ES2633353T3 - Péptidos derivados de lactoferrina humana y su uso - Google Patents
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- ES2633353T3 ES2633353T3 ES12701866.1T ES12701866T ES2633353T3 ES 2633353 T3 ES2633353 T3 ES 2633353T3 ES 12701866 T ES12701866 T ES 12701866T ES 2633353 T3 ES2633353 T3 ES 2633353T3
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- 108090000765 processed proteins & peptides Proteins 0.000 title abstract description 77
- 102000004196 processed proteins & peptides Human genes 0.000 title abstract description 21
- 101000798114 Homo sapiens Lactotransferrin Proteins 0.000 title 1
- 102000050459 human LTF Human genes 0.000 title 1
- 210000004027 cell Anatomy 0.000 description 14
- 238000012216 screening Methods 0.000 description 8
- 230000003110 anti-inflammatory effect Effects 0.000 description 6
- 239000012091 fetal bovine serum Substances 0.000 description 6
- 206010052428 Wound Diseases 0.000 description 5
- 208000027418 Wounds and injury Diseases 0.000 description 5
- 239000002158 endotoxin Substances 0.000 description 5
- 229920006008 lipopolysaccharide Polymers 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 4
- 230000000845 anti-microbial effect Effects 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 239000007621 bhi medium Substances 0.000 description 4
- 239000006161 blood agar Substances 0.000 description 4
- 230000000875 corresponding effect Effects 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 210000001616 monocyte Anatomy 0.000 description 4
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 2
- 239000007995 HEPES buffer Substances 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- 238000000692 Student's t-test Methods 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 231100000673 dose–response relationship Toxicity 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 229940105132 myristate Drugs 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 229940054269 sodium pyruvate Drugs 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 208000034309 Bacterial disease carrier Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 206010053317 Hydrophobia Diseases 0.000 description 1
- 102000010445 Lactoferrin Human genes 0.