the aortic outflow (AO).
- AO aortic outflow
- FIG. 18 demonstrates the lesser curvature of the aortic arch of the control ApoE mice compared to the lesser curvature of the aortic arch of mice treated with the 15-mer peptide, in accordance with the steps outlined in this example.
- FIG. 19 demonstrates the reduction of the total area achieved by peptide treatment.
- the total area of the 2.5 mm segment (as described in FIG. 17 ) was substantially reduced.
- FIG. 20 demonstrates the reduction of number of plaque, including early lesions and advanced plaque.
- the total number of plaque was significantly decreased in the treatment group. Plaque was subdivided based on morphology of early lesions (observable as fatty streaks containing macrophage derived foam cells with varying degrees of lipid accumulation) compared with more advanced fibroatheromas (containing varying degrees of lipid or necrotic core and fibrous caps). All plaque within the designated 2.5 mm segment were included. Both the total number and type of plaque were significantly decreased in those subjects treated with the peptide.
- This example demonstrates administration of the KGYY 15 (15-mer—SEQ ID NO:7) augments cap formation while reducing advancement of existing disease.
- mice received a normal chow diet from 0 to 20 weeks of age. At 20 weeks of age, three mice were randomly assigned to receive dose of 1 mg/kg of KGYY 15 (15-mer—SEQ ID NO:7) by IV tail injection, once a week for a period of 4 weeks. Control mice received vehicle only. After 4 weeks of treatment, animals were euthanized then perfused through cardiac puncture with 4% paraformaldehyde. Aortic arches were dissected in surcrose gradient and flash frozen. Approximately fifty, 8 um longitudinal sections were obtained per mouse. Slides were treated with trichrome stain and analyzed using cellSens software for measurements. Total plaque was measured including 2.5 mm lesser curvature and innominate artery.
- FIG. 20 shows the number of individual early plaques and advanced plaques were reduced in the treated subjects compared to the control subjects.
- FIG. 21 shows that the cap to core ratio of advanced plaques was reduced in the treated subjects compared to the control subjects.
- FIG. 22 shows the average cap width (cap size) was greater in the treated subjects compared to the control subjects.
- FIG. 23 shows the average core width (core size/plaque size) was decreased in the subjects treated with the peptide compared to the control subjects.
- TID shares with atherosclerosis the CD40-CD154 dyad which drives autoimmune inflammation.
- Th40 cell levels in peripheral blood of NOD mice, human TID patients, and ApoE ⁇ / ⁇ mice.
- Th40 cells infiltrate the aortic wall and are found within the plaque of ApoE ⁇ / ⁇ mice.
- Th40 cells produce the inflammatory cytokine IFN ⁇ at a level greater than that of other cells and this drives inflammation.
- the KGYY 15 peptide targets Th40 cells.
- the specified peptide furthermore abrogates and modulates atherosclerosis which may be due to Th40 blockade or general blockade of CD40.
- the administration of the specified peptide trends toward more stable plaque types.
- FIG. 24 provides the results of clot studies observed in humans.
- mice received a normal chow diet from 0 to 20 weeks of age. At 20 weeks of age, three mice were randomly assigned to receive dose of 1 mg/kg of KGYY 6 (6-mer—SEQ ID NO:29) by IV tail injection, once a week for a period of 4 weeks. Control mice received vehicle only.
- FIG. 28A is an image of KGYY6 treated aortic en-face Sudan IV staining
- FIG. 28B is an image of control (untreated) aortic en-face Sudan staining
- FIG. 29 is a graph demonstrating the reduction of lesion areas of Sudan IV staining.
- FIG. 31 characterization of plaque composition or aortic samples was performed using trichrome staining techniques and these results are presented in graph format in FIG. 31 . Indeed, this FIG. 31 data, which was generated using Image Pro Plus software analysis, quantifies and shows that plaque compositional changes occurred, including increased collagen and reduced smooth muscle content.
- FIG. 32A is an image of trichrome stained cells of the cross-sections of the aorta of the KGYY 6 treated subject.
- FIG. 32B is an image of trichrome stained cells of the cross-sections of the control subjects.
- 50 identifies areas characteristic of plaque formation.
- 60 identifies aortic valve leaflets.
- the control (untreated) 50 areas characteristic of plaque are greater than those in the subjects treated with the KGYY 6 peptide.
- ApoE ⁇ / ⁇ mice were fed a 60% high fat diet (research diet) for 1 week. A select population of mice were then injected with 6-mer peptide at a dose of 1 mg/kg and others were untreated and tracked as controls. Glucose tolerance testing (GTT) was performed by fasting 6 hours, followed by intraperitoneal injection of 1 g/kg body weight glucose in water. Both blood glucose and blood serum insulin were measured at 0, 15 minutes, 30 minutes, 60 minutes, and 2 hours. The results of this study are shown in FIGS. 33( a ) and 33( b ) . Peptide treated ApoE deficient mice demonstrated significantly increased glucose tolerance as well as significantly improved insulin sensitivity and lowered plasma insulin levels compared to control.
- This example demonstrates administration of the KGYY 6 (6-mer—SEQ ID NO:29) affects the inflammatory cytokine production of IL2, INF ⁇ , and IL17a.
- Spleens from ApoE mice and C57BL/6 mice were processed for lymphocytes.
- ApoE ⁇ / ⁇ and C57BL/6 mice were fed a 60% high fat diet (research diet) for 1 week.
- Splenic lymphocytes were treated with 6-mer in vitro for 24 hours. Cells were placed in media overnight in the presence of varying concentrations of 6-mer peptides. The following morning, Brefeldin A was administered for 4 hours. All cells were stained for CD3, CD4, CD40 (Th40 cells) and measured by use of flow cytometry for their production of IL2, INF ⁇ , and IL17a. The results of this study are shown in FIG. 35 .
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Cell Biology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Description
- This application contains a Sequence Listing submitted as an electronic text file entitled “US_16/240,630_Sequence_Listing.txt” having a size in bytes of 11 kb and created May 1, 2019. The information contained in this electronic file is hereby incorporated by reference in its entirety pursuant to 37 CFR 1.52(e)(5).
- The present disclosure relates to methods or uses for prevention, modulation, and reduction of cardiovascular disease and/or atherosclerosis in a subject in need of a therapeutically effective amount of a peptide that inhibits the interaction of CD40 and CD154, and the use of such compounds in modulating T-cell activity and in treating disease. Furthermore, the present disclosure relates to methods of preventing, modulating, reducing, treating and/or reversing of
type 2 diabetes mellitus and/or auto-inflammatory disease, via administration of a therapeutically effective amount of a CD40-binding peptide that inhibits, influences, disrupts, blocks, and/or changes the interaction of CD40 and CD154 are disclosed. - Autoimmune diseases are conditions arising from an abnormal immune response to a normal body part. More than 80 diseases occur because of the immune system attacking the body's own organs, tissues, and cells.
Type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosus, and inflammatory bowel disease are common autoimmune diseases that affect a wide range of people across entire populations. Significantly, the autoimmune disorders mentioned above afflict substantial number of people affecting their daily lives and routines and require significant monetary and healthcare resources, time and care from healthcare providers. - According to the World Health Organization (WHO), an estimated 17.7 million people died from cardiovascular diseases (CVDs) in 2015, which represented 31% of all global deaths. CVDs, may also be known as heart and blood vessel disease, and includes numerous problems, many of which are related to a process called atherosclerosis. Moreover, according to the Healthcare Cost and Utilization Project (HCUP) which is sponsored by the Agency for Healthcare Research and Quality (AHRQ), in 2011, coronary atherosclerosis alone accounted for more than $10.4 billion in hospital costs. Accordingly, in 2011, coronary atherosclerosis alone was one of the ten most expensive conditions for inpatient hospitalizations in the United States.
- Cardiovascular Disease and Atherosclerosis
- Atherosclerosis is defined by arterial plaque formation that may lead to heart attack and stroke. Arterial plaque formation is caused by the deposition of cells, substances, waste products, and cellular debris including, but not limited to: cholesterol, dead cells, dendritic cells, foam cells, macrophages, mast cells, monocytes, smooth muscle cells, T-cells, collagen, calcium, and fibrin. Inflammatory changes within the arterial wall and plaque may play a crucial and causative role in atherosclerotic disease development. Consequently, the concept of atherosclerosis as an autoimmune and inflammatory disease has been investigated; however, a therapeutic control has not been established. The importance of controlling inflammation is highlighted by current clinical trials targeting other aspects of autoimmune, inflammation, and cardiovascular disease and death.
- For example, CIRT (Cardiovascular Inflammation Reduction Trial) is attempting to use methotrexate to target interleukin-6 (IL-6) to test whether methotrexate will reduce rates of myocardial infarction, stroke, and cardiovascular death among patients with coronary artery disease patients with
type 2 diabetes. Another example includes, CANTOS (Canakinumab Anti-inflammatory Thrombosis Outcomes Study) which is studying whether canakinumab can block the pro-inflammatory cytokine interleukin-1β (IL-1β) to reduce rates of recurrent myocardial infarction, stroke, and cardiovascular death rates in heart attack patients who remain at a high risk. This risk is demarcated by elevated levels of the inflammatory biomarker high sensitivity C-reactive protein (hsCRP). These studies acknowledge that inflammation plays a critical role in atherothrombosis and atherosclerosis; however, these studies also recognize that is unknown whether inhibition of inflammation per se will lower vascular event rates. - Mammalian and human atherosclerotic lesions are characterized as a chronic inflammatory-fibroproliferative disease of the blood vessel wall containing monocytes, macrophages, endothelial cells, smooth muscle cells, platelets, and T-cells. Each of these cell types can express either or both of the CD40/CD154 costimulatory pair. This dyad is responsible for enhancing the immune response and may contribute to many chronic inflammatory diseases including rheumatoid arthritis, multiple sclerosis, and
type 1 diabetes (T1D). However, no viable therapy exists for this highly atherogenic dyad. - Inflammation may occur when inflammatory cells, such as neutrophils, eosinophils, basophils, mast cells, macrophages, platelets, and endothelial cells, respond to inflammatory events or harmful stimuli, such as, invading microorganisms, damages cells, or other irritants. The body's inflammatory response is beneficial because for example, in the case of invading microorganisms, the inflammatory response is an important step in localizing the infecting agent for removal by the immune system. However, in autoimmunity there is no infection, yet severe inflammation is present or persistent. The inflammation in this case, referred to as aseptic chronic inflammation (ACI), is detrimental since it destroys normal tissues. The results of this aseptic inflammation are life-altering and in some cases life-threatening. Moreover, as with acute inflammation, this process is mediated by immune cells, including T-cells.
- A major concern for modern medicine is how to control ACI such as that which occurs during autoimmune diseases, as well as how to control acute inflammation resulting from trauma. Inflammation, both chronic and acute, leads to tissue degeneration and eventual loss of function of major organs. ACI is not limited to a single disease, but is instrumental in numerous autoimmune diseases, including, but not limited to:
type 1 diabetes (T1D), multiple sclerosis (MS), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Crohn's disease, inflammatory bowel disease (IBS), chronic obstructive pulmonary disease (COPD) including types of autoimmune asthma, atherosclerosis, vasculitis, hypertension, thyroiditis including Hashimoto's and Graves diseases, primary biliary cirrhosis, Paget's disease, Addison's disease, acute respiratory distress syndrome (ARDS), acute lung injury, and aseptic chronic inflammation (ACI) associated with organ transplantation. - Autoimmune disorders are classified into two types: organ-specific (directed mainly at one organ) and non-organ-specific (widely spread throughout the body). Examples of organ-specific autoimmune disorders are insulin-
dependent Type 1 diabetes (T1D) which affects the pancreas; Hashimoto's thyroiditis and Graves' disease, which affect the thyroid gland; pernicious anemia, which affects the blood; Addison's disease, which affects the adrenal glands; chronic active hepatitis, which affects the liver; myasthenia gravis which affects the muscle; and multiple sclerosis (MS), which affects tissue of the nervous system. An example of a non-organ-specific autoimmune disorders is rheumatoid arthritis (RA). Autoimmune diseases are often chronic, debilitating, and life-threatening. The National Institutes of Health (NIH) estimates that up to 23.5 million Americans suffer from autoimmune disease and that the prevalence is rising. It has been estimated that autoimmune diseases are among the ten leading causes of death among women in all age groups up to 65 years. - Acute inflammation, as observed during trauma or sepsis, is also immune cell mediated. While a comprehensive, complete, and exhaustive list of the molecular mediators in this process have not yet been identified, a prominent role for T-cells, lymphocytes, neutrophils, macrophages, monocytes, neutrophils, eosinophils, basophils, mast cells, and other inflammatory cells is strongly implicated. Therefore, a process to modulate these cell types may control the inflammatory response.
-
Type 2 Diabetes - The CD40-CD154 dyad constitutes a major inflammatory pathway (Schonbeck U, et al. Cell Mol. Life Sci. (2001) 58(1):4-43) that plays a significant role in
type 1 diabetes (Waid D M. et al. Clin. Immunol. (2007) 124(2):138-148).Type 2 diabetes (T2D) has historically and scientifically been primarily categorized as a metabolic disorder; however,type 2 diabetes is in the process of being redefined as an autoimmune disease rather than just a metabolic disorder (Winer, D, et al. Nature Medicine (2011) 17:610-617). - According to the United States Centers for Disease Control National Diabetes Statistics Report for 2017 (available at https://www.cdc.gov/diabetes/pdfs/data/statistics/national-diabetes-statistics-report.pdf), an estimated 30.3 million people of all ages—or 9.4% of the U.S. population had diabetes in 2015. An estimated 23 million people—or 7.2% of the U.S. population had been diagnosed with diabetes mellitus, and about 95% of those diagnosed with diabetes have
type 2 diabetes. Based on fasting plasma glucose levels, one third to one half of cases oftype 2 diabetes are undiagnosed and untreated (Harris M I, Diabetes Care. (1998) 21 [Suppl. 3: C11-C14]; Howard B V, et al., Circulation. 2002; 105:e132-e137; Grundy S M, et al., Circulation. 1999; 100: 1134-1146). - Although
type 2 diabetes most often develops in people over the age of 45,type 2 diabetes increased 21% in American youth from 2001 to 2009 and a large study called SEARCH for Diabetes in Youth found that newly diagnosed cases ofType 2 diabetes in children and teens increased by about 4.8 percent in each year of the study's period between 2002 and 2012 (“Rates of new diagnosed cases oftype 1 andtype 2 diabetes on the rise among children, teens” National Institutes of Health, Apr. 13, 2017, available at https://www.nih.gov/news-events/news-releases/rates-new-diagnosed-cases-type-1-type-2-diabetes-rise-among-children-teens). - Major comorbidities complicate diabetes, the most common being cardiovascular disease (70.4 per 1,000 persons) including those with ischemic heart disease and stroke. Overall, for the year 2012, the American Diabetes Association estimates that the total direct and indirect estimated cost in the United States was $245 billion, including $176 billion in direct medical costs and $69 billion in reduced productivity (Yang, W. American Diabetes Association, 2013, Diabetes Care, 36 (4): 1033-46).
- Both
type 1 andtype 2 diabetes are powerful and independent risk factors for coronary artery disease (CAD), stroke, and peripheral arterial disease (Schwartz C J, et al. Diabetes Care. (1992) 15:1156-1167; Stamler, J. et al. Diabetes Care. (1993) 16:434-444; Beckman J A, et al., Diabetes and atherosclerosis: epidemiology, pathophysiology, and management. JAMA (2002) 287:2570-2581). Atherothrombosis accounts for 65% to 80% of all deaths among North American patients with diabetes, compared with about 33% of all deaths in the general North American population (American Diabetes Association. Diabetes Care. (1993) 16:72-78). Therefore, a therapeutic regimen that is effective and can be tolerated for long periods of time would be beneficial to individuals and from a public health perspective. An ideal anti-diabetic agent may be an agent which corrects hyperglycemia, prevents macrovascular complications, and corrects the pathophysiological disturbances responsible forType 2 Diabetes (“T2D”). Insulin resistance is basic to T2D, but β-cell failure eventually occurs with imbalance between insulin resistance and insulin secretion being a further complication. Therefore, therapeutically beneficial treatment approaches may aim to reverse insulin resistance and improve β-cell function. - T2D often conincides with obesity however genetic and environmental factors recently have been described as disease contributors (Comuzzie A G, Best Pract. Res. Clin. Endocrinol. Metab. (2002) 16(4):611-21. PubMed PMID: 12468410; van Tilberg J., et al. J. Med. Genet. (2001) 38(9):569-78. Pub Med PMID: 11546824; PMCID: PMC 1734947). Moreover, additional research has emerged that indicates that T2D, like T1D, has prominent inflammation component that is a contributing and/or driving factor of the T2D disease commencement, development and progression. The CD40-CD154 inflammatory dyad may act as a molecular driver to propel auto-immune inflammation and influence excessive levels of the dyad may be an unappreciated but contributing factor to T2D. (Hseih C J, et al., Cir. J. (2009) 73(5) 948-54; Kutlu M, et al., Clin. Invest. Med. (2009) 32(6): E244; Santilli F, et al., J. Am. Coll. Cardiol. (2006) 47(2):391-7; Santini, E, et al. J. Endocrinol Invest. 2008; 31(7):660-5; Varo N, et al., Circulation. 2003; 107(21):2664-9).
- Generally, inflammation may occur when inflammatory cells, such as neutrophils, eosinophils, basophils, mast cells, macrophages, platelets, endothelial cells, and lymphocytes, including but not limited to T cells and B cells respond to inflammatory events or harmful stimuli, such as, invading microorganisms, damaged cells, or other irritants. The body's inflammatory response is beneficial because, for example, in the case of invading microorganisms, the inflammatory response is an important step in localizing the infecting agent for removal by the immune system. However, in autoimmunity there is no infection, yet severe inflammation is present or persistent. The inflammation in this case, referred to as aseptic chronic inflammation (ACI), is detrimental since it destroys normal tissues. The results of this aseptic inflammation are life-altering and in some cases life-threatening. Moreover, as with acute inflammation, this process is mediated by immune cells, including T-cells.
- A major concern for modern medicine is how to control ACI such as that which occurs during autoimmune diseases, as well as how to control acute inflammation resulting from trauma. Inflammation, both chronic and acute, leads to tissue degeneration and eventual loss of function of major organs. ACI is not limited to a single disease, but is instrumental in numerous autoimmune diseases, including, but not limited to: type 1 diabetes (T1D), multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, Crohn's disease, inflammatory bowel disease, chronic obstructive pulmonary disease including types of autoimmune asthma, atherosclerosis, vasculitis, hypertension, thyroiditis including Hashimoto's and Graves diseases, primary biliary cirrhosis, Paget's disease, Addison's disease, acute respiratory distress syndrome, acute lung injury, and ACI associated with organ transplantation.
- Autoimmune disorders are classified into two types: organ-specific (directed mainly at one organ) and non-organ-specific (widely spread throughout the body). Examples of organ-specific autoimmune disorders are insulin-
dependent Type 1 diabetes (T1D) which affects the pancreas; Hashimoto's thyroiditis and Graves' disease, which affect the thyroid gland; pernicious anemia, which affects the blood; Addison's disease, which affects the adrenal glands; chronic active hepatitis, which affects the liver; myasthenia gravis which affects the receptors at the junction between nerves and muscles; and multiple sclerosis, which affects tissue of the nervous system. An example of a non-organ-specific autoimmune disorders is rheumatoid arthritis. Autoimmune diseases are often chronic, debilitating, and life-threatening. The National Institutes of Health (NIH) estimates that up to 23.5 million Americans suffer from autoimmune disease and that the prevalence is rising. It has been estimated that autoimmune diseases are among the ten leading causes of death among women in all age groups up to 65 years. - Acute inflammation, as observed during trauma or sepsis, is also immune cell mediated. While a comprehensive, complete, and exhaustive list of the molecular mediators in this process have not yet been identified, a prominent role for T-cells, lymphocytes, neutrophils, macrophages, monocytes, neutrophils, eosinophils, basophils, mast cells, and other inflammatory cells is strongly implicated. Therefore, a process to modulate these cell types may control the inflammatory response.
- A unique T cell subset has been shown to be instrumental in the development of autoimmune disease. These cells are phenotypically characterized as CD4loCD40+ (Waid, D. M., et al., Eur. J. of Immunol., 34:1488, 2004; Vaitaitis, G. M., et al., Cutting Edge, J. Immunol., 170:3455, 2003; Wagner, D. H., Jr., et al., Proc. Nat'l. Acad. Sci. USA, 99:3782, 2002; Wagner, D. H., Jr., et al., Int'l J. of Mol. Med. 4:231, 1999), and are referred to as Th40 cells. (Waid, D. M., et al. (2004) Eur. J. of Immunol. 34:1488; Vaitaitis, G. M., et al., Cutting Edge, J. Immunol. 170:3455, 2003; Wagner, D. H., Jr., et al., Proc. Nat'l Acad. Sci. USA 99:3782, 2002; Wagner, D. H., Jr., et al., Int'l J. of Mol. Med. 4:231, 1999). CD40 expression typically is associated with antigen presenting cells and the majority of prior art describes CD40 as being expressed on B cells, macrophages, monocytes, and other cells; however, CD40 proteins are also expressed on T cells (Waid, D. M., et al., 2004. Eur. J. of Immunol., 34:1488, 2004; Vaitaitis, G. M., et al., Cutting Edge, J. Immunol., 170:3455, 2003; Wagner, D. H., Jr., et al., Proc. Nat'l Acad. Sci. USA, 99:3782, 2002; Wagner, D. H., et al., Int'l. J. of Mol. Med., 4:231, 1999; Bourgeois, C., et al., Science, 297:2060, 2002; Fanslow, W. C., et al., J. of Immun., 152:4262, 1994; Ramsdell, F., et al., J. of Immunol. 152:2190, 1994; Grabstein, K. H., et al., J. of Immunol., 150:3141, 1993; Armitage, R. J., et al., Sem. in Immun., 5:401, 1993; Cooper, C. J., et al., J of Immunol., 173:6532, 2004). While Th40 cells comprise a proportion of the peripheral CD4+ compartment in naïve, non-autoimmune mice (Waid, D. M., et al., Eur. J. of Immunol., 34:1488, 2004; Wagner, D. H., Jr., et al., Int'l J. of Mol. Med., 4:231, 1999), and in humans (Waid. D. M., et al., Clin. Immunol. 124:138, 2007), this proportion is drastically expanded to as much as 50% of the CD4+ compartment in autoimmune prone mice (Waid, D. M., et al., Eur. J. of Immunol. 34:1488, 2004; Wagner, D. H., Jr., et al., Proc. Nat'l Acad. Sci. USA 99:3782, 2002; Wagner, D. H., et al., Int'l J. of Mol. Med., 4:231, 1999) and humans (Waid, D. M., et al., Eur. J. of Immunol. 34:1488, 2004; Waid. D. M., et al., Clin. Immunol. 124:138, 2007; Waid. D. M., et al., Clin. Immunol. 124:138, 2007). These T cells do not express early activation markers and occur in the naïve phenotype of non-challenged mice.
- In NOD (non-obese diabetic) mice, Th40 cells occur at exaggerated levels in spleen, lymph nodes and the pancreas, even prior to diabetes onset (Waid, D. M., et al., Eur. J. of Immunol. 34:1488, 2004; Wagner, D. H., Jr., et al., Proc. Nat'l Acad. Sci. USA 99:3782, 2002). An elevated number and percentage of these T cells are seen in peripheral blood of
type 1 diabetic (T1D) patients when compared to non-autoimmune controls andtype 2 diabetic patients (Waid. D. M., et al., Clin. Immunol., 124:138, 2007). - The observed increase in Th40 cells could mean that those T cells are antigen responsive or that CD40 expression is activation induced. Furthermore, several diabetogenic T cell clones are CD40+ (Wagner, D. H., Jr., et al., Proc. Nat'l Acad. Sci. USA 99:3782, 2002). Purified primary Th40 cells from NOD mice and from pre-diabetic NOD (12-weeks of age) mice successfully transfer
type 1 diabetes to NOD/scid (Non-Obese Diabetic/Severe Combined Immunodeficiency) recipient mice, directly demonstrating pathogenicity of the Th40 T cell subset (Waid, D. M., et al., Eur. J. of Immunol. 34:1488, 2004; Wagner, D. H., Jr., et al., 2002. Proc. Nat'l Acad. Sci. USA, 99:3782, 2002). It has been shown that Th40 cells infiltrate islet beta cells destroying insulin production thus suggesting islet antigen specificity (Waid, D. M., et al., Eur. J. of Immunol. 34:1488, 2004; Wagner, D. H., Jr., et al., Proc. Nat'l Acad. Sci. USA 99:3782, 2002). It has also been shown that Th40 cells are required for diabetes transfer. Peripheral (spleen and regional lymph node) T cells that were CD40 depleted, then CD25, Treg, depleted were not capable of transferring diabetes to Scid (Severe Combined Immunodeficiency) recipients. Even though Treg cells were removed, if the auto-aggressive CD40+ T cells subset is absent, disease transfer does not occur. - While Th40 cells are important in the development of autoimmunity, another important factor is expression of the CD40-Ligand, CD154. CD154 is temporally induced on activated T-cells in response to CD3/TCR stimulation (Lederman, S. et al., J. of Exp. Med., 175:1091, 1992). CD154 expression has also been demonstrated on platelets, monocytes, basophils, eosinophils, dendritic cells, fibroblasts, smooth muscle, and endothelial cells (Russo, S. et al., J. Immunol. 171:5489, 2003; Stumpf, C., et al., Eur. J. Heart Fail., 5:629, 2003; Schonbeck, U., et al., Cell Mol. Life Sci. 58:4, 2001). CD154 is a member of the tumor necrosis factor (TNF) super-family and a soluble form of CD154 (sCD154) has been described (Russo, S., et al., J. Immunol. 171:5489 2003; Stumpf, C., et al., Eur. J. Heart Fail 5:629, 2003; Toubi, E., et al., Autoimmunity 37:457, 2004). Therefore, sCD154 may act like a cytokine (Stumpf, C., et al., Eur. J. Heart Fail. 5:629, 2003). Even though CD154 has not been genetically linked in T1D studies, sCD154 is significantly elevated in T1D and may play a role in the disease process (Varo, N. et al., Circulation 107:2664, 2003; Cipollone, F., et al., Diabetologia 48:1216, 2005; Devaraj, S., et al., Diabetes 55:774, 2006). The importance of CD40-CD154 interaction in autoimmunity has been established (Wagner, D. H., Jr., et al., Proc. Nat'l Acad. Sci. USA 99:3782, 2002; Kobata, T., et al., Rev. Immunogenet. 2:74, 2000; Homann, D., et al., Immunity 16:403, 2002; Goodnow, C. C., et al., Lancet 357:2115, 2001; Balasa, B., et al., J. of Immunol. 159:4620, 1997). Blocking CD40-CD154 interaction may prevent collagen induced arthritis, (Durie, F. H., et al., Science 281:1328, 1993) experimental autoimmune encephalitis (Howard, L. M., et al., Autoimmunity 37:411, 2004), prostatitis (Grossman, M. E., et al., J. Immunother. 24:237, 2001), and type-1 diabetes in the NOD mouse model (Durie, F. H. et al., Science 281:1328, 1993; Balasa, B. et al., Journal of Immunology 159:4620, 1997; Howard, L. M., et al., Autoimmunity 37:411, 2004; Grossman, M. E. et al., J. Immunother. 24:237, 2001). In the diabetes model, it was essential to administer a CD154 blocking antibody to NOD mice at 3-weeks of age because at 9-weeks, blocking antibodies had no effect on diabetes prevention (Balasa, B. et al., J. of Immunol. 159:4620, 1997).
- Previous work has also demonstrated that the Th40 cell subset induces RAG1 and RAG2 (Recombination-Activating Genes) transcription, translation and nuclear translocation (Vaitaitis, G. M., et al., Cutting Edge, J. Immunol. 170:3455, 2003) when CD40 is engaged (Vaitaitis, G. M. et al., Cutting Edge, J. Immunol. 170:3455, 2003). CD3 engagement does not induce RAG1 or RAG2 in T-cells (Vaitaitis, G. M., et al., Cutting Edge, J. Immunol. 170:3455, 2003). Subsequent to RAG1/RAG2 induction, CD40-mediated T-cell receptor (TCR) revision occurs in peripheral T cells (Vaitaitis, G. M. et al., Cutting Edge, J. Immunol. 170:3455, 2003). CD40 induction of TCR revision is RAG dependent. T cells isolated from a TCR-Tg mouse undergo TCR revision when CD40 engaged, but T-cells from the TCR-Tg.RAG−/− mouse do not TCR revise when CD40 engaged (Wagner, D. H., Jr. et al., Int'l J. of Mol. Med. 4:231, 1999).
- CD40 is a 50-kDa integral membrane protein of the tumor necrosis factor receptor (TNF-R) family. It is constitutively expressed as a homotrimer (Foy T M, et al., Ann. Rev. of Immunol., 14:591, 1996). In general, stimulation of all CD40-expressing cell types induces operations which contribute to inflammation, such as enhancement of costimulatory and adhesion molecules, and up-regulation of proteolytic enzymes (Mach, F. et al., Atherosclerosis. 137 Suppl:S89-95, 1998).
- CD40's ligand—CD154—is a 39-kDa protein that belongs to the tumor necrosis factor (TNF) family. CD40 forms a trimer that binds CD154 at the interface of the three monomers. CD154 is expressed commonly on cells beyond the surface-expressed CD154, as CD154 may also exist in a soluble biologically active form (sCD154) that is shed from the cell surface after activation. The main source of sCD154 is platelets. (Foy T M, et al., Ann. Rev. of Immunol., 14:591, 1996).
- Genetically manipulated mouse models are utilized for research and development concerning atherosclerosis and cardiovascular disease because wild type mice are generally highly resistant to development and progression of atherosclerosis. Prior studies have attempted to block the CD40/CD154 interaction by using monoclonal antibodies and this approach has proven efficacious in several mouse model studies utilizing the Apoe−/− or LDLr deficient atherosclerotic models. Additionally, these same mouse models built with a deletion of CD154 saw significant reductions in overall plaque formation and may have also contributed to production of a more stable plaque phenotype. Clinically, stable plaques are identifiable and denoted by increased collagen and smooth muscle content, a thick fibrous cap, and an observable decrease in T cell, macrophage, and lipid accumulation.
- Genetically manipulated mouse models are utilized for research and development concerning T2D because mouse models can portray insulin resistance and the inability of the beta cell to sufficiently compensate, which are characteristic of T2D in humans. Many animal models, including mouse models for T2D are obese, reflecting the human condition where obesity is closely linked to T2D development.
- Multiple treatment options have been put forward to address and control both chronic and acute inflammation. Many approaches use non-steroidal anti-inflammatory drugs (NSAIDS) that attack the production of leukotrienes and prostaglandins, cellular products that cause localized inflammation. Other approaches use more powerful immunosuppressant drugs such as cyclophosphamide, methotrexate and azathioprine that suppress the immune response and stop the progression of the disease. Still other treatments involve the use of monoclonal antibodies (mAb) designed to alter the immune responses to self-tissues, as occurs during autoimmune diseases. However, all of these treatments often have severe, long-term side effects.
- Current immune-modulatory therapies may rely upon monoclonal antibody treatments that may give rise to complications. For example, antibodies administered to a subject may cross-react with unintended targets and cause severe nephritic complications and those that specifically act against CD154 may cause embolic complications. Further, the CD40-CD154 interaction may play an important role in antibody generation which may indicate that administration of a monoclonal antibody could induce auto-antibody generation and further complications, which may inhibit the restoration of normal immune function (see generally Banchereau, J. et al., Annu. Rev. of Immunol. 12:881, 1994).
- Other studies have demonstrated that blocking the CD154 interaction by using monoclonal antibodies, or limiting the CD40 receptor by monoclonal antibodies may abrogate atherosclerosis, and may confer a more favorable plaque phenotype characterized by lower inflammation and higher fibrosis. These studies additionally demonstrated that neointimal formation and restenosis may be limited by blocking the CD154 interaction. Studies concerning lupus nephritis may have demonstrated that blocking CD40 mediated signals can reduce anti-double-stranded DNA (anti-dsDNA) antibodies. Moreover, these studies may demonstrate that the reduction of anti-dsDNA was associated with increased serum complement levels and reduced glomerular inflammation, which may be viewed positively from a clinical perspective. However, the use of monoclonal antibodies to target the CD40/CD154 dyad was abandoned due to thromboembolic events which may have been related to the functioning of CD154 in thrombus stabilization. It is postulated that CD154 stabilize thrombi by interaction with the integrin αIIbβ3, and by inhibiting CD154, thrombi may be less stable, and as a consequence shed emboli causing thrombotic events.
- Studies of small molecules have also been conducted to attempt to inhibit the important CD40-CD154 costimulatory interaction required for T cell activation and the development of an effective immune response. For example, suramin, a symmetric polysulfonated napthylamine-benzamide urea derivative was studied for its ability to inhibit CD154 binding to its receptor and prevented the CD154-induced proliferation of human B cells; however, its numerous reversible toxicities (lethargy, rash, fatigue, anemia, hyperglycemia, hypocalcemia, coagulapathies, neutropenia, renal and hepatic complications) (Kaur, M. et al. (2002) Invest. New Drugs 20(2):209-19), loss of activity in protein-rich medium, and its interference with positive costimulatory interaction (Margolles-Clark, E. et al. (2009) Biochem. Pharmacol. 77(7):1236-45) made this and other related small-molecule candidates unlikely sources for effective therapy for the CD40-CD154 dyad.
- Uncertainty exists regarding the primary lesion and the relative importance of the different tissues, metabolic defects in the liver and the peripheral tissues such as fat, muscle, and pancreatic β-cells likely all contribute to type 2 diabetes. Furthermore, although the cause of T2D is incompletely understood, complex and confounded by both genetic and environmental influences, hyperglycemia itself is believed to hinder pancreatic beta-cell function (Cernea, S., et al. Biochem. Med. (Zagreb) 2013; 23(3):266-80).
- Accordingly,
type 2 diabetes in humans typically develops through a progressive series of increasingly disruptive phases or stages. Initially, pre-diabetes is characterized by impaired glucose tolerance, wherein the body has difficulty clearing glucose after a meal (postprandial hyperglycemia) and/or the body may have decreased sensitivity to insulin. In a second phase or stage, postprandial hyperglycemia and basal hyperglycemia occur while insulin producing beta cells of the pancreas become dysfunctional at an increasing rate. During the next phase of the disease progression, hyperglycemia occurs even after fasting and at a cellular level significant beta cell atrophy takes place. Ultimately, in the final phase or end stage of disease progression, beta cells can no longer produce and/or release insulin and the patient requires insulin replacement therapy. - T2D is clinically characterized by hyperglycemia and pathologically by insulin resistance with relative insulin secretory impairment. Individuals who are genetically prone to developing T2D may experience insulin resistance (the earliest detectable metabolic defect) between 15 and 25 years or more before the clinical onset of overt diabetes. (Kahn, C R, Diabetes. (1994) 43:1066-1084). T2D classically has been associated with age and obesity; however, the increased diagnosis of youth with T2D has demonstrated that these two factors alone are not the sole reliable predictors of the disease. Body mass index (BMI) is also clearly associated with T2D, but both genetic and environmental factors are now identified as contributory as well (Wu Y, et al. Int. J. Med. Sci. (2014) 11(11):1185-200. Epub 2014/09/06; Cefalu, W T, Diabetes. (2009) 58(2):307-8; Donath, M Y, et al., Nat. Rev. Immunol. (2011) 11(2):98-107). Obesity creates inflammatory conditions, and sustained inflammation resulting from obesity or other conditions may be important in T2D development (Donath, M Y, et al., Nat. Rev. Immunol. (2011) 11(2):98-107; Donath, M Y, Nat. Rev. Drug Discov. (2014) 13(6):465-76).
- Thus, there exists a need in the art for safer and more effective methods for treatment and prevention of T2D implicated by the autoimmune and inflammatory pathway and dyad related to CD40-CD154. The present developments may address this need by describing methods for treatment of T2D by administration of a therapeutically effective amount of CD40-binding peptide.
- Thus, there exists a need in the art for safer and more effective methods for treatment and prevention of cardiovascular diseases (CVDs) and T2D implicated by aseptic chronic inflammation (ACI). The present developments may address this need by describing methods for treatment of atherosclerosis by administration of a therapeutically effective amount of a peptide that affects, regulates, blocks, inhibits, or modulates the CD40-CD154 dyad. Further, the present developments may provide the added benefit of preventing auto-antibody generation, and thus allow the resumption of normal immune function.
- This statement of background is for information purposes only and is not intended to be a complete or exhaustive explication of all potentially relevant background.
- The present developments may provide novel methods for preventing, modulating, and/or reducing atherosclerosis that arises in a corporeal body. Atherosclerosis may arise as a result of chronic inflammatory response of white blood cells in the walls of arteries. It is postulated that the chronic inflammatory response and the subsequent buildups of plaque in arteries may be caused by elevated levels of cholesterol and triglycerides in the blood, high blood pressure, and cigarette smoking.
- The present developments are based on the knowledge that interaction of CD40-ligand (CD154 protein) with CD40 protein expressed on T-cells (Th40 cells), may be important in the development of atherosclerosis and autoimmune disease. The present developments may be based on the elucidation of the critical residues in CD40 and CD154 that may be important for this interaction. The present developments relate to blocking the interaction between a CD40 protein and a CD154 protein through the use of small peptides that interact with the CD40 protein at a site where the CD154 protein would normally bind. The present developments also relate to using such peptides to reduce the level of Th40 cells, thereby reducing the severity of disease.
- One embodiment of the present developments is a method for preventing atherosclerosis comprising contacting the CD40 protein with a peptide that interacts with the CD40 protein. Preferred peptides may be those that are less than 25 amino acids in length, and that bind to a CD40 protein, thereby inhibiting its interaction with a CD154 protein; however, the length of the peptide should not be considered a limitation on the developments herein as there are numerous other factors that may affect the ability of the peptide to perform its intended and desired result.
- One embodiment of the present developments is a method for preventing, modulating, and/or reducing atherosclerosis, the method comprising inhibiting interaction between a CD40 protein and a CD154 protein with a peptide that interacts with the CD40 protein. Preferred peptides interact with the CD40 protein at the CD154-binding site. Preferably such peptides are less than 25 amino acids in length. Even more preferred peptides are those amino acid sequences selected from SEQ ID NOs 3-9 and 25-30.
- One embodiment of the present developments is a method for preventing, modulating, and/or reducing atherosclerosis, the method comprising administering to a subject in need thereof, a therapeutically effective amount of a peptide that affects the interaction of CD40 with CD154/CD40-ligand. An aspect of this development may be that the peptide binds to CD40. In this embodiment, the peptide may bind to a CD40 protein with a Kd of greater than 10−6. Further, in this embodiment, the peptide may affect the interaction between CD40 and CD154. Additionally, a preferred embodiment may inhibit the binding of CD40 to CD154. Moreover, in this embodiment, the peptide binds to CD40 at the site where CD40 interacts with CD154. In this embodiment, the peptide affects the interaction of CD40 with CD154 in such a manner as to prevent the expansion of Th40 cells. In this embodiment, the peptide affects the interaction of CD40 with CD154 in such a manner as to reduce the number of Th40 cells. In this embodiment, the peptide affects the interaction of CD40 with CD154 in such a manner as to alter the cytokine expression profile of a cell population, treated with said peptide.
- One embodiment of the present developments is a method to modulate and/or reduce atherosclerosis in an animal, the method comprising administering to the animal, a peptide that interacts with a CD40 protein in such a manner as to modulate IFNγ (interferon gamma). Preferred peptides are those that interact with the CD40 protein at the CD154-binding site, thereby modulating IFNγ. Preferred peptides modulate inflammation by reducing the level of Th40 cells to no more than 25% of the total T-cell population. Such methods can be used to prevent and/or reduce atherosclerosis and symptoms that may accompany cardiovascular diseases, more generally.
- One embodiment of the present developments is a method to identify a patient at risk for developing cardiovascular disease and/or atherosclerosis, the method comprising obtaining a sample containing T-cells from a patient to be tested, contacting the sample with a peptide that binds the CD40 protein, detecting the CD-40 bound peptide, and determining the level of Th40 cells from the amount of CD40 bound, wherein a level of Th40 cells greater than 25% of the total T-cell population indicates the patient is at risk for developing cardiovascular disease and/or developing atherosclerosis.
- Yet, another embodiment of the present developments is a method to prevent, modulate, or reduce calcium buildup, or calcification of vessel walls, the method comprising administering to the subject in need thereof, a therapeutically effective amount of a peptide which specifically binds to a CD40 presenting cells at the CD154 binding site.
- Another embodiment of the present developments, is a method to administer a CD40-binding peptide to prevent, modulate, and/or reduce atherosclerosis, comprising selecting a peptide that interacts with a CD40 protein and CD154 binding site, selecting a delivery method selected from the group comprising intramuscular (IM) delivery, intravenous (IV) delivery, subcutaneous (SC) delivery, oral delivery, gavage delivery, emollient/skin delivery, or transdermal patch.
- Another embodiment of the present developments, is a method to administer a CD40-binding peptide to prevent, modulate, and/or reduce atherosclerosis in an animal, comprising selecting a peptide that interacts with a CD40 protein and CD154 binding site, using an extended delivery method selected from the group comprising an implantable device, a hydrophilic polymer formulation, a permeable polymeric membrane, injectable gel implants, solvent extraction system, phase inversion system, thermosensitive gels, pH dependent in situ gels, microparticles, microspheres, nanoparticles, nanospheres, bio-degradable implants, or photoactivated depot.
- Another embodiment of the present developments, is a method to lower LDL cholesterol in a subject, the method comprising administering to the subject in need thereof, a therapeutically effective amount of a peptide which specifically binds to a CD40 presenting cells at the CD154 binding site.
- The present developments may provide novel methods for preventing, modulating, reducing, treating and/or reversing T2D that arises in a corporeal body. Moreover, the developments disclosed herein are therapeutic methods which may additionally be used for the prevention, control, and treatment of diseases, disorders, and conditions, in particular immune and inflammatory diseases.
- The present developments are based on the knowledge that interaction of CD40-ligand (CD154 protein) with CD40 protein expressed on T-cells (Th40 cells), may be important in the development of
type 2 diabetes and autoimmune diseases. The present developments may be based on the elucidation of the critical residues in CD40 and CD154 that may be important for this interaction. The present developments relate to blocking and/or disrupting the interaction between a CD40 protein and a CD154 protein through the use of small synthesized peptides that interact and/or associate with the CD40 protein at a site where the CD154 protein would normally bind. The present developments also relate to using such peptides to reduce the level of Th40 cells, thereby reducing the severity of disease. The peptides of the current developments may bind directly and/or alternatively associate with the CD40 molecule in such a way as to alter CD40 function. - In autoimmune diseases and conditions, CD40 engagement may promote inflammation. Accordingly, in one embodiment of the present developments the peptide may alter CD40 signals to no longer be inflammatory. Thus, in one embodiment the peptides of the current development may block, disrupt, interfere, and/or inhibit CD40 function at sites including but not limited to Th40 cells, pancreas beta cells, endothelial cells, B cells, monocytes, and/or macrophages. The current developments contemplate the use of the small interfering peptides (SIPs) disclosed herein to interfere with the CD40-signaling pathway of any cells that may present with CD40. These peptides may be able to interfere at specific sites depending on the route of administration.
- One embodiment of the present developments is a method for preventing, modulating, reducing, and/or reversing
type 2 diabetes comprising contacting the CD40 protein with a peptide that interacts with the CD40 protein. Preferred peptides are those that are less than 25 amino acids in length, and that bind to a CD40 protein, thereby inhibiting its interaction with a CD154 protein. - One embodiment of the present developments is a method for preventing, modulating, reducing and/or reversing
type 2 diabetes, the method comprising inhibiting interaction between a CD40 protein and a CD154 protein with a peptide that interacts with the CD40 protein. Preferred peptides interact with the CD40 protein at the CD154-binding site. Preferably such peptides are less than 25 amino acids in length. Even more preferred peptides are those amino acid sequences selected from SEQ ID NOs 3-9 and 25-30. - In one aspect the present development provides a method for modulating and/or increasing glucose transport protein 4 (GLUT4) the method comprising administering to a patient a therapeutically sufficient dose of a peptide selected from SEQ ID NOs 3-9 and 25-30.
- One embodiment of the present developments is a method to prevent, modulate, reduce and/or
reverse type 2 diabetes in an animal, the method comprising administering to the animal, a peptide that interacts with a CD40 protein in such a manner as to modulate glucose transport protein 4 (“GLUT4”). Preferred peptides are those that interact with the CD40 protein at the CD154-binding site, thereby modulating GLUT4. Preferred peptides may modulate, upregulate, or increase GLUT4 in both adipose and muscle tissue compared to untreated populations. Such methods can be used to prevent and/or reduce T2D and symptoms that may accompany T2D and autoimmune related inflammation, more generally. - Another embodiment of the present developments, is a method to administer a CD40-binding peptide to prevent, modulate, reduce and/or
reverse type 2 diabetes, comprising selecting a peptide that interacts with a CD40 protein and CD154 binding site, selecting a delivery method selected from the group comprising intramuscular (IM) delivery, intravenous (IV) delivery, subcutaneous (SC) delivery, oral delivery, gavage delivery, emollient/skin delivery, transdermal patch, or nasal administration. - Another embodiment of the present developments, is a method to administer a CD40-binding peptide to prevent, modulate, reduce and/or
reverse type 2 diabetes in an animal, comprising selecting a peptide that interacts with a CD40 protein and CD154 binding site, using an extended delivery method selected from the group comprising an implantable device, a hydrophilic polymer formulation, a permeable polymeric membrane, injectable gel implants, solvent extraction system, phase inversion system, thermosensitive gels, pH dependent in situ gels, microparticles, microspheres, nanoparticles, nanospheres, bio-degradable implants, or photoactivated depot. - Another embodiment of the present developments, is a method of modulating, controlling, and/or increasing GLUT4 in a subject, the method comprising administering to the subject in need thereof, a therapeutically effective amount of a peptide selected from SEQ ID NOs 3-9 and 25-30.
- Another embodiment of the present developments, is a method to modulate, affect and/or reduce interleukin-2 signaling in a cell or a subject comprising administering a peptide selected from SEQ ID NOs 3-9 and 25-30, in an amount sufficient to reduce or inhibit interleukin-2 signaling, wherein, the interleukin-2 signaling is associated with a condition selected from the group comprising type I diabetes, multiple sclerosis, systemic lupus erythematosa, rheumatoid arthritis, Crohn's disease, inflammatory bowel disease, chronic obstructive pulmonary disease, asthma, atherosclerosis, vasculitis, hypertension, thyroiditis, primary biliary cirrhosis, Paget's disease, Addison's disease, acute respiratory distress syndrome, acute lung injury, and/or aseptic chronic inflammation, more generally.
- Another embodiment of the present developments, is a method to modulate and/or reduce interleukin-2 signaling in a cell or a subject comprising administering a peptide selected from SEQ ID NOs 3-9 and 25-30 in an amount sufficient to reduce or inhibit interleukin-2 signaling, wherein, the interleukin-2 signaling is associated with
type 2 diabetes. - Another embodiment of the present developments is a method to modulate and/or reduce IFN-γ in a cell or a subject comprising administering a peptide selected from SEQ ID NOs 3-9 and 25-30, in an amount sufficient to reduce or inhibit IFN-γ signaling, wherein, the IFN-γ signaling is associated with
type 2 diabetes. - Another embodiment of the present developments, is a method to modulate, affect, and/or induce changes of interleukin-21 (IL-21), interleukin-22 (IL-22), IFNγ, TNFα, interleukin-6 (IL-6), granulocyte-macrophage colongy-stimulating factor (GM-CSF), interleukin-4 (IL-4), interleukin-10 (IL-10) and transforming growth factor beta (TGFβ) in a cell or subject comprising administering a peptide selected from SEQ ID NOs 3-9 and 25-30, in an amount sufficient to change said interleukin-21 (IL-21), interleukin-22 (IL-22), IFNγ, TNFα, interleukin-6 (IL-6), granulocyte-macrophage colongy-stimulating factor (GM-CSF), interleukin-4 (IL-4), interleukin-10 (IL-10) and transforming growth factor beta (TGFβ), wherein the said interleukin-21 (IL-21), interleukin-22 (IL-22), IFNγ, TNFα, interleukin-6 (IL-6), granulocyte-macrophage colongy-stimulating factor (GM-CSF), interleukin-4 (IL-4), interleukin-10 (IL-10) and transforming growth factor beta (TGFβ) signaling is associated with a condition selected from group comprising type I diabetes, multiple sclerosis, systemic lupus erythematosa, rheumatoid arthritis, Crohn's disease, inflammatory bowel disease, chronic obstructive pulmonary disease, asthma, atherosclerosis, vasculitis, hypertension, thyroiditis, primary biliary cirrhosis, Paget's disease, Addison's disease, acute respiratory distress syndrome, acute lung injury, type 2 diabetes, and/or aseptic chronic inflammation, more generally.
- Another embodiment of the present developments includes a method to modulate and/or reduce interleukin 17 (IL-17) in a cell or a subject comprising administering a peptide selected from SEQ ID NOs 3-9 and 25-30 in an amount sufficient to reduce or inhibit interleukin 17 (IL-17) signaling, wherein, the IL-17 signaling is associated with a condition selected from the group comprising type I diabetes, multiple sclerosis, systemic lupus erythematosa, rheumatoid arthritis, Crohn's disease, inflammatory bowel disease, chronic obstructive pulmonary disease, asthma, atherosclerosis, vasculitis, hypertension, thyroiditis, primary biliary cirrhosis, Paget's disease, Addison's disease, acute respiratory distress syndrome, acute lung injury, and/or aseptic chronic inflammation, more generally.
- Another embodiment of the present developments includes a method to modulate and/or reduce interleukin 17 (IL-17) in a cell or a subject comprising administering a peptide selected from SEQ ID NOs 3-9 and 25-30 in an amount sufficient to reduce or inhibit interleukin 17 (IL-17) signaling, wherein, the IL-17 is associated with
type 2 diabetes. - One embodiment of the present developments is a method to identify a patient at risk for developing
type 2 diabetes, the method comprising obtaining a sample containing T-cells from a patient to be tested, contacting the sample with a peptide that binds the CD40 protein, detecting the CD-40 bound peptide, and determining the level of Th40 cells from the amount of CD40 bound, wherein a level of Th40 cells greater than 25% of the total T-cell population indicates the patient is at risk for developingtype 2 diabetes. - One embodiment of the present developments is the composition of matter of small interfering peptides of those in SEQ ID NOs: 4, 27, 28, 29, and 30. These embodiments of the current developments may be used for the for the treatment of disease selected from the group comprising type I diabetes, multiple sclerosis, systemic lupus erythematosa, rheumatoid arthritis, Crohn's disease, inflammatory bowel disease, chronic obstructive pulmonary disease, asthma, atherosclerosis, vasculitis, hypertension, thyroiditis, primary biliary cirrhosis, Paget's disease, Addison's disease, acute respiratory distress syndrome, acute lung injury,
type 2 diabetes, and/or aseptic chronic inflammation, more generally. -
FIG. 1 is a chart of the effect of various peptides of CD154 on the development of diabetes in NOD mice. The 8-mer (SEQ ID NO: 5), 10-mer (SEQ NO: 24), 13-mer (SEQ ID NO:25), 15-mer (SEQ ID NO: 7), and 24-mer (SEQ ID NO:26) were tested. -
FIG. 2A is a chart of the effect of a 15-mer peptide from CD154 on the CD4/CD8 ratio in NOD mice. -
FIG. 2B is a chart of the effect of 15-mer peptide on beta-islet infiltration in treated versus control pancreata excised, examined, and scored. -
FIG. 3 is a graph of reversal of diabetes in NOD mice using a 15-mer peptide from CD154. -
FIG. 4 is a dot-plot of the detection of Th40 cells using a SIP-15-mer peptide from CD154. -
FIG. 5 is a dot-plot of a screening of B cells using a SIP-15-mer peptide from CD154. -
FIG. 6 is a chart demonstrating a comparison of Th40 cell levels in diabetic and non-diabetic mice. -
FIG. 7 is a chart demonstrating the effect of treatment with the 15-mer peptide on insulin granulation of the pancreas. -
FIG. 8 is a graph that shows the effect of mutations in the 15-mer peptide on the ability of the 15-mer peptide to inhibit development of diabetes in NOD mice. -
FIG. 9 is a chart showing the number of cells (×106) of CD3+CD4+CD40+ in different mice models. -
FIG. 10 is a chart showing the percentage of Th40 cells in the peripheral blood in human subjects in control and diabetic subjects. -
FIG. 11 is a dot-plot comparing CD4+ and CD40 cell data obtained through flow cytometry. Th40 cells (CD4+CD40+) are in the upper right quadrant. -
FIG. 12 is a chart showing the percentage of Th40 cells of CD3+CD4+ population of mice models. -
FIG. 13 is a chart that shows Th40 Cell percentage of CD3+CD4+ population within the murine aorta of C57B-6, young non-diabetic NOD mice, and diabetic NOD mice. -
FIG. 14 is an image at 200× magnification showing Th40 cells in the shoulder region of plaque in the ApoE−/− mouse model. -
FIG. 15 is a graph of interferon gamma control of Th40 proliferation. -
FIG. 16 is a plot of CD40 stimulated proliferation of Th40 cells in the absence/presence of anti-interferon gamma antibody (αIFNγ) demonstrating that interferon gamma mediates CD40 induced proliferation. -
FIG. 17 is a sample of stains of aortic arch of the lesser curvature of the aortic arch. -
FIG. 18 is a stain of lesser curvature of aortic arch in control and treated subjects. -
FIG. 19 is a chart of area measurements of the lesser curvature of aortic arches and plaques. -
FIG. 20-23 are charts of treated and control ApoE mice subjects. -
FIG. 24 is a table of blood clot data from human blood in the presence of 15-mer peptide compared to normal clotting. -
FIG. 25A is a table providing the relative peptide stability assessed by ExPASy analysis. -
FIG. 25B is a table providing the relative peptides stability assessed by ExPASy analysis. -
FIG. 26 is a western blot comparing control and treated samples from subject mice. -
FIG. 27 is a graph of LDL cholesterol measured in treated and untreated subjects. -
FIG. 28A is an image of KGYY6 treated aortic en-face Sudan IV staining. -
FIG. 28B is an image of control aortic en-face Sudan IV staining. -
FIG. 29 is a graph demonstrating the reduction of lesion areas of Sudan IV staining. -
FIG. 30 is a graph of plaque volume reduction for area under the curve. -
FIG. 31 is a graph of plaque composition for KGYY6 (SEQ ID NO:29) treated and control subject mice. -
FIG. 32A is an image of trichrome stained sections of KGYY6 (SEQ ID NO:29) treated subject. -
FIG. 32B is an image of trichrome stained sections of control subjects. -
FIG. 33(a)-(b) are graphs that show a statistically significant improvement in glucose tolerance (GTT) and insulin sensitivity in response to SEQ ID NO: 29. -
FIG. 34 is a western blot analysis of GLUT4 (insulin regulated glucose transport protein) comparing adipose and muscle tissue of treated (with SEQ ID NO: 29) and untreated mice. -
FIG. 35 is a graph of the percentage change of in-vitro lymphocyte cytokines measured in spleen cells from ApoE−/− and C57BL/6 mice as measured by flow cytometry. - The present subject matter is based on the discovery that a unique subset of T-cells, which express CD40 protein, and thus are referred to as Th40 cells, may be instrumental in autoimmune inflammation. Moreover, involvement of Th40 cells in the autoimmune process may be dependent on the interaction between CD40 protein expressed on the surface of the T-cell, and CD154 protein. Interaction of CD40 and CD154 results in activation signals being delivered between the cells, and subsequent activation of the Th40 cell. Such activation results in propagation of the Th40 cell and an increase in inflammation (e.g., an increase in the number of immune cells and immunoregulatory molecules, present in the system). Accordingly, inhibition of the CD40/CD154 interaction can modulate Th40 cell activity, and thereby affect inflammation. Thus the present subject matter relates to the peptides, and administration thereof, that may affect the interaction between a CD40 protein and a CD154 protein, thereby modulating inflammation. Moreover, the present subject matter relates to peptides that affect the interaction between CD40 protein expressed on the surface of a T-cell, and a CD154 protein, thereby affecting T-cell activity, controlling inflammation, and consequently preventing, modulating, and reducing atherosclerosis. The present subject matter also encompasses the use of such peptides to detect Th40 cells.
- Before the present development is further described, it is to be understood that this invention is not strictly limited to particular embodiments described, as such may of course vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the claims.
- It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. It should further be understood that as used herein, the term “a” entity or “an” entity refers to one or more of that entity. For example, a nucleic acid molecule refers to one or more nucleic acid molecules. As such, the terms “a”, “an”, “one or more” and “at least one” can be used interchangeably. Similarly, the terms “comprising”, “including” and “having” can be used interchangeably.
- Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.
- It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.
- It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.
- Furthermore, as used herein the term animal refers to a vertebrate, preferably a mammal, more preferably a human. Suitable mammals on which to use the methods of the present invention include but are not limited farm animals, sports animals, pets, primates, mice, rats, horses, dogs, cats, and humans. The term animal can be used interchangeably with the terms subject or patient.
- One embodiment of the present subject matter is a peptide that interacts with a CD40 protein in such a manner as to prevent atherosclerosis. As used herein, the terms interact, interaction, and the like, mean that two molecules come into sufficient physical proximity such that they cause a modulation of inflammation. One such type of interaction is a binding interaction. In such an interaction the peptide associates with CD40 to form a complex. An example of complex formation is the association of an antigen with an antibody. According to the present subject matter, binding of a peptide hereof to a CD40 protein can be reversible (e.g., non-covalent binding interactions) or non-reversible (e.g., covalent binding interactions). Moreover, a reversible interaction can be strong or weak, the strength of the interaction being determined by the forces (e.g., ionic charges, hydrogen binding, van der Walls interactions, etc.) exerted by each protein on the other protein in the complex. Factors affecting the strength of an interaction between two molecules are known to those skilled in the art. One useful measure of the strength of binding between two molecules, such as a peptide and a protein, is the dissociation constant (Kd). Preferred peptides of the present invention are those that bind to a CD40 protein with a Kd of no more than about 1×10−6 M, about 1×10−7 M, or about 1×10−8 M. Particularly preferred peptides are those having a Kd of less than about 1×10−9 M. In one embodiment, a peptide hereof binds to a CD40 protein with a Kd of less than 100 nM, less than 50 nM, less than 25 nM, less than 10 nM, less than 5 nM, less than 3 nM, less than 2 nM, or less than 1 nM. Methods of measuring and analyzing binding interactions between a peptide and a CD40 protein are known by those of skill in the art.
- As used herein, the change the level of Th40 cells present in an animal, or in a culture of T cells may be indicative of modulation of inflammation. As used herein, the terms level, number, count and concentration can be used interchangeably. Modulation of inflammation may mean an increase or decrease in the number of Th40 cells present in the inflammatory environment; however, the modulation of inflammation should not be limited to cell numbers or counts.
- Consequently, modulation can be referred to as positive or negative. Positive modulation (also referred to as up-regulation) of inflammation may result in an increase in the number of Th40 cells in the inflammatory environment. Negative modulation (also referred to as down-regulation) of inflammation may result in a reduction in the number of Th40 cells present in the inflammatory environment. The level, count, or concentration of Th40 cells may not be indicative of inflammation in the inflammatory environment. A preferred peptide may be one that down-regulates inflammation, thereby reducing the number of Th40 cells present in the inflammatory environment. Positive and negative modulation of inflammation may or may not result in a change in the type and amount of immunoregulatory molecules present in the inflammatory environment. In some instances, it is possible that the Th40 levels will not change but the activity of those cells is altered such that they are no longer exerting or increasing inflammatory cytokines and other biomarkers of inflammation. Therefore, as used herein modulating inflammation may refer to changes in the Th40 levels, numbers, or concentration in an corporeal body or sample, and may also refer to changes in inflammation more generally that may be associated with disease, inflammatory cytokines, and/or cell derived inflammatory mediator molecules.
- It will be appreciated by those skilled in the art that both a cell culture system and the immune system of an animal comprise basal levels of immune cells and immunoregulatory molecules. The phrases basal level and normal level can be used interchangeably. With regard to the immune system of an animal, as used herein, the basal level of a type of immune cell (e.g., Th40 cell), or a immunoregulatory molecule, refers to the average number of that cell type, or immunoregulatory molecule, present in a population of individuals considered healthy (i.e., free of metabolic, autoimmune, or infectious disease). With regard to a cell culture system, as used herein, the basal level of a type of immune cell, or an immunoregulatory molecule, refers to the average level of that cell type, or immunoregulatory molecule, present in a population of cells that is non-activated. Those skilled in the art are capable of determining if a T-cell, or a population of such cells, is activated. For example, the expression of CD69, CD25 and/or CD154 proteins by a cell indicates that the cell has been activated.
- The basal level of a cell or molecule can be a specific amount (e.g., a specific concentration) or it can encompass a range of amounts. Basal levels, or ranges, of immune cells and immunoregulatory molecules are known to those in the art. For example, in a healthy individual, the normal level of CD4+ T-cells present in human blood is 500-1500 cells/ml. Variability in this measurement can result from differences in the method used to determine the cell count. Furthermore, normal levels of cells can also be reported as a percentage of a total cell population. For example, in a healthy individual, Th40 cells make up less than 25% of the total T cell population. Thus, as used herein, the term inflammation refers to an inflammatory environment in which Th40 cells make up greater than about 25%, greater than about 30%, greater than about 35%, greater than about 40%, greater than about 45%, greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, or greater than about 80% of the total T-cell population. Moreover, a preferred peptide herein is one that reduces the level of Th40 cells to less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 27%, or equal to about 25% of the total T-cell population. Methods of measuring different types of T-cells in the T-cell population are known to those skilled in the art. Furthermore, a novel method for detecting Th40 cells using peptides hereof is disclosed herein.
- As used herein, the phrase inflammatory environment refers to the overall population of immune cells, and related immunoregulatory molecules, that are present in a culture of cells, or in the body of an animal. As such, the phrase inflammatory environment encompasses the types, and/or the relative amounts of immune cells and immunoregulatory molecules (e.g., cytokines) present in a culture of cells, or in an animal, which are involved in affecting an inflammatory reaction. Examples of cells encompassed by the term inflammatory environment include, but are not limited to, T cells, neutrophils, macrophages, granulocytes, and the like. The inflammatory environment relates to cells and molecules that mediate both acute and chronic inflammation. It will be appreciated by those skilled in the art that the inflammatory environment refers to the system to which peptides hereof are administered. In one embodiment, the system is a cell culture system. In one embodiment, the system is a whole animal.
- A preferred peptide hereof is one that selectively interacts with a CD40 protein in solution, as determined using an assay such as an immunosorbent assay, or on the surface of a T-cell. As used herein, the terms selectively, selective, specific, and the like, indicate the peptide has a greater affinity for a CD40 protein than it does for proteins unrelated to the CD40 protein. More specifically, the terms selectively, selective, specific, and the like indicate that the affinity of the peptide for CD40 is statistically significantly higher than its affinity for a negative control (e.g., an unrelated protein such as albumin) as measured using a standard assay (e.g., ELISA). Suitable techniques for assaying the ability of a peptide to selectively interact with a CD40 protein are known to those skilled in the art. Such assays can be in vitro or in vivo assays. Examples of useful assays include, but are not limited to, an enzyme-linked immunoassay, a competitive enzyme-linked immunoassay, a radioimmunoassay, a fluorescence immunoassay, a chemiluminescent assay, a lateral flow assay, a flow-through assay, an agglutination assay, a particulate-based assay (e.g., using particulates such as, but not limited to, magnetic particles or plastic polymers, such as latex or polystyrene beads), an immunoprecipitation assay, an immunoblot assay (e.g., a western blot), a phosphorescence assay, a flow-through assay, a chromatography assay, a polyacrylamide gel electrophoresis (PAGE)-based assay, a surface plasmon resonance assay, a spectrophotometric assay, a particulate-based assay, an electronic sensory assay and a flow cytometric assay. Methods of performing such assays are well known to those skilled in the art. In one embodiment, an assay can be performed using cells in culture, or it can be performed in a whole animal. Assays can be designed to give qualitative, quantitative or semi-quantitative results, depending on how they are used and the type of result that is desired.
- Cardiovascular Disease (CVD), Atherosclerosis, and Cholesterol Related Developments
- One embodiment hereof is a peptide that interacts with a CD40 protein in such a manner as to affect the interaction of the CD40 protein with a CD154 protein, thereby modulating inflammation. The effect of the peptide on the CD40/CD154 interaction can be positive or it can be negative. For example, the peptide can interact with the CD40 protein in such a manner that the strength of the interaction between the CD40 protein and a CD154 protein is increased. Alternatively, the peptide can interact with the CD40 protein such that the strength of the interaction between the CD40 protein and a CD154 protein is decreased. Methods of measuring the strength of binding between the peptide and a CD40 protein are known to those skilled in the art. A preferred peptide hereof is one that reduces the strength of the interaction between a CD40 protein and a CD154 protein. Preferred peptides hereof reduce the strength of binding between a CD40 protein and a CD154 protein by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. A particularly preferred peptide is one that completely inhibits binding of CD40 to CD154. Complete inhibition of binding between CD40 and CD154 means that when a peptide hereof is brought into proximity with a CD40 protein and a CD154 protein under conditions that would normally allow the interaction of CD40 and CD154, no such interaction occurs and activation signals are not stimulated in the CD40-expressing cell. Consequently CD40/CD154 mediated modulation of inflammation does not occur. In one embodiment, the peptide interacts with the CD40 protein in such a manner as to reduce the level of inflammation in the system. In one embodiment, the peptide interacts with the CD40 protein in such a manner as to inhibit the development of inflammation in the system.
- While peptides hereof can interact with any site on the CD40 protein, preferred peptides interact with the CD40 protein at a location that overlaps with the CD154 binding site. In one embodiment, a peptide hereof interacts with the CD40 protein at the CD154 binding site. An example of such a peptide is a CD40 ligand competitive antagonist. As used herein, peptides that interfere with, or inhibit, the binding of a CD154 protein to a CD40 protein are referred to as small interfering peptides (SIPs). As used herein a small interfering peptide is a peptide that, through physio-chemical properties, interferes with the interaction of a CD40 protein with a CD154 protein, thereby preventing activation signals from being delivered to the CD40-bearing cell, thus limiting the activation of the CD40-bearing cell, and consequently, inflammation. As demonstrated herein, the consequences of such interference are prevention of T-cell activation and propagation, and a prevention or reduction of inflammation. As demonstrated herein, in some instances the results of such inhibition or prevention of interaction between CD40 and CD154 may include observable data that demonstrates that atherosclerosis, and characteristics of diseases associated therewith, are prevented, modulated, and/or reduced.
- Additionally, a small interfering peptide, may, through its physio-chemical properties, interfere with the interaction of a CD40 protein with a CD154 protein, thereby preventing activation signals from being delivered to the CD40-bearing cell, thus limiting the activation of the CD40-bearing cell, and consequently, modulating, inhibiting, and preventing atherosclerosis. As demonstrated herein, the consequences of such interference are prevention of T cell activation and propagation, and a prevention, reduction, or modulation of atherosclerotic developments.
- A peptide useful for practicing methods of the present developments should be of a size sufficient to interact with CD40 protein in such a manner as to modulate atherosclerosis. It is understood by those skilled in the art that preferred peptides are relatively short since they are easier and less expensive to produce. Preferred peptides may be those that are less than 25 amino acids in length; however, the length of the peptide may be longer than 25 amino acids in some instances. A preferred peptide may be one that is 4, 6, 8, 10, 13, 15, or 24 amino acids in length. In one embodiment, the peptide is an amino acid selected from the group of SEQ ID NO:3 (Core-sequence see Table 1), SEQ ID NO:4 (6-mer see Table 1), SEQ ID NO:5 (8-mer mouse see Table 1), SEQ ID NO:6 (8-mer human see Table 1), SEQ IN NO:7 (15-mer see Table 1), SEQ ID NO:8 (15-mer human see Table 1), SEQ ID NO:9 (24-mer see Table 1), SEQ ID NO: 24 (10-mer see Table 1), SEQ ID NO: 25 (13-mer see Table 1), SEQ ID NO: 26 (24-mer see Table 1), SEQ ID NO: 27 (6-mer (Form 2) see Table 1), SEQ ID NO: 28 (6-mer (Form 3) see Table 1), SEQ ID NO: 29 (6-mer (Form 4) see Table 1), SEQ ID NO: 30 (6-mer (Form 4) see Table 1) and SEQ ID NO:32 (24-mer-mouse (Form 2)). The sequences of such peptides are shown below in Table 1.
-
TABLE 1 SEQ ID NO SEQUENCE Description 1 MIETYSQPSP RSVATGLPAS MKIFMYLLTV SwissPro FLITQMIGSV LFAVYLHRRL DKVEEEVNLH 27548.2 Mouse EDFVFIKKLK RCNKGEGSLS LLNCEEMRRQ CD40 Ligand FEDLVKDITL NKEEKKENSF EMQRGDEDPQ (CD154 IAAHVVSEAN SNAASVLQWA KKGYYTMKSN Protein) LVMLENGKQL TVKREGLYYV YTQVTFCSNR EPSSQRPFIV GLWLKPSSGS ERILLKAANT HSSSQLCEQQ SVHLGGVFEL QAGASVFVNV TEASQVIHRV GFSSFGLLKL 2 MIETYNQTSP RSAATGLPIS MKIFMYLLTV SwissPro FLITQMIGSA LFAVYLHRRL DKIEDERNLH 29965 Human EDFVFMKTIQ RCNTGERSLS LLNCEEIKSQ CD40 Ligand FEGFVKDIML NKEETKKENS FEMQKGDQNP (CD154 QIAAHVISEA SSKTTSVLQW AEKGYYTMSN Protein) NLVTLENGKQ LTVKRQGLYY IYAQVTFCSN REASSQAPFI ASLCLKSPGR FERILLRAAN THSSAKPCGQ QSIHLGGVFE LQPGASVFVN VTDPSQVSHG TGFTSFGLLK L 3 KGYY Core-sequence 4 KKGYYT 6-mer 5 AKKGYYTM 8-mer-mouse 6 AEKGYYTM 8-mer human 7 VLQWAKKGYYTMKSN 15-mer-mouse 8 VLQWAEKGYYTMSNN 15-mer human 9 NAASVLQWAKKGYYTMKSNLVMLE 24-mer 10 ISQAVHAAHAEINEAGR 15-mer from ovalbumin; control peptide 11 G-L-Q-W-A-K-K-G-Y-Y-T-M-K-S-N Gly-1 12 V-G-Q-W-A-K-K-G-Y-Y-T-M-K-S-N Gly-2 13 V-L-G-W-A-K-K-G-Y-Y-T-M-K-S-N Gly-3 14 V-L-Q-G-A-K-K-G-Y-Y-T-M-K-S-N Gly-4 15 V-L-Q-W-G-K-K-G-Y-Y-T-M-K-S-N Gly-5 16 V-L-Q-W-A-G-K-G-Y-Y-T-M-K-S-N Gly-6 17 V-L-Q-W-A-K-G-G-Y-Y-T-M-K-S-N Gly-7 18 V-L-Q-W-A-K-K-G-G-Y-T-M-K-S-N Gly-8 19 V-L-Q-W-A-K-K-G-Y-G-T-M-K-S-N Gly-9 20 V-L-Q-W-A-K-K-G-Y-Y-G-M-K-S-N Gly-10 21 V-L-Q-W-A-K-K-G-Y-Y-T-G-K-S-N Gly-11 22 ISQAVHAAHAEINEAGR 15-mer from ovalbumin; control peptide 23 YVQGKANLKSKLMYT Scrambled peptide 24 WAKKGYYTMK 10-mer mouse 25 VLQWAKKGYYTMK 13-mer mouse 26 AASVLQW AKKGYYTMKSNLVVLEN 24-mer mouse 27 KGYYTM 6-mer (Form 2) 28 AEKGYY 6-mer (Form 3) 29 AKKGYY 6-mer (Form 4) 30 AKGYYT 6-mer (Form 5) 31 YKNVKQMAYWLTGKS Scrambled peptide 32 AASVLQWAKKGYYTMKSNLVMLEN 24-mer-mouse (Form 2) - Interaction of a CD40 protein and a CD154 protein has been shown to occur at particular regions within each protein. The inventors have now shown that, surprisingly, a peptide comprising only a short portion of the CD154 region that interacts with CD40 is capable of binding to a CD40 protein, thereby modulating atherosclerosis. Thus one embodiment hereof is a peptide that comprises at least a portion of the amino acid sequence of a CD154 protein such that the peptide interacts with CD40 protein in such a manner as to modulate atherosclerosis. In one embodiment, interaction of the peptide with CD40 protein results in negative modulation of atherosclerosis. In one aspect, the peptide comprises at least a portion of SEQ ID NO:1 or SEQ ID NO:2.
- In one aspect, the peptide is as short as possible yet comprises enough of the CD154 protein to allow interaction with a CD40 protein in such a manner as to modulate atherosclerosis. In one embodiment, a peptide hereof comprises 6, 13 or 15 contiguous amino acids from SEQ ID NO:1 or SEQ ID NO:2, and interacts with CD40 in such a manner as to modulate atherosclerosis. A preferred peptide comprises a core sequence of lysine-glycine-tyrosine-tyrosine (KGYY; SEQ ID NO:3), which corresponds to amino acids 142-145 of SEQ ID NO:1 and amino acids 143-146 of SEQ ID NO:2. Useful peptides can comprise additional regions of sequence from SEQ ID NO:1 or SEQ ID NO:2 that are adjacent to the core sequence, so long as the peptide is capable of modulating atherosclerosis. In one embodiment hereof, a peptide comprises at least one sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:32, so long as the peptide interacts with CD40 protein in such a manner as to modulate atherosclerosis. In one embodiment of the present subject matter, a peptide hereof is a sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:32. In one embodiment of the present subject matter, a peptide hereof is a sequence selected from SEQ ID NOs 3-9, 25-30, and 32.
- While peptides of the present subject matter may be selected entirely of or from sequences that are responsible for the interaction of the peptide with a CD40 protein, they may additionally contain amino acid sequences that do not interact with a CD40 protein, but which have other useful functions. Any useful, additional amino acid sequence can be added to the CD40-interacting sequence, so long as the additional sequences do not have an unwanted effect on the ability of the CD40 interacting sequence to interact with a CD40 protein. For example, in addition to the amino acid sequence responsible for interacting with a CD40 protein, a peptide hereof can contain amino acid sequences that are useful for visualizing or purifying the peptide. Such sequences act as labels (e.g., enzymes) or tags (antibody binding sites). Examples of such labels and tags include, but are not limited to, B-galactosidase, luciferase, glutathione-s-transferase, thioredoxin, HIS-tags, biotin tags, and fluorescent tags. Other useful sequences for labeling and tagging proteins are known to those of skill in the art.
- Likewise, peptides hereof can be modified, so long as such modification does not significantly affect the ability of the peptide to modulate atherosclerosis. Such modifications can be made, for example, to increase the stability, solubility or absorbability of the protein. Examples of such modifications include, but are not limited to pegylation, glycosylation and chemical modification of the peptide.
- Peptides hereof may be obtained from nature (e.g., obtained from plants, animals or microorganisms) or they may be produced in a laboratory (e.g., recombinantly or synthetically). Preferred peptides are those that are synthesized. Also encompassed are peptides that are combinations of natural and synthetic molecules. General methods for producing and isolating recombinant or synthetic peptides are known to those skilled in the art. It should be noted that, as used herein, an isolated, or biologically pure, molecule, is one that has been removed from its natural milieu. As such the terms isolated, biologically pure, and the like, do not necessarily reflect the extent to which the protein has been purified.
- As has been described herein, interaction of the CD40 protein and the CD154 protein are necessary for involvement of Th40 cells in atherosclerosis. Consequently, inhibition of the interaction between a CD40 and CD154 protein using peptides hereof is a useful method of affecting atherosclerosis. Thus one embodiment is a method to reduce the interaction between a CD40 protein and a CD154 protein comprising introducing into an environment containing a CD40 protein and a CD154 protein, a peptide, that interacts with the CD40 protein in such a manner as to reduce the interaction between the CD40 protein and the CD154 protein. In one aspect hereof, the peptide reduces the interaction between the CD40 protein and the CD154 protein by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In one embodiment, the peptide reduces the interaction between the CD40 protein and the CD154 protein by a factor of at least 10, at least 100, at least 1,000, at least 10,000. Methods of measuring the strength of the interaction between the CD40 protein and the CD154 protein have been discussed previously, and are also know to those of skill in the art.
- One embodiment hereof is a method to modulate atherosclerosis comprising contacting a CD40 protein with a peptide that interacts to the CD40 protein in such a manner as to modulate inflammation. In one aspect, interaction of the peptide with the CD40 protein decreases the number of Th40 cells by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In one embodiment, interaction of the peptide with the CD40 protein decreases the number of Th40 cells by a factor of at least 10, at least 100, at least 1,000, at least 10,000.
- One aspect of the current developments hereof is a method to modulate atherosclerosis, cardiovascular disease, and/or cholesterol levels comprising contacting a CD40 protein with a peptide that interacts with the CD40 protein in such a manner as to ameliorate inflammation. One aspect of this alternative embodiment is that the Th40 levels do not change in response to contacting said CD40 protein with a peptide that interacts with the CD40 protein. In one aspect, Th40 cells treated with peptide may stop, slow, reduce, or retard production of inflammatory cytokines. In this aspect the number of Th40 levels may remain relatively unchanged.
- One aspect is a method to reduce atherosclerosis in a patient, the method comprising administering a peptide hereof to the patient. In one embodiment, the peptide comprises an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:32. In one embodiment, the peptide is an amino acid sequence selected from SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30 and SEQ ID NO:32. In a preferred embodiment, interaction of the peptide with the CD40 protein decreases the number of Th40 cells by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In another embodiment, interaction of the peptide with the CD40 protein decreases the number of Th40 cells by a factor of at least 10, at least 100, at least 1,000, at least 10,000. In a preferred embodiment, the level of Th40 cells is reduced so that Th40 cells comprise no more than about 20%, about 25%, about 30%, about 35%, or about 40% of the total T-cell population.
- Peptides and methods hereof are suitable for use in cell culture as well as for treating a patient. As used herein the term patient refers to any animal in need of such treatment. The animal can be a human or a non-human animal. A preferred animal to treat is a mammal. A peptide can be administered or applied per se, or as pharmaceutical compositions. A peptide hereof, or a pharmaceutical composition thereof, can be administered to a patient by a variety of routes, including, but limited to, by injection (e.g., intravenous, intramuscular, subcutaneous, intrathecal, intraperitoneal), by inhalation, by oral (e.g., in a pill, tablet, capsule, powder, syrup, solution, suspension, thin film, dispersion or emulsion.), transdermal, transmucosal, pulmonary, buccal, intranasal, sublingual, intracerebral, intravaginal rectal or topical administration or by any other convenient method known to those of skill in the art.
- The amount of a peptide hereof and/or a pharmaceutical composition thereof that will be effective can be determined by standard clinical techniques known in the art. Such an amount is dependent on, among other factors, the patient being treated, including, but not limited to the weight, age, and condition of the patient, the intended effect of the compound, the manner of administration and the judgment of the prescribing physician. Also, in this context, it should be noted that in treating a patient exhibiting a disorder of interest, a therapeutically effective amount of an agent or agents such as these is administered. A therapeutically effective dose refers to that amount of the compound that results in amelioration of one or more symptoms or a prolongation of survival in a patient.
- A peptide hereof, or a pharmaceutical composition thereof, can be administered alone or in combination with one or more other pharmaceutical agents, including other compounds of the present disclosure. The specific pharmaceutical composition depends on the desired mode of administration, as is well known to the skilled artisan.
- Because the developments have demonstrated that Th40 cells are intimately involved in the development of autoimmune diseases, atherosclerosis, and cardiovascular disease, the peptides and methods disclosed herein can be used to affect atherosclerosis resulting from such diseases. Thus, one embodiment hereof is a method to treat atherosclerotic disease in a patient in need of such treatment, the method comprising administering to a patient a peptide that interacts with the CD40 protein, thereby reducing atherosclerosis. In one embodiment the peptide interacts with the CD40 protein in such a manner as to affect the interaction of CD40 and CD154, thereby reducing atherosclerosis. In a preferred embodiment, interaction of the peptide with the CD40 protein reduces the number of Th40 cells in a patient to a level equal to that observed in subjects that do not have cardiovascular disease. The present developments are suitable for treating any patient having an autoimmune disease and/or cardiovascular disease, the development of which is dependent on Th40 cells. More specifically, peptides hereof are suitable for reducing the level of Th40 cells in such patients. In a preferred embodiment, a peptide hereof reduces the level of Th40 cells in a patient suffering from a cardiovascular disease to no more than about 25% of the total T-cell population. In another embodiment, a peptide hereof reduces inflammatory cytokine levels while having no effect on the Th40 levels in a patient.
- One example of a disease that is particularly amenable to treatment using a peptide of the present developments may be atherosclerosis. In atherosclerosis, inflammatory changes of the arterial wall occur resulting in the formation and buildup of arterial plaque. Consequently, control of inflammatory cells and cell signaling via CD40-CD154 interaction may be able to be used to control, modulate, and/or reduce atherosclerotic lesions that are characterized as chronic inflammatory-fibroproliferative disease of the vessel wall. Several murine models of T2D and/or atherosclerosis have been developed. The progression of lesion formation is observable in Apolipoprotein E (ApoE) deficient transgenic mice and can be observed by measurement of the aortic arch, the number and type of plaque, and characterized in accordance with the American Heart Association's staging of atherosclerosis, ranging from AHA type I to AHA type
V. AHA type 1, may be characterized by early or initial lesions, may be comprised of histologically “normal” cells, macrophage infiltration, and isolated foam cells. AHA type V, may be characterized by advanced or complicated legions, including but not limited to increased endothelial dysfunction characterized by surface defects, hematoma, hemorrhage, and/or thrombosis. Thus, one embodiment of the present developments is a method to prevent atherosclerosis in an individual at risk for developing atherosclerosis, the method comprising administering to the individual a peptide to selectively bind to a CD40 expressing cell. - Moreover, atherosclerosis may be particularly amenable to treatment using a peptide of the current development. The risk for atherosclerosis may result from familial factors (e.g., inheritance) or from other factors, such as the physical condition of the individual. The level of atherosclerotic activity and disease may vary from individual to individual depending on numerous factors such as level of activity, diet, smoking status, and other variable factors that are dynamic such as levels of inflammation. Some methods of risk assessment for atherosclerosis are known to those skilled in the art. Accordingly, in one embodiment of the present development, the method of treatment comprises administering to a patient in need thereof, a peptide that comprises an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 7, SEQ ID NO:8, and SEQ ID NO:9, so long as the peptide can down-regulate inflammation. In one embodiment, the peptide is an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30 and SEQ ID NO:32.
- The developments herein also show that, surprisingly, peptides hereof can be used to reverse the disease process in individuals already showing signs of atherosclerosis. Thus, one aspect of the present subject matter is a method to reverse atherosclerosis comprising administering to a patient diagnosed as having atherosclerosis, a peptide hereof. In one embodiment, the peptide comprises an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 7, SEQ ID NO:8, and SEQ ID NO:9, so long as the peptide can down-regulate inflammation. In one embodiment, the peptide is an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9. As used herein the phrase to reverse atherosclerosis means to reduce the aortic-arch infiltration, plaque, and lesions of an individual with atherosclerosis to a level comparable to that observably lower levels or in some instances to levels that may be more common in a non-atherosclerotic individual.
- As has been described, peptides of the present invention selectively bind to a CD40 expressing cell. Consequently, peptides of the present subject matter can be used to identify Th40 cells. Thus one embodiment hereof is a method to detect Th40-dependent atherosclerosis, said method comprising contacting a T-cell population with a peptide hereof. In a preferred embodiment, the peptide is labeled with a detectable marker, such as, for example, luciferase or alkaline phosphatase. Such detection can be performed using assay techniques known to those skilled in the art. In general, an assay for detecting Th40 cells using a peptide hereof comprises (a) obtaining a sample of cells; (b) contacting a peptide hereof with said cells under condition suitable to allow binding of the peptide to Th40 cells, if present; (c) washing said cells using conditions that disrupt non-specific interactions, and that remove unbound peptide; and (d) detecting peptide bound to cells. Detection of bound peptide can be achieved directly or indirectly. For example, direct detection can be achieved using a peptide labeled using a detectable marker, as disclosed herein. Following the wash step listed above, the cells are then simply screened for the presence of detectable marker. The presence of detectable marker in the cell sample indicates the presence of Th40 cells, and thus Th40-dependent atherosclerosis. Alternatively, indirect detection involves the use of a second molecule, such as an antibody, that binds to the peptide. In an indirect detection assay, following the wash step listed above, a detection molecule that binds the peptide is added to the cell sample. The detection molecule is labeled with a detectable marker. After washing away unbound detection molecule, the cells are screened for the presence of detectable marker. The presence of detectable marker in the cell sample indicates the presence of Th40 cells. It should be understood that the assays described herein are meant as examples of useful assays, and other assay techniques can be employed. Suitable assay techniques are known to those skilled in the art, and are also disclosed in, for example, Molecular Cloning: A Laboratory Manual, Sambrook, J., Fritsch, E. F., and Maniatis, T, Cold Spring Harbor Laboratory Press; 2nd Edition (December 1989). All references cited herein are incorporated herein in their entirety.
- The assay technology described above can also be used to identify other molecules that affect the interaction of a CD40 protein with a CD154 protein. Examples of such molecules include, but are not limited to, proteins, peptides and small molecules. For example, assays can be designed that test the ability of molecules to compete with a peptide of the present developments for binding to a Th40 cell. For instance, a peptide labeled with a detectable marker, can be mixed with a test molecule and a population of cells known to contain Th40 cells, under conditions that allow binding of the peptide to the Th40 cells. Following an appropriate incubation period, the cells are washed to remove unbound peptide, and the cells screened for the presence of detectable marker. Alternatively, the labeled peptide could be bound to Th40 cells first, and after a wash step to remove unbound peptide, the test molecule could be added to the cells containing bound peptide. Following an incubating period and a wash step to remove unbound molecule, or released peptide, the cells are screened for the presence of detectable marker. In either case, absence of the detectable marker in the cell sample indicates the test molecule is able to compete with the peptide for binding to the Th40 cells, while presence of the detectable marker would indicate the test molecule does not inhibit binding of the peptide to Th40 cells. Inhibition of binding need not be 100%, as such assay would also be useful for identifying molecules that partially inhibit binding of the peptide to Th40 cells. It is understood by those skilled in the art that such assays would involve the use of positive controls (e.g., unlabeled peptide) and negative controls (e.g., a protein/molecule that is known not to bind to Th40 cells).
- Because increased levels of Th40 cells are associated with the development of autoimmune disease, the present developments can be used to identify patients at risk for developing autoimmune disease and autoimmune related atherosclerosis and/or cardiovascular disease more generally. Thus, one embodiment of the present developments is a method to identify a patient at risk for developing autoimmune related atherosclerosis. In one embodiment, patients at risk for developing atherosclerosis are identified by obtaining a sample from a patient to be tested, contacting the T-cell portion of said sample with a peptide hereof, and determining the level of Th40 cells present in the sample, wherein a level of Th40 cells greater than about 25% of the total T-cell population indicates the patient is at risk for developing atherosclerotic disease. In one embodiment, the peptide comprises an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:7, SEQ ID NO:9 SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:32 so long as the peptide binds to the CD40 protein. In one embodiment, the peptide is an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:32. In a preferred embodiment the peptide is labeled with a suitable detectable marker such as, for example, luciferase or alkaline phosphatase.
- The present developments also comprise kits useful for practicing the methods disclosed herein, the kit comprising a peptide that interacts with a CD40 protein in such a manner as to modulate atherosclerosis. In one embodiment, the peptide comprises an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, so long as the peptide can down-regulate atherosclerosis. In one embodiment, the peptide is an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9. Another embodiment is a kit for determining the level of Th40 cells, the kit comprising a peptide that interacts with a CD40 protein, and methods for detecting CD40-bound peptide. Kits can also contain associated reagents and components, such as, but not limited to, buffers, labels, containers, inserts, tubing, vials, syringes, and the like.
-
Type 2 Diabetes Related Developments - The present subject matter is based on the discovery that a unique subset of T-cells, which express CD40 protein, and thus are referred to as Th40 cells, that may be instrumental in autoimmune inflammation, including conditions such as
type 2 diabetes. Moreover, involvement of Th40 cells in the autoimmune process may be dependent on the interaction between CD40 protein expressed on the surface of the T-cell, and CD154 protein. Interaction of CD40 and CD154 results in activation signals being delivered between the cells, and subsequent activation of the Th40 cell. Such activation results in propagation of the Th40 cell and an increase in inflammation (e.g., an increase in the number of immune cells and immunoregulatory molecules, present in the system). Accordingly, inhibition of the CD40/CD154 interaction can modulate Th40 cell activity, and thereby affect inflammation. Thus the present subject matter relates to the peptides, and administration thereof, that may affect the interaction between a CD40 protein and a CD154 protein, thereby modulating inflammation. Moreover, the present subject matter relates to peptides that affect the interaction between CD40 protein expressed on the surface of a T-cell, and a CD154 protein, thereby affecting T-cell activity, controlling inflammation, and consequently preventing, modulating, reducing and/or reversingtype 2 diabetes. The present subject matter also encompasses the use of such peptides to detect Th40 cells. - One embodiment of the present subject matter is a peptide that interacts with a CD40 protein in such a manner as to prevent
type 2 diabetes. As used herein, the terms interact, interaction, and the like, mean that two molecules come into sufficient physical proximity such that they cause a modulation of inflammation. One such type of interaction is a binding interaction. In such an interaction the peptide associates with CD40 to form a complex. An example of complex formation is the association of an antigen with an antibody. According to the present subject matter, binding of a peptide hereof to a CD40 protein can be reversible (e.g., non-covalent binding interactions) or non-reversible (e.g., covalent binding interactions). Moreover, a reversible interaction can be strong or weak, the strength of the interaction being determined by the forces (e.g., ionic charges, hydrogen binding, van der Walls interactions, etc.) exerted by each protein on the other protein in the complex. Factors affecting the strength of an interaction between two molecules are known to those skilled in the art. One useful measure of the strength of binding between two molecules, such as a peptide and a protein, is the dissociation constant (Kd). Preferred peptides of the present invention are those that bind to a CD40 protein with a Kd of no more than about 1×10−6 M, about 1×10−7 M, or about 1×10−8 M. Particularly preferred peptides are those having a Kd of less than about 1×10−9 M. In one embodiment, a peptide hereof binds to a CD40 protein with a Kd of less than 100 nM, less than 50 nM, less than 25 nM, less than 10 nM, less than 5 nM, less than 3 nM, less than 2 nM, or less than 1 nM. Methods of measuring and analyzing binding interactions between a peptide and a CD40 protein are known by those of skill in the art. - As used herein, the change the level of Th40 cells present in an animal, or in a culture of T cells may be indicative of modulation of inflammation. As used herein, the terms level, number, count and concentration can be used interchangeably. Modulation of inflammation may mean an increase or decrease in the number of Th40 cells present in the inflammatory environment; however, the modulation of inflammation should not be limited to cell numbers or counts. Consequently, modulation can be referred to as positive or negative. Positive modulation (also referred to as up-regulation) of inflammation may result in an increase in the number of Th40 cells in the inflammatory environment. Negative modulation (also referred to as down-regulation) of inflammation may result in a reduction in the number of Th40 cells present in the inflammatory environment. The level, count, or concentration of Th40 cells may not be indicative of inflammation in the inflammatory environment. A preferred peptide may be one that down-regulates inflammation, thereby reducing the number of Th40 cells present in the inflammatory environment. Positive and negative modulation of inflammation may or may not result in a change in the type and amount of immunoregulatory molecules present in the inflammatory environment. In some instances, it is possible that the Th40 levels will not change but the activity of those cells is altered such that they are no longer exerting or increasing inflammatory cytokines and other biomarkers of inflammation. Therefore, as used herein modulating inflammation may refer to changes in the Th40 levels, numbers, or concentration in a corporeal body or sample, and may also refer to changes in inflammation more generally that may be associated with disease, inflammatory cytokines, and/or cell derived inflammatory mediator molecules.
- It will be appreciated by those skilled in the art that both a cell culture system and the immune system of an animal comprise basal levels of immune cells and immunoregulatory molecules. The phrases basal level and normal level can be used interchangeably. With regard to the immune system of an animal, as used herein, the basal level of a type of immune cell (e.g., Th40 cell), or a immunoregulatory molecule, refers to the average number of that cell type, or immunoregulatory molecule, present in a population of individuals considered healthy (i.e., free of metabolic, autoimmune, or infectious disease). With regard to a cell culture system, as used herein, the basal level of a type of immune cell, or an immunoregulatory molecule, refers to the average level of that cell type, or immunoregulatory molecule, present in a population of cells that is non-activated. Those skilled in the art are capable of determining if a T-cell, or a population of such cells, is activated. For example, the expression of CD69, CD25 and/or CD154 proteins by a cell indicates that the cell has been activated.
- The basal level of a cell or molecule can be a specific amount (e.g., a specific concentration) or it can encompass a range of amounts. Basal levels, or ranges, of immune cells and immunoregulatory molecules are known to those in the art. For example, in a healthy individual, the normal level of CD4+ T-cells present in human blood is 500-1500 cells/ml. Variability in this measurement can result from differences in the method used to determine the cell count. Furthermore, normal levels of cells can also be reported as a percentage of a total cell population. For example, in a healthy individual, Th40 cells make up less than 25% of the total T cell population. Thus, as used herein, the term inflammation refers to an inflammatory environment in which Th40 cells make up greater than about 25%, greater than about 30%, greater than about 35%, greater than about 40%, greater than about 45%, greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, or greater than about 80% of the total T-cell population. Moreover, a preferred peptide herein is one that reduces the level of Th40 cells to less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 27%, or equal to about 25% of the total T-cell population. Methods of measuring different types of T-cells in the T-cell population are known to those skilled in the art. Furthermore, a novel method for detecting Th40 cells using peptides hereof is disclosed herein.
- As used herein, the phrase inflammatory environment refers to the overall population of immune cells, and related immunoregulatory molecules, that are present in a culture of cells, or in the body of an animal. As such, the phrase inflammatory environment encompasses the types, and/or the relative amounts of immune cells and immunoregulatory molecules (e.g., cytokines) present in a culture of cells, or in an animal, which are involved in affecting an inflammatory reaction. Examples of cells encompassed by the term inflammatory environment include, but are not limited to, T cells, neutrophils, macrophages, granulocytes, and the like. The inflammatory environment relates to cells and molecules that mediate both acute and chronic inflammation. It will be appreciated by those skilled in the art that the inflammatory environment refers to the system to which peptides hereof are administered. In one embodiment, the system is a cell culture system. In one embodiment, the system is a whole animal.
- A preferred peptide hereof is one that selectively interacts with a CD40 protein in solution, as determined using an assay such as an immunosorbent assay, or on the surface of a T-cell. As used herein, the terms selectively, selective, specific, and the like, indicate the peptide has a greater affinity for a CD40 protein than it does for proteins unrelated to the CD40 protein. More specifically, the terms selectively, selective, specific, and the like indicate that the affinity of the peptide for CD40 is statistically significantly higher than its affinity for a negative control (e.g., an unrelated protein such as albumin) as measured using a standard assay (e.g., ELISA). Suitable techniques for assaying the ability of a peptide to selectively interact with a CD40 protein are known to those skilled in the art. Such assays can be in vitro or in vivo assays. Examples of useful assays include, but are not limited to, an enzyme-linked immunoassay, a competitive enzyme-linked immunoassay, a radioimmunoassay, a fluorescence immunoassay, a chemiluminescent assay, a lateral flow assay, a flow-through assay, an agglutination assay, a particulate-based assay (e.g., using particulates such as, but not limited to, magnetic particles or plastic polymers, such as latex or polystyrene beads), an immunoprecipitation assay, an immunoblot assay (e.g., a western blot), a phosphorescence assay, a flow-through assay, a chromatography assay, a polyacrylamide gel electrophoresis (PAGE)-based assay, a surface plasmon resonance assay, a spectrophotometric assay, a particulate-based assay, an electronic sensory assay and a flow cytometric assay. Methods of performing such assays are well known to those skilled in the art. In one embodiment, an assay can be performed using cells in culture, or it can be performed in a whole animal. Assays can be designed to give qualitative, quantitative or semi-quantitative results, depending on how they are used and the type of result that is desired.
- One embodiment hereof is a peptide that interacts with a CD40 protein in such a manner as to affect the interaction of the CD40 protein with a CD154 protein, thereby modulating inflammation. The effect of the peptide on the CD40/CD154 interaction can be positive or it can be negative. For example, the peptide can interact with the CD40 protein in such a manner that the strength of the interaction between the CD40 protein and a CD154 protein is increased. Alternatively, the peptide can interact with the CD40 protein such that the strength of the interaction between the CD40 protein and a CD154 protein is decreased. Methods of measuring the strength of binding between the peptide and a CD40 protein are known to those skilled in the art. A preferred peptide hereof is one that reduces the strength of the interaction between a CD40 protein and a CD154 protein. Preferred peptides hereof reduce the strength of binding between a CD40 protein and a CD154 protein by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. A particularly preferred peptide is one that completely inhibits binding of CD40 to CD154. Complete inhibition of binding between CD40 and CD154 means that when a peptide hereof is brought into proximity with a CD40 protein and a CD154 protein under conditions that would normally allow the interaction of CD40 and CD154, no such interaction occurs and activation signals are not stimulated in the CD40-expressing cell. Consequently CD40/CD154 mediated modulation of inflammation does not occur. In one embodiment, the peptide interacts with the CD40 protein in such a manner as to reduce the level of inflammation in the system. In one embodiment, the peptide interacts with the CD40 protein in such a manner as to inhibit the development of inflammation in the system. In one aspect, the peptide may alter the way the cells may interact with other molecules that normally occur during cell-to-cell interactions. Such cell-to-cell interactions may be those that result in inflammation. The peptides of the developments hereof, may disrupt the inflammasome complex, which may promote the maturation of pro-inflammatory cytokines interleukin-1β (IL-1β) and interleukin-18 (IL-18). Furthermore, one aspect of the peptides hereof is that these peptides may disrupt the inflammasome complex of
caspase 1, PYCARD, NALP,caspase 5, nucleotide-binding oligomerization domain and leucine-rich repeat-containing receptors (NLRs) and ALRs (AIM2-like receptors). - One aspect of the peptides described herein is that administration of the peptide may alter the way the cell may interact with other molecules that normally occur during cell to cell interactions which may be indicative of or result in inflammation. One aspect of the peptides described herein is that these peptide(s) may disrupt the inflammasome and thus alter inflammatory outcomes.
- While peptides hereof can interact with any site on the CD40 protein, preferred peptides interact with the CD40 protein at a location that overlaps with the CD154 binding site. In one embodiment, a peptide hereof interacts with the CD40 protein at the CD154 binding site. An example of such a peptide is a CD40 ligand competitive antagonist. As used herein, peptides that interfere with, or inhibit, the binding of a CD154 protein to a CD40 protein are referred to as small interfering peptides (SIPs). As used herein a small interfering peptide is a peptide that, through physio-chemical properties, interferes with the interaction of a CD40 protein with a CD154 protein, thereby preventing activation signals from being delivered to the CD40-bearing cell, thus limiting the activation of the CD40-bearing cell, and consequently, inflammation. As demonstrated herein, the consequences of such interference are prevention of T-cell activation and propagation, and a prevention or reduction of inflammation.
- Additionally, a small interfering peptide, may, through its physio-chemical properties, interfere with the interaction of a CD40 protein with a CD154 protein, thereby preventing activation signals from being delivered to the CD40-bearing cell, thus limiting the activation of the CD40-bearing cell, and consequently, modulating, inhibiting, preventing, and/or reversing
type 2 diabetes. As demonstrated herein, the consequences of such interference are prevention of T cell activation and propagation, and a prevention, reduction, modulation, and reversal oftype 2 diabetic developments. - A peptide useful for practicing methods of the present developments should be of a size sufficient to interact with CD40 protein in such a manner as to modulate
type 2 diabetes. It is understood by those skilled in the art that preferred peptides are relatively short since they are easier and less expensive to produce. Preferred peptides are those that are less than 20 amino acids in length. A preferred peptide is one that is 4, 6, 8, 10, 13, 15, or 24 amino acids in length. In one embodiment, the peptide is an amino acid selected from the group of SEQ ID NO:3 (Core-sequence see Table 1), SEQ ID NO:4 (6-mer see Table 1), SEQ ID NO:5 (8-mer mouse see Table 1), SEQ ID NO:6 (8-mer human see Table 1), SEQ ID NO:7 (15-mer mouse see Table 1), SEQ ID NO:8 (15-mer human see Table 1), SEQ ID NO:9 (24-mer see Table 1), SEQ ID NO: 24 (10-mer see Table 1), SEQ ID NO: 25 (13-mer see Table 1), SEQ ID NO: 26 (24-mer see Table 1), SEQ ID NO: 27 (6-mer (Form 2) see Table 1), SEQ ID NO: 28 (6-mer (Form 3) see Table 1), SEQ ID NO: 29 (6-mer (Form 4) see Table 1), SEQ ID NO: 30 (6-mer (Form 4) see Table 1) and SEQ ID NO:32 (24-mer-mouse (Form 2)). The sequences of such peptides are shown below in Table 1 (which is an exact duplicate of the previously listed Table 1, but repeated here for the convenience of the reader). -
TABLE 1 SEQ ID NO SEQUENCE Description 1 MIETYSQPSP RSVATGLPAS MKIFMYLLTV SwissPro FLITQMIGSV LFAVYLHRRL DKVEEEVNLH 27548.2 Mouse EDFVFIKKLK RCNKGEGSLS LLNCEEMRRQ CD40 Ligand FEDLVKDITL NKEEKKENSF EMQRGDEDPQ (CD154 IAAHVVSEAN SNAASVLQWA KKGYYTMKSN Protein) LVMLENGKQL TVKREGLYYV YTQVTFCSNR EPSSQRPFIV GLWLKPSSGS ERILLKAANT HSSSQLCEQQ SVHLGGVFEL QAGASVFVNV TEASQVIHRV GFSSFGLLKL 2 MIETYNQTSP RSAATGLPIS MKIFMYLLTV SwissPro FLITQMIGSA LFAVYLHRRL DKIEDERNLH 29965 Human EDFVFMKTIQ RCNTGERSLS LLNCEEIKSQ CD40 Ligand FEGFVKDIML NKEETKKENS FEMQKGDQNP (CD154 QIAAHVISEA SSKTTSVLQW AEKGYYTMSN Protein) NLVTLENGKQ LTVKRQGLYY IYAQVTFCSN REASSQAPFI ASLCLKSPGR FERILLRAAN THSSAKPCGQ QSIHLGGVFE LQPGASVFVN VTDPSQVSHG TGFTSFGLLK L 3 KGYY Core-sequence 4 KKGYYT 6-mer 5 AKKGYYTM 8-mer-mouse 6 AEKGYYTM 8-mer human 7 VLQWAKKGYYTMKSN 15-mer-mouse 8 VLQWAEKGYYTMSNN 15-mer human 9 NAASVLQWAKKGYYTMKSNLVMLE 24-mer 10 ISQAVHAAHAEINEAGR 15-mer from ovalbumin; control peptide 11 G-L-Q-W-A-K-K-G-Y-Y-T-M-K-S-N Gly-1 12 V-G-Q-W-A-K-K-G-Y-Y-T-M-K-S-N Gly-2 13 V-L-G-W-A-K-K-G-Y-Y-T-M-K-S-N Gly-3 14 V-L-Q-G-A-K-K-G-Y-Y-T-M-K-S-N Gly-4 15 V-L-Q-W-G-K-K-G-Y-Y-T-M-K-S-N Gly-5 16 V-L-Q-W-A-G-K-G-Y-Y-T-M-K-S-N Gly-6 17 V-L-Q-W-A-K-G-G-Y-Y-T-M-K-S-N Gly-7 18 V-L-Q-W-A-K-K-G-G-Y-T-M-K-S-N Gly-8 19 V-L-Q-W-A-K-K-G-Y-G-T-M-K-S-N Gly-9 20 V-L-Q-W-A-K-K-G-Y-Y-G-M-K-S-N Gly-10 21 V-L-Q-W-A-K-K-G-Y-Y-T-G-K-S-N Gly-11 22 ISQAVHAAHAEINEAGR 15-mer from ovalbumin; control peptide 23 YVQGKANLKSKLMYT Scrambled peptide 24 WAKKGYYTMK 10-mer mouse 25 VLQWAKKGYYTMK 13-mer mouse 26 AASVLQW AKKGYYTMKSNLVVLEN 24-mer mouse 27 KGYYTM 6-mer (Form 2) 28 AEKGYY 6-mer (Form 3) 29 AKKGYY 6-mer (Form 4) 30 AKGYYT 6-mer (Form 5) 31 YKNVKQMAYWLTGKS Scrambled peptide 32 AASVLQWAKKGYYTMKSNLVMLEN 24-mer-mouse (Form 2) - Interaction of a CD40 protein and a CD154 protein has been shown to occur at particular regions within each protein. The inventors have now shown that, surprisingly, a peptide comprising only a short portion of the CD154 region that interacts with CD40 is capable of binding to a CD40 protein, thereby modulating
type 2 diabetes. Thus one embodiment hereof is a peptide that comprises at least a portion of the amino acid sequence of a CD154 protein such that the peptide interacts with CD40 protein in such a manner as to modulatetype 2 diabetes. In one embodiment, interaction of the peptide with CD40 protein results in negative modulation, reduction, or inhibition oftype 2 diabetes. In one aspect, the peptide comprises at least a portion of SEQ ID NO:1 or SEQ ID NO:2. - In one aspect, the peptide is as short as possible yet comprises enough of the CD154 protein to allow interaction with a
CD 40 protein in such a manner as to modulatetype 2 diabetes. In one embodiment, a peptide hereof comprises 6, 13 or 15 contiguous amino acids from SEQ ID NO:1 or SEQ ID NO:2, and interacts with CD40 in such a manner as to modulatetype 2 diabetes. A preferred peptide comprises a core sequence of lysine-glycine-tyrosine-tyrosine (KGYY; SEQ ID NO:3), which corresponds to amino acids 142-145 of SEQ ID NO:1 and amino acids 143-146 of SEQ ID NO:2. Moreover, another preferred peptide comprises a core sequence of lysine-glycine-tyrosine-tyrosine-threonine-methionine (KGYYTM; SEQ ID NO:27), which corresponds to amino acids 142-147 of SEQ ID NO:1 and amino acids 143-148 of SEQ ID NO:2. Useful peptides can comprise additional regions of sequence from SEQ ID NO:1 or SEQ ID NO:2 that are adjacent to the core sequence, so long as the peptide is capable of modulatingtype 2 diabetes. In one embodiment hereof, a peptide comprises at least one sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30 and SEQ ID NO:32 so long as the peptide interacts with CD40 protein in such a manner as to modulatetype 2 diabetes. In one embodiment of the present subject matter, a peptide hereof is a sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30 and SEQ ID NO:32. - While peptides of the present subject matter can be selected entirely of or from sequences that are responsible for the interaction of the peptide with a CD40 protein, they may additionally contain amino acid sequences that do not interact with a CD40 protein, but which have other useful functions. Any useful, additional amino acid sequence can be added to the CD40-interacting sequence, so long as the additional sequences do not have an unwanted effect on the ability of the CD40 interacting sequence to interact with a CD40 protein. For example, in addition to the amino acid sequence responsible for interacting with a CD40 protein, a peptide hereof can contain amino acid sequences that are useful for visualizing or purifying the peptide. Such sequences act as labels (e.g., enzymes) or tags (antibody binding sites). Additionally, the developments presented herein demonstrate that substitutions of amino acids at any position other than the position-7 (K) of the 15-mer and the position-9 (Y) of the 15-mer may be made and the integrity and function of the peptide may be maintained (see
FIG. 8 ). Accordingly, these developments may contemplate that numerous substitutions may be possible while still maintaining the beneficial aspects of the peptide. - Any useful, additional amino acid sequence can be added to the CD40-interacting sequence, so long as the additional sequences do not have an unwanted effect on the ability of the CD40 interacting sequence to interact with a CD40 protein. For example, in addition to the amino acid sequence responsible for interacting with a CD40 protein, a peptide hereof can contain amino acid sequences that are useful for visualizing or purifying the peptide. Examples of such labels and tags include, but are not limited to, B-galactosidase, luciferase, glutathione-s-transferase, thioredoxin, HIS-tags, biotin tags, and fluorescent tags. Moreover, acetyl groups and amides may be appended on the N-terminus or C-terminus, and the developments hereof contemplate these and other variations that may enhance stability or other traits desired of such a peptide. Other useful sequences for labeling and tagging proteins are known to those of skill in the art.
- Likewise, peptides hereof can be modified, so long as such modification does not significantly affect the ability of the peptide to modulate
type 2 diabetes. Such modifications can be made, for example, to increase the stability, solubility or absorbability of the protein. Examples of such modifications include, but are not limited to pegylation, glycosylation and chemical modification of the peptide. - Peptides hereof may possibly be derived from nature (e.g., obtained from plants, animals or microorganisms) or they can be produced in a laboratory (e.g., recombinantly or synthetically). Preferred peptides are those that are synthesized. Also encompassed are peptides that are combinations of natural and synthetic molecules. General methods for producing and isolating recombinant or synthetic peptides are known to those skilled in the art. It should be noted that, as used herein, an isolated, or biologically pure, molecule, is one that has been removed from its natural milieu. As such the terms isolated, biologically pure, and the like, do not necessarily reflect the extent to which the protein has been purified.
- The peptides hereof do not arise naturally in a corporeal body, but rather must be constructed and synthesized to obtain the small interfering peptides. Certain aspects of the design and synthesis may affect the peptides stability and ability to perform its intended use. The peptides hereof may vary in length from four amino acids in length as in SEQ ID NO:3, five amino acids in length, six amino acids in length as in SEQ ID NOs: 4, 27, 28, 29, and 30, seven amino acids in length, eight amino acids in length as in SEQ ID NOs: 5 and 6, nine amino acids in length, ten amino acids in length as in SEQ ID NOs: 24, eleven amino acids in length, twelve amino acids in length, thirteen amino acids in length as in SEQ ID NO: 25, fourteen amino acids in length, fifteen amino acids in length as in SEQ ID NOs: 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, and 20, sixteen amino acids in length, seventeen amino acids in length, eighteen amino acids in length, nineteen amino acids in length, twenty amino acids in length, twenty-one amino acids in length, twenty-two amino acids in length, twenty-three amino acids in length, twenty-four amino acids in length as in SEQ ID NO: 26 and 32, and twenty-five amino acids in length. In some instances, the embodiments of this development may be up to fifty or more amino acids in length and in such instances, repeats of the core sequence of SEQ ID NO: 3, may occur one, two, three, four, five, six, seven, eight, nine, ten, eleven or more times in such a peptide. In other instances, the embodiments of this development may be up to fifty or more amino acids in length, and in such instances repeats of the core sequence of SEQ ID NO:3, may be varied with other sequences known to provide the desired effect such as those of SEQ ID NOS: 4-9, 25-30, and 32. The repeats and sequences of said variations are countless; however, the developments herein contemplate a peptide that maintains its ability to perform its intended use of interacting with CD40 in such a manner as change, control, or affect inflammation in the subject.
- As has been described herein, interaction of the CD40 protein and the CD154 protein are necessary for involvement of Th40 cells in
type 2 diabetes. Consequently, inhibition of the interaction between a CD40 and CD154 protein using peptides hereof is a useful method of affectingtype 2 diabetes. Thus, one embodiment is a method to reduce the interaction between a CD40 protein and a CD154 protein comprising introducing into an environment containing a CD40 protein and a CD154 protein, a peptide, that interacts with the CD40 protein in such a manner as to reduce the interaction between the CD40 protein and the CD154 protein. In one aspect hereof, the peptide reduces the interaction between the CD40 protein and the CD154 protein by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In one embodiment, the peptide reduces the interaction between the CD40 protein and the CD154 protein by a factor of at least 10, at least 100, at least 1,000, at least 10,000. Methods of measuring the strength of the interaction between the CD40 protein and the CD154 protein have been discussed previously, and are also know to those of skill in the art. - One embodiment hereof is a method to modulate
type 2 diabetes comprising contacting a CD40 protein with a peptide that interacts to the CD40 protein in such a manner as to modulate inflammation. In one aspect, interaction of the peptide with the CD40 protein increases the number of Th40 cells by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In one embodiment, interaction of the peptide with the CD40 protein increases the number of Th40 cells by a factor of at least 10, at least 100, at least 1,000, at least 10,000. - One aspect is a method to reduce
type 2 diabetes in a patient, the method comprising administering a peptide hereof to the patient. In one embodiment, the peptide comprises an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO:30 and SEQ ID NO:32. In one embodiment, the peptide is an amino acid sequence selected from SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:27, SEQ ID NO: 28, and SEQ ID NO: 29. In a preferred embodiment, interaction of the peptide with the CD40 protein decreases the number of Th40 cells by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In another embodiment, interaction of the peptide with the CD40 protein decreases the number of Th40 cells by a factor of at least 10, at least 100, at least 1,000, at least 10,000. In a preferred embodiment, the level of Th40 cells is reduced so that Th40 cells comprise no more than about 20%, about 25%, about 30%, about 35%, or about 40% of the total T-cell population. - Peptides and methods hereof are suitable for use in cell culture as well as for treating a patient. As used herein the term patient refers to any animal in need of such treatment. The animal can be a human or a non-human animal. A preferred animal to treat is a mammal. A peptide can be administered or applied per se, or as pharmaceutical compositions. A peptide hereof, or a pharmaceutical composition thereof, can be administered to a patient by a variety of routes, including, but limited to, by injection (e.g., intravenous, intramuscular, subcutaneous, intrathecal, intraperitoneal), by inhalation, by oral (e.g., in a pill, tablet, capsule, powder, syrup, solution, suspension, thin film, dispersion or emulsion.), transdermal, transmucosal, pulmonary, buccal, intranasal, sublingual, intracerebral, intravaginal rectal or topical administration or by any other convenient method known to those of skill in the art.
- The amount of a peptide hereof and/or a pharmaceutical composition thereof that will be effective can be determined by standard clinical techniques known in the art. Such an amount is dependent on, among other factors, the patient being treated, including, but not limited to the weight, age, and condition of the patient, the intended effect of the compound, the manner of administration and the judgment of the prescribing physician. Also, in this context, it should be noted that in treating a patient exhibiting a disorder of interest, a therapeutically effective amount of an agent or agents such as these is administered. A therapeutically effective dose refers to that amount of the compound that results in amelioration of one or more symptoms or a prolongation of survival in a patient.
- A peptide hereof, or a pharmaceutical composition thereof, can be administered alone or in combination with one or more other pharmaceutical agents, including other compounds of the present disclosure. The specific pharmaceutical composition depends on the desired mode of administration, as is well known to the skilled artisan.
- Because the inventors have discovered that Th40 cells are intimately involved in the development of autoimmune diseases and
type 2 diabetes, the peptides and methods disclosed herein can be used to affect conditioning resulting from such diseases. Thus, one embodiment hereof is a method to treattype 2 diabetes in a patient in need of such treatment, the method comprising administering to a patient a peptide that interacts with the CD40 protein, thereby reducingtype 2 diabetes. In one embodiment the peptide interacts with the CD40 protein in such a manner as to affect the interaction of CD40 and CD154, thereby reducingtype 2 diabetes. In a preferred embodiment, interaction of the peptide with the CD40 protein reduces the number of Th40 cells in a patient to a level equal to that observed in subjects that do not havetype 2 diabetes. In another embodiment, interaction of the peptide with the CD40 protein reduces the inflammatory cytokine levels in a patient. In another embodiment, interaction of the peptide with the CD40 protein reduces the inflammatory cytokine levels in a patient to a level equal or similar to those observed in subjects that do not havetype 2 diabetes. The present developments are suitable for treating any patient having an autoimmune disease and/or cardiovascular disease, the development of which may be related to, correlated with, or dependent on Th40 cells. The present developments may also be suitable for treating any patient having an autoimmune disease and/or cardiovascular disease, the development of which is not related to, correlated with, or dependent on Th40 cell counts, levels, and or concentrations. - In one embodiment, more specifically, peptides hereof may be suitable for reducing the level of Th40 cells in such patients. In this embodiment, a peptide hereof may reduce the level of Th40 cells in a patient suffering from an autoimmune disease to no more than about 25% of the total T-cell population.
- One example of a disease that is particularly amenable to treatment using a peptide of the present developments may be
type 2 diabetes. Intype 2 diabetes, glucose tolerance is reduced, insulin sensitivity is decreased, and plasma insulin levels are increased. Consequently, control of inflammatory cells and cell signaling via CD40-CD154 interaction may be able to be used to control, modulate, reduce and/orreverse type 2 diabetes symptoms that are characterized as by glucose intolerance, insulin resistance, and increased plasma insulin levels. Several murine models of T2D and/or atherosclerosis have been developed. For initial studies, ApoE−/− mice were selected due to their ability to developtype 2 diabetes from a high fat diet. Glucose tolerance and insulin testing were performed on all of the mice. The mice were administered the 6-mer peptide (SEQ ID NO:29) at a rate of 1 mg/kg weekly via I.V. injection and monitored. Peptide treated ApoE deficient mice demonstrated significantly increased glucose tolerance as well as significantly improved insulin sensitivity, improve insulin resistance, and lowered plasma insulin levels compared to controls. Thus, one embodiment of the present developments is a method to preventtype 2 diabetes in an individual at risk for developingtype 2 diabetes, the method comprising administering to the individual a peptide to selectively bind to a CD40 expressing cell. In such an embodiment, the peptide may be selected from SEQ ID NOS: 3-9 and 24-30. - The developments hereof have also shown that, surprisingly, peptides hereof can be used to reverse the disease process in individuals already showing signs of
type 2 diabetes. Thus, one aspect of the present subject matter is a method to reversetype 2 diabetes comprising administering to a patient diagnosed as havingtype 2 diabetes, a peptide hereof. In one embodiment, the peptide comprises an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, so long as the peptide can down-regulate or reduce inflammation. In one embodiment, the peptide is an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30. As used herein the phrase to reversetype 2 diabetes means to increase glucose tolerance, decrease insulin resistance, and decrease plasma insulin levels to levels closer to or more comparable to those observed in individuals who do not havetype 2 diabetes. - In yet another development hereof has shown that, surprisingly, peptides hereof can be used to reverse the disease process in individuals already showing signs of
type 2 diabetes. Thus, one aspect of the present subject matter is a method to reversetype 2 diabetes comprising administering to a patient diagnosed as havingtype 2 diabetes, a peptide hereof. In one embodiment, the peptide comprises an amino acid sequence selected from SEQ ID NOS: 3-9 and 24-30, so long as the peptide can control, modulate, reduce and/or reverse inflammation. In one embodiment, the peptide is an amino acid sequence selected from SEQ ID NOS: 3-9 and 24-30, so long as the peptide can control, modulate, reduce and/orreverse type 2 diabetes. Furthermore, in one aspect of this embodiment a peptide hereof may reduce the level of Th40 cells in a patient suffering from an autoimmune disease to no more than about 25% of the total T-cell population. In an alternative embodiment, interaction of the peptide with the CD40 protein reduces the number of Th40 cells in a patient to a level equal to that observed in subjects that do not havetype 2 diabetes. In another embodiment, interaction of the peptide with the CD40 protein reduces the inflammatory cytokine levels in a patient. In another embodiment, interaction of the peptide with the CD40 protein reduces the inflammatory cytokine levels in a patient to a level equal or similar to those observed in subjects that do not havetype 2 diabetes. The present developments may be suitable for treating any patient having an autoimmune disease and/or cardiovascular disease, the development of which may be related to, correlated with, or dependent on Th40 cells. The present developments may also be suitable for treating any patient having an autoimmune disease and/or cardiovascular disease, the development of which is not related to, correlated with, or dependent on Th40 cell counts, levels, and or concentrations. - As has been described, peptides of the present invention selectively bind to a CD40 expressing cell. Consequently, peptides of the present subject matter can be used to identify Th40 cells. Thus one embodiment hereof is a method to detect Th40-
dependent type 2 diabetes, said method comprising contacting a T-cell population with a peptide hereof. In a preferred embodiment, the peptide is labeled with a detectable marker, such as, for example, fluorescein, luciferase or alkaline phosphatase. Such detection can be performed using assay techniques known to those skilled in the art. In general, an assay for detecting Th40 cells using a peptide hereof comprises (a) obtaining a sample of cells; (b) contacting a peptide hereof with said cells under condition suitable to allow binding of the peptide to Th40 cells, if present; (c) washing said cells using conditions that disrupt non-specific interactions, and that remove unbound peptide; and (d) detecting peptide bound to cells. Detection of bound peptide can be achieved directly or indirectly. For example, direct detection can be achieved using a peptide labeled using a detectable marker, as disclosed herein. Following the wash step listed above, the cells are then simply screened for the presence of detectable marker. The presence of detectable marker in the cell sample indicates the presence of Th40 cells, and thus Th40-dependent type 2 diabetes. Alternatively, indirect detection involves the use of a second molecule, such as an antibody, that binds to the peptide. In an indirect detection assay, following the wash step listed above, a detection molecule that binds the peptide is added to the cell sample. The detection molecule is labeled with a detectable marker. After washing away unbound detection molecule, the cells are screened for the presence of detectable marker. The presence of detectable marker in the cell sample indicates the presence of Th40 cells. It should be understood that the assays described herein are meant as examples of useful assays, and other assay techniques can be employed. Suitable assay techniques are known to those skilled in the art, and are also disclosed in, for example, Molecular Cloning: A Laboratory Manual, Sambrook, J., Fritsch, E. F., and Maniatis, T, Cold Spring Harbor Laboratory Press; 2nd Edition (December 1989). All references cited herein are incorporated herein in their entirety. - The assay technology described above can also be used to identify other molecules that affect the interaction of a CD40 protein with a CD514 protein. Examples of such molecules include, but are not limited to, proteins, peptides and small molecules. For example, assays can be designed that test the ability of molecules to compete with a peptide of the present developments for binding to a Th40 cell. For instance, a peptide labeled with a detectable marker, can be mixed with a test molecule and a population of cells known to contain Th40 cells, under conditions that allow binding of the peptide to the Th40 cells. Following an appropriate incubation period, the cells are washed to remove unbound peptide, and the cells screened for the presence of detectable marker. Alternatively, the labeled peptide could be bound to Th40 cells first, and after a wash step to remove unbound peptide, the test molecule could be added to the cells containing bound peptide. Following an incubating period and a wash step to remove unbound molecule, or released peptide, the cells are screened for the presence of detectable marker. In either case, absence of the detectable marker in the cell sample indicates the test molecule is able to compete with the peptide for binding to the Th40 cells, while presence of the detectable marker would indicate the test molecule does not inhibit binding of the peptide to Th40 cells. Inhibition of binding need not be 100%, as such assay would also be useful for identifying molecules that partially inhibit binding of the peptide to Th40 cells. It is understood by those skilled in the art that such assays would involve the use of positive controls (e.g., unlabeled peptide) and negative controls (e.g., a protein/molecule that is known not to bind to Th40 cells).
- The assay technology above can also be used to identify other molecules that affect the interaction of a CD40 protein with a CD154 proteins. Examples of such molecules include, but are not limited to, proteins, peptides and small molecules. For example, assays can be designed that test the ability of molecules to compete with a peptide of the present developments for binding to a CD40 protein of cells other than T cells, such as neutrophils, eosinophils, basophils, mast cells, macrophages, platelets, endothelial cells, and lymphocytes, including natural killer cells and B cells. For instance, a peptide labeled with a detectable marker, can be mixed with a test molecule and a population of cells known to contain CD40 containing cells, under conditions that allow binding of the peptide to the CD40 bearing cells. Following an appropriate incubation period, the cells are washed to remove unbound peptide, and the cells screened for the presence of detectable marker. Alternatively, the labeled peptide could be bound to CD40 bearing cells first, and after a wash step to remove unbound peptide, the test molecule could be added to the cells containing bound peptide. Following an incubating period and a wash step to remove unbound molecule, or released peptide, the cells are screened for the presence of detectable marker. In either case, absence of the detectable marker in the cell sample indicates the test molecule is able to compete with the peptide for binding to the CD40 bearing cells, while presence of the detectable marker would indicate the test molecule does not inhibit binding of the peptide to CD40 bearing cells. Inhibition of binding need not be 100%, as such assay would also be useful for identifying molecules that partially inhibit binding of the peptide to CD40 bearing cells. It is understood by those skilled in the art that such assays would involve the use of positive controls (e.g., unlabeled peptide) and negative controls (e.g., a protein/molecule that is known not to bind to CD40 bearing cells).
- Because increased levels of Th40 cells are associated with the development of autoimmune disease, the present developments can be used to identify patients at risk for developing autoimmune disease and autoimmune
related type 2 diabetes. Thus, one embodiment of the present developments is a method to identify a patient at risk for developing autoimmunerelated type 2 diabetes. In one embodiment, patients at risk for developingtype 2 diabetes are identified by obtaining a sample from a patient to be tested, contacting the T-cell portion of said sample with a peptide hereof, and determining the level of Th40 cells present in the sample, wherein a level of Th40 cells greater than about 25% of the total T-cell population indicates the patient is at risk for developingtype 2 diabetes. In one embodiment, the peptide comprises an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, so long as the peptide binds to the CD40 protein. In one embodiment, the peptide is an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30. In a preferred embodiment the peptide is labeled with a suitable detectable marker such as, for example, fluorescein, luciferase or alkaline phosphatase. In yet another embodiment, the peptide comprises an amino acid sequence selected from SEQ ID NOs: 4-9, 24-30, and 32, so long as the peptide binds to the CD40 protein. - The present developments also comprise kits useful for practicing the methods disclosed herein, the kit comprising a peptide that interacts with a CD40 protein in such a manner as to modulate, reduce, prevent, treat, or otherwise improve symptoms of
type 2 diabetes. In one embodiment, the peptide comprises an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, so long as the peptide can modulatetype 2 diabetes. In one embodiment, the peptide is an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30. Another embodiment is a kit for determining the level of Th40 cells, the kit comprising a peptide that interacts with a CD40 protein, and methods for detecting CD40-bound peptide. Kits can also contain associated reagents and components, such as, but not limited to, buffers, labels, containers, inserts, tubing, vials, syringes, and the like. - The present developments also comprise kits useful for practicing the methods disclosed herein, the kit comprising a peptide that interacts with a CD40 protein in such a manner as to modulate, reduce, prevent, treat, or otherwise improve symptoms of
type 2 diabetes. In one embodiment, the peptide comprises an amino acid sequence selected from SEQ ID NOs: 3-9 and 24-30, so long as the peptide can modulatetype 2 diabetes. In one embodiment, the peptide is an amino acid sequence selected from SEQ ID NOs: 3-9 and 24-30. Another embodiment is a kit for determining the level of Th40 cells, the kit comprising a peptide that interacts with a CD40 protein, and methods for detecting CD40-bound peptide. Kits can also contain associated reagents and components, such as, but not limited to, buffers, labels, containers, inserts, tubing, vials, syringes, and the like. - The following examples are provided for the purposes of illustration and are not intended to limit the scope of the present invention.
- This Example demonstrates the effect of various peptide fragments of CD154 on CD4/CD8 ratios and the development of diabetes in NOD mice.
- Peptides were designed based on the amino acid sequence of mouse CD154 protein (SEQ ID NO:1) in the SwissPro database. The peptides (8-mer (SEQ ID NO: 5; SEQ ID NO: 6), 10-mer (SEQ ID NO:24), 13-mer (SEQ ID NO:25), 15-mer (SEQ ID NO: 7), 24-mer (SEQ ID NO:26), scrambled (SEQ ID NO: 23), and RGD (arginylglycylaspartic acid) were then ordered from New England Peptide. The RGD peptide is a 15-amino acid sequence from the CD154 sequence that does not include the CD40 binding motif. The lyophilized peptides were suspended in sterile saline at 1 mg/ml. 25 ug in 100 ul (1 mg/kg) of a particular peptide was then injected into the tail vein of 6-week old NOD mice. Control mice received 100 ul of sterile saline. This is well before the onset of diabetes (and atherosclerosis), but after damage to pancreatic islets has begun. Weekly after the initial injection, another 25 ug of peptide (or 100 ul of saline in the case of the Control mice) was injected into the tail vein. At 10 weeks of age, mice were monitored for diabetes, as indicated by a blood glucose level greater than 250 mg/dL for three consecutive days. The results of this study are shown in
FIG. 1 . During this time, blood was also taken from the tail vein, or by sub-mandibular venal puncture, and the level of CD4+ and CD8+ cells determined by flow cytometry using antibodies for CD4 protein and CD8 protein. The results of this analysis are shown inFIG. 2A . - Pancreata were excised and examined by histology for cellular infiltrates and assigned scores based on observable, measurable, and quantifiable data: 0=no infiltrate; 1=one pole infiltrate; 2=peri-insulitis, bi-polar-infiltrates; 3=75% infiltrate and 4=full infiltration. The results of this analysis are shown in
FIG. 2B . - The results demonstrate that treatment with a peptide unrelated to the CD154 protein did not reduce the development of diabetes in NOD mice. In contrast, treatment of mice with a 15-mer peptide derived from the CD154 protein prevented the onset of diabetes. Further, the 13-mer peptides derived from the CD154 protein had significant effects on the development of diabetes. In addition, the data demonstrates that the 15-mer peptide did not result in compromise of the immune system, as determined by the CD4/CD8 ratio.
- This Example demonstrates the effect of the 15-mer peptide on hyperglycemia in newly diabetic NOD mice.
- Six mice from Example 1 that were not treated were allowed to subsequently develop diabetes. These mice were injected intravenously with 100 ug of the 15-mer peptide. These mice were then given weekly injections of the 15-mer peptide into their tail veins, and their blood glucose levels monitored twice-weekly. The 15-mer peptide was administered for a total of ten weeks, after which the treatment was stopped. The results of this study are shown in
FIG. 3 . - This study demonstrates that injection of the 15-mer peptide into already diabetic mice can reverse hyperglycemia. It also demonstrates that cessation of the treatment results in return of hyperglycemia within 5 weeks.
- This study demonstrates the ability of the 15-mer peptide to bind to Th40 cell and B cells.
- Total lymphocytes were isolated from 9 week old NOD mice. The lymphocytes were incubated with anti-CD4, anti-CD8, anti-CD40 and an FITC-labeled 15-mer peptide, and then analyzed by flow cytometry. Cells were gated for CD4 (both CD4hi and CD4lo populations were included) and CD40 versus the 15-mer peptide. The results of this analysis are shown in
FIG. 4 . - B cells were isolated from the spleens of NOD mice. Sorted MHC-II+ cells were purified from total lymphocytes. Cells were stained with FITC-labeled 15 mer peptide, anti-CD40, and B cell markers CD19 and CD21. MHC-II+ cells were gated for CD19+ and CD21+ and then 15-mer peptide versus CD40 antibody was measured. The results of this study are shown in
FIG. 5 . - This study shows that a substantial majority, 90% of CD40+ T-cells, also bind the 15-mer peptide, thereby demonstrating that the 15-mer peptide is highly specific for CD40+ cells. It also shows that while 90% of B cells were CD40 positive, only 8% of B cells bound the 15-mer peptide.
- This example demonstrates the level of CD40 positive cells in the blood of type-I diabetic subjects and non-diabetic (control) subjects.
- 1 ml of whole blood was obtained from each individual and incubated with biotin-conjugated, 15-mer peptide. The cells were then exposed to horseradish peroxidase (HRP)-avidin, washed and the absorbance at 405 nm determined using a spectrophotometer. The results of this study are shown in
FIG. 6 . - This study demonstrates that blood cells from patients having type-I diabetes had higher 15-mer peptide binding activity than cells from non-diabetic controls.
- This example demonstrates the level of insulin granulation observed in the pancreas of NOD mice treated with either the 15-mer peptide or a peptide from ovalbumin.
- At the onset of diabetes, six NOD mice were injected with 100 ug/ml of the 15-mer peptide, resulting in the reversal of hyperglycemia in 80% of the recipients. Six weeks after reversal of hyperglycemia, mice were sacrificed, and the pancreas removed for analysis. The pancreas was fixed, sectioned and then stained using an aldehyde/fuschsin stain that allows detection of insulin granules. Granulation of the tissue was scored as follows: 4=completely granulated; 3=75% of islet granulated; 2=50% of islet granulated, and peri-insulitis; 1=25% of islet granulated; 0=no insulin granules detected. The results of this analysis are shown in
FIG. 7 . - This analysis demonstrates that the 15-mer peptide preserved insulin granules in the majority of the mice, and was significantly improved in peptide-reversed diabetic mice compared to diabetic mice that received an irrelevant peptide.
- This example demonstrates the effect of mutations in the 15-mer peptide on its ability to prevent the onset of diabetes.
FIG. 8 provides results related to this Example 6. - Peptide were designed and produced as described in Example 1. Variant peptides were produced so that in each variant, a glycine was substituted for an amino acid corresponding to an amino acid in positions 1-7 or 9-12 of SEQ ID NO:7, as follows:
-
Gly-1 (SEQ ID NO: 11) G-L-Q-W-A-K-K-G-Y-Y-T-M-K-S-N Gly-2 (SEQ ID NO: 12) V-G-Q-W-A-K-K-G-Y-Y-T-M-K-S-N Gly-3 (SEQ ID NO: 13) V-L-G-W-A-K-K-G-Y-Y-T-M-K-S-N Gly-4 (SEQ ID NO: 14) V-L-Q-G-A-K-K-G-Y-Y-T-M-K-S-N Gly-5 (SEQ ID NO: 15) V-L-Q-W-G-K-K-G-Y-Y-T-M-K-S-N Gly-6 (SEQ ID NO: 16) V-L-Q-W-A-G-K-G-Y-Y-T-M-K-S-N Gly-7 (SEQ ID NO: 17) V-L-Q-W-A-K-G-G-Y-Y-T-M-K-S-N Gly-9 (SEQ ID NO: 18) V-L-Q-W-A-K-K-G-G-Y-T-M-K-S-N Gly-10 (SEQ ID NO: 19) V-L-Q-W-A-K-K-G-Y-G-T-M-K-S-N Gly-11 (SEQ ID NO: 20) V-L-Q-W-A-K-K-G-Y-Y-G-M-K-S-N Gly-12 (SEQ ID NO: 21) V-L-Q-W-A-K-K-G-Y-Y-T-G-K-S-N - NOD mice were placed in groups of 10, and the mice in each group injected IV weekly with 25 ug of either wild-type (WT; Legend) peptide or a variant peptide (in PBS, ph 7.2) listed above. The development of diabetes was monitored by measuring blood glucose levels on a weekly basis. Mice were considered “diabetic” when blood glucose was 250 mg/dl or greater for 3 consecutive readings. Injections began at 6 weeks of age=pre-diabetes.
- This example demonstrates that substitution of a glycine at any of positions 1-7, or 9-12, reduces the ability of the 15-mer peptide to inhibit the development of diabetes. It also shows that such mutations do not completely abolish the ability of the mutated 15-mer peptide to inhibit the development of diabetes. Substitutions at the
Tyr position 9,Thr position 11, andMet position 12 proved differential for hyperglycemic prevention activity, as shown inFIG. 8 . Therefore it is also postulated that a second 6-mer derivative—SEQ ID NO:27-6-mer (Form 2), SEQ ID NO:28-6-mer (Form 3), SEQ ID NO:29-6-mer (Form 4), and SEQ ID NO:30-6-mer (Form 5) may also provide increased therapeutic efficacy. - This example demonstrates that the same elevation of Th40 cell levels in the ApoE deficient mouse model of atherosclerosis is also notably elevated in
human Type 1 Diabetes (T1D). - The peripheral blood was measured was measured for total count of CD3+CD4+CD40+ cell numbers in NOD, NOR (non-obese diabetic resistant), and BALB/c (control) mice as in
FIG. 9 . This was compared to the percentage of Th40 cells in peripheral blood in human subjects for control, diabetic/new onset, and long term diabetic populations as inFIG. 10 . Further, lymphocytes were isolated from 9-week old NOD mice. The lymphocytes were incubated with anti-CD, anti-CD8, and an FITC-labeled 15-mer peptide, and then analyzed by flow cytometry. Cells were gated for CD4 (both CD4hi and CD4lo populations were included) and CD4 versus the 15-mer peptide. These results are displayed inFIG. 11 . - ApoE deficient mice on a normal chow diet were selected to receive a dose of 1 mg/kg of the 15-mer peptide (SEQ ID NO: 7) by IV tail injection, three times a week over a period of 26 weeks, beginning at 9 weeks of age and also utilized a control. At 25 weeks, the animals were euthanized, weighed, and then had blood, spleen, and pancreas removed for analysis. The subjects were then perfused through cardiac puncture with 4% paraformaldehyde. Aortic arches were dissected, dehydrated in sucrose gradient and then flash frozen. Approximately thirty-five 8 um longitudinal sections were obtained per mouse for various staining procedures. Flow cytometry was performed utilizing a MACSQuant® Analyzer 10 (Miltenyi Biotec Inc.). Additional analysis was performed using FlowJo® (FlowJo, LLC wholly owned by BectonDickinson, Inc.) single-cell flow cytometry software.
- C57BL/6 and ApoE−/− mice demonstrated increased levels of Th40 cells relative to all CD3+CD4+ cells prior to hyperglycemia as demonstrated in
FIG. 12 . - Further, NOD mice tested in this study demonstrated significant Th40 infiltration in the aorta compared with control and young non-diabetic NOD mice populations, as shown in
FIG. 13 . - An exemplar aortic plaque of one of the longitudinal sections was observed at 200× magnification using oil-red-0, trichrome stain and immune-fluorescence, and is shown in
FIG. 14 . This microscopy and stain of the aorta showed that not only are the Th40 cells increased in the aorta similarly to the peripheral blood as demonstrated inFIGS. 10-14 , but also the Th40 cells are found within the shoulder region of plaque in the ApoE−/−model (the growth region of plaque/atherosclerosis). InFIGS. 14 , 10 and 20 identify cells that represent CD3+, CD4+, and CD40+ (Th40 cells) that have significant intracellular CD40. 30 demonstrates Th40 cell with extracellular expression and no demonstrative CD40 intracellularly. 40 identifies CD3+, CD4+, CD40neg cell. - This example demonstrates that CD3+CD4+CD40+ cells appear to produce interferon gamma (INFγ) in abundance. Additionally, interferon gamma controls Th40 proliferation.
- ApoE deficient mice on a normal chow diet were selected to receive a dose of 1 mg/kg of the 15-mer peptide (SEQ ID NO: 7) by IV tail injection, three times a week over a period of 26 weeks, beginning at 9 weeks of age and also utilized a control. At 25 weeks, the animals were euthanized, weighed, and then had blood, spleen, and pancreas removed for analysis. The subjects were then perfused through cardiac puncture with 4% paraformaldehyde. Aortic arches were dissected, dehydrated in sucrose gradient and then flash frozen. Approximately thirty-five 8 m longitudinal sections were obtained per mouse for various staining procedures. Flow cytometry was performed utilizing a MACSQuant® Analyzer 10 (Miltenyi Biotec Inc.). Additional analysis was performed using FlowJo® (FlowJo, LLC wholly owned by BectonDickinson, Inc.) single-cell flow cytometry software.
- As demonstrated in
FIG. 14 through confocal microscopy, CD40 can be internal or external to the CD3+CD4+ cell. Flow cytometry was further performed and demonstrated that while most CD3+ cells appear to have ability to produce CD40, the CD3+CD4+CD40+ cells appear to produce interferon gamma (IFNγ) in abundance. This flow cytometry study incorporated both the external and internal staining of CD3, CD4, CD40, and IFNγ. -
FIG. 16 demonstrates that interferon gamma controls Th40 proliferation. Isolated Th40 cells were cross-linked by antibody to CD40. The graph inFIG. 16 denotes proliferation of CD40 stimulated (CD40XL: activated) Th40 cells in the absence/presence of antibody to INFγ (αIFNγ) and non-stimulated controls (UN). Additionally, by blocking IFNγ, activated Th40 cells do not proliferate. - This example demonstrates that KGYY15 (15-mer—SEQ ID NO:7) and KGYY6 (6-mer—SEQ ID NO:29) abrogates atherosclerosis.
- ApoE deficient mice on a normal chow diet were selected to receive a dose of 1 mg/kg of the 15-mer peptide (SEQ ID NO: 7) by IV tail injection, three times a week over a period of 26 weeks, beginning at 9 weeks of age and also utilized a control. At 25 weeks, the animals were euthanized, weighed, and then had blood, spleen, and pancreas removed for analysis. The subjects were then perfused through cardiac puncture with 4% paraformaldehyde. Aortic arches were dissected, dehydrated in sucrose gradient and then flash frozen.
-
FIG. 17 provides an example of the lesser curvature of the aortic arch, defined proximally from the aortic outflow (AO). Of the segments seen in the trichrome stain, an intimal distance of 2.4 mm was measured distally. The aortic-arch wall area subtended by this 2.5 mm stretch of the intima was calculated for each section of all mice, with maximal area of the inner-aortic-arch wall of each mouse used to compute averages per group. The luminal surface (L), aortic arch (AO), and innominate artery (I) are labelled in thisFIG. 17 . -
FIG. 18 demonstrates the lesser curvature of the aortic arch of the control ApoE mice compared to the lesser curvature of the aortic arch of mice treated with the 15-mer peptide, in accordance with the steps outlined in this example. -
FIG. 19 demonstrates the reduction of the total area achieved by peptide treatment. The total area of the 2.5 mm segment (as described inFIG. 17 ) was substantially reduced. -
FIG. 20 demonstrates the reduction of number of plaque, including early lesions and advanced plaque. The total number of plaque was significantly decreased in the treatment group. Plaque was subdivided based on morphology of early lesions (observable as fatty streaks containing macrophage derived foam cells with varying degrees of lipid accumulation) compared with more advanced fibroatheromas (containing varying degrees of lipid or necrotic core and fibrous caps). All plaque within the designated 2.5 mm segment were included. Both the total number and type of plaque were significantly decreased in those subjects treated with the peptide. - This study demonstrates that administration of the peptide abrogates atherosclerosis.
- This example demonstrates administration of the KGYY15 (15-mer—SEQ ID NO:7) augments cap formation while reducing advancement of existing disease.
- In this study, six ApoE−/− mice received a normal chow diet from 0 to 20 weeks of age. At 20 weeks of age, three mice were randomly assigned to receive dose of 1 mg/kg of KGYY15 (15-mer—SEQ ID NO:7) by IV tail injection, once a week for a period of 4 weeks. Control mice received vehicle only. After 4 weeks of treatment, animals were euthanized then perfused through cardiac puncture with 4% paraformaldehyde. Aortic arches were dissected in surcrose gradient and flash frozen. Approximately fifty, 8 um longitudinal sections were obtained per mouse. Slides were treated with trichrome stain and analyzed using cellSens software for measurements. Total plaque was measured including 2.5 mm lesser curvature and innominate artery.
-
FIG. 20 shows the number of individual early plaques and advanced plaques were reduced in the treated subjects compared to the control subjects. -
FIG. 21 shows that the cap to core ratio of advanced plaques was reduced in the treated subjects compared to the control subjects. -
FIG. 22 shows the average cap width (cap size) was greater in the treated subjects compared to the control subjects. -
FIG. 23 shows the average core width (core size/plaque size) was decreased in the subjects treated with the peptide compared to the control subjects. - This example demonstrates that administration of the KGYY15 (15-mer—SEQ ID NO:7) augments cap formation while reducing advancement of existing disease. Moreover, this study further demonstrates that administration of the KGYY15 peptide trends toward results of plaque stability.
- From the foregoing, it is readily apparent that TID shares with atherosclerosis the CD40-CD154 dyad which drives autoimmune inflammation. There are increased Th40 cell levels in peripheral blood of NOD mice, human TID patients, and ApoE−/− mice. Th40 cells infiltrate the aortic wall and are found within the plaque of ApoE−/− mice. Th40 cells produce the inflammatory cytokine IFNγ at a level greater than that of other cells and this drives inflammation. The KGYY15 peptide targets Th40 cells. The specified peptide furthermore abrogates and modulates atherosclerosis which may be due to Th40 blockade or general blockade of CD40. Moreover, the administration of the specified peptide trends toward more stable plaque types.
- Whole human blood was administered the peptide in accordance with similar dosing levels to those used for murine studies.
-
FIG. 24 provides the results of clot studies observed in humans. - This study demonstrates that the KGYY15 peptide when administered to humans does not modify or change the clotting of whole human blood significantly outside of normally recognized levels.
- This example demonstrates that ApoE mice that have been genetically modified to obtain atherosclerosis and fed a high fat diet and treated with the 6-mer peptide may have the levels of LDL cholesterol values decreased compared to untreated subjects.
- In this study, six ApoE−/− mice received a normal chow diet from 0 to 20 weeks of age. At 20 weeks of age, three mice were randomly assigned to receive dose of 1 mg/kg of KGYY6 (6-mer—SEQ ID NO:29) by IV tail injection, once a week for a period of 4 weeks. Control mice received vehicle only.
- Data obtained from untreated mice and compared to those in treated mice showed statistically significant reduction (>50%) in LDL cholesterol values. This data is provided in
FIG. 27 . - This example demonstrates atherosclerotic changes that ApoE −/− mice experienced when treated with KGYY6 (6-mer—SEQ ID NO: 29). ApoE −/− mice were fed a high fat diet for 16 weeks. Mice were randomly assigned to receive dose of 1 mg/kg of KGYY6 (6-mer—SEQ ID NO:29) by IV tail injection, once a week for a period of 4 weeks. Control mice received vehicle only. Atherosclerosis was investigated by several methods. En-face analysis utilizing Sudan IV stain (lipid stain) demonstrated a significant reduction in lesion areas.
FIG. 28A is an image of KGYY6 treated aortic en-face Sudan IV staining andFIG. 28B is an image of control (untreated) aortic en-face Sudan staining.FIG. 29 is a graph demonstrating the reduction of lesion areas of Sudan IV staining. - Further, measurement of plaque area and morphology was performed using the Paigen method. This method obtains sequential 5 m aortic cross sections from the aortic root beginning at the valve leaflets into the ascending aorta. At 50 μm intervals, slides are stained after which the area of atherosclerotic lesion is measured. Individual plaque area measurements are plotted against the micrometer intervals and a curve is established, with the area under the curve (AUC) giving the total volume of plaque. These results are presented in graph format in
FIG. 30 , which demonstrates that mice treated with KGYY6 (SEQ ID NO: 29) showed reduction in plaque volume under the curve. - Moreover, characterization of plaque composition or aortic samples was performed using trichrome staining techniques and these results are presented in graph format in
FIG. 31 . Indeed, thisFIG. 31 data, which was generated using Image Pro Plus software analysis, quantifies and shows that plaque compositional changes occurred, including increased collagen and reduced smooth muscle content. -
FIG. 32A is an image of trichrome stained cells of the cross-sections of the aorta of the KGYY6 treated subject.FIG. 32B is an image of trichrome stained cells of the cross-sections of the control subjects. InFIG. 32A , 50 identifies areas characteristic of plaque formation. 60 identifies aortic valve leaflets.FIG. 32B , the control (untreated) 50 areas characteristic of plaque (area under the curve) are greater than those in the subjects treated with the KGYY6 peptide. - This example demonstrates that KGYY6 (6-mer—SEQ ID NO:29) results in significant improvement in glucose tolerance and insulin sensitivity.
- ApoE −/− mice were fed a 60% high fat diet (research diet) for 1 week. A select population of mice were then injected with 6-mer peptide at a dose of 1 mg/kg and others were untreated and tracked as controls. Glucose tolerance testing (GTT) was performed by
fasting 6 hours, followed by intraperitoneal injection of 1 g/kg body weight glucose in water. Both blood glucose and blood serum insulin were measured at 0, 15 minutes, 30 minutes, 60 minutes, and 2 hours. The results of this study are shown inFIGS. 33(a) and 33(b) . Peptide treated ApoE deficient mice demonstrated significantly increased glucose tolerance as well as significantly improved insulin sensitivity and lowered plasma insulin levels compared to control. - Western analysis was performed on adipose and muscle tissue of the treated and untreated mice, demonstrating an increase in expression of the glucose transport protein (GLUT4) in white adipose tissue, as shown in
FIG. 34 . Moreover, the western analysis also demonstrated that the muscle tissue showed increased expression of the GLUT4 protein. GLUT4 is responsible for glucose uptake in response to insulin and known to have reduced expression intype 2 diabetes. - This study demonstrates that administration of the peptide affects glucose tolerance, insulin sensitivity, and GLUT4 levels, each of which may be important to treating
type 2 diabetic subjects. - This example demonstrates administration of the KGYY6 (6-mer—SEQ ID NO:29) affects the inflammatory cytokine production of IL2, INFγ, and IL17a.
- Spleens from ApoE mice and C57BL/6 mice were processed for lymphocytes. ApoE −/− and C57BL/6 mice were fed a 60% high fat diet (research diet) for 1 week. Splenic lymphocytes were treated with 6-mer in vitro for 24 hours. Cells were placed in media overnight in the presence of varying concentrations of 6-mer peptides. The following morning, Brefeldin A was administered for 4 hours. All cells were stained for CD3, CD4, CD40 (Th40 cells) and measured by use of flow cytometry for their production of IL2, INFγ, and IL17a. The results of this study are shown in
FIG. 35 . - From the foregoing, it is readily apparent that new and useful implementations of the methods have been herein described and illustrated which fulfill numerous desiderata in remarkably unexpected fashions. It is, of course, understood that such modifications, alterations and adaptations as may readily occur to the artisan confronted with this disclosure are intended within the spirit of this disclosure, which is limited only by the scope of the claims appended hereto.
Claims (114)
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US16/240,630 US20190263888A1 (en) | 2010-10-19 | 2019-01-04 | Therapeutic peptides and methods for treating autoimmune related disease |
US17/365,881 US20210332104A1 (en) | 2010-10-19 | 2021-07-01 | Therapeutic peptides and methods for treating autoimmune related disease |
Applications Claiming Priority (9)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US39469910P | 2010-10-19 | 2010-10-19 | |
PCT/US2011/056860 WO2012054584A2 (en) | 2010-10-19 | 2011-10-19 | Peptides for modulating t-cell activity and uses thereof |
US201313880387A | 2013-05-23 | 2013-05-23 | |
US15/423,822 US11130795B2 (en) | 2010-10-19 | 2017-02-03 | CD154 peptides and methods of inhibiting CD40 interactions with CD154 |
US201762584595P | 2017-11-10 | 2017-11-10 | |
US201862614262P | 2018-01-05 | 2018-01-05 | |
US201862669918P | 2018-05-10 | 2018-05-10 | |
US16/184,129 US20190194290A1 (en) | 2010-10-19 | 2018-11-08 | Methods for preventing, modulating and/or reducing cardiovascular disease |
US16/240,630 US20190263888A1 (en) | 2010-10-19 | 2019-01-04 | Therapeutic peptides and methods for treating autoimmune related disease |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/184,129 Continuation-In-Part US20190194290A1 (en) | 2010-10-19 | 2018-11-08 | Methods for preventing, modulating and/or reducing cardiovascular disease |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US17/365,881 Continuation US20210332104A1 (en) | 2010-10-19 | 2021-07-01 | Therapeutic peptides and methods for treating autoimmune related disease |
Publications (1)
Publication Number | Publication Date |
---|---|
US20190263888A1 true US20190263888A1 (en) | 2019-08-29 |
Family
ID=78221890
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/240,630 Abandoned US20190263888A1 (en) | 2010-10-19 | 2019-01-04 | Therapeutic peptides and methods for treating autoimmune related disease |
US17/365,881 Pending US20210332104A1 (en) | 2010-10-19 | 2021-07-01 | Therapeutic peptides and methods for treating autoimmune related disease |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US17/365,881 Pending US20210332104A1 (en) | 2010-10-19 | 2021-07-01 | Therapeutic peptides and methods for treating autoimmune related disease |
Country Status (1)
Country | Link |
---|---|
US (2) | US20190263888A1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11130795B2 (en) | 2010-10-19 | 2021-09-28 | The Regents Of The University Of Colorado | CD154 peptides and methods of inhibiting CD40 interactions with CD154 |
US11793854B2 (en) * | 2019-03-21 | 2023-10-24 | Op-T Llc | Methods for reducing symptoms of multiple sclerosis using a six-amino acid long peptide that inhibits CD40-CD150 interaction |
-
2019
- 2019-01-04 US US16/240,630 patent/US20190263888A1/en not_active Abandoned
-
2021
- 2021-07-01 US US17/365,881 patent/US20210332104A1/en active Pending
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11130795B2 (en) | 2010-10-19 | 2021-09-28 | The Regents Of The University Of Colorado | CD154 peptides and methods of inhibiting CD40 interactions with CD154 |
US11793854B2 (en) * | 2019-03-21 | 2023-10-24 | Op-T Llc | Methods for reducing symptoms of multiple sclerosis using a six-amino acid long peptide that inhibits CD40-CD150 interaction |
Also Published As
Publication number | Publication date |
---|---|
US20210332104A1 (en) | 2021-10-28 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20220106381A1 (en) | Cd154 peptides and methods of inhibiting cd40 interactions with cd154 | |
CA3086193A1 (en) | Therapeutic peptides and methods for treating autoimmune related disease | |
Gurion et al. | Systemic arthritis in children: a review of clinical presentation and treatment | |
US11744875B2 (en) | Peptides and methods for treating disease | |
US20210332104A1 (en) | Therapeutic peptides and methods for treating autoimmune related disease | |
US20060025335A1 (en) | Netrin compositions and methods of using the same | |
US20240115651A1 (en) | Peptides for treating disease and reducing symptoms related to multiple sclerosis | |
Charafeddine et al. | Platelet-derived CD154: ultrastructural localization and clinical correlation in organ transplantation | |
CN114828890A (en) | Treatment of diabetes with stem cell migration agent | |
US20200072837A1 (en) | Methods for preventing, modulating and/or reducing cardiovascular disease | |
US20190194290A1 (en) | Methods for preventing, modulating and/or reducing cardiovascular disease | |
WO2019094581A1 (en) | Methods for preventing, modulating and/or reducing cardiovascular disease | |
WO2021105137A1 (en) | Trem-1 inhibitors for the treatment of vaso-occlusions and tissue injuries in patients suffering from sickle cell disease | |
WO2006118085A1 (en) | Agent for promoting hepatic cell replication and agent for improving insulin resistance | |
WO2021075535A1 (en) | Diabetes therapy targeting abnormal stem cells | |
WO2022148816A1 (en) | Inhibition of t-cell activity | |
JP4913742B2 (en) | Methods and means | |
Jongbloed | Characterisation of dendritic cell subsets in inflammatory arthritis |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: OP-T LLC, COLORADO Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:WAGNER, DAVID HAL;YUSSMAN, MARTIN GLENN;HENRY, CHARLES W.;SIGNING DATES FROM 20190103 TO 20190104;REEL/FRAME:048268/0216 |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |
|
AS | Assignment |
Owner name: WAGNER, JR., DAVID H., COLORADO Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:OP-T LLC;REEL/FRAME:065584/0289 Effective date: 20230531 Owner name: OP-T LLC, COLORADO Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:THE REGENTS OF THE UNIVERSITY OF COLORADO, A BODY CORPORATE;REEL/FRAME:065584/0519 Effective date: 20230925 Owner name: THE REGENTS OF THE UNIVERSITY OF COLORADO, A BODY CORPORATE, COLORADO Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:WAGNER, DAVID;REEL/FRAME:065584/0415 Effective date: 20230721 |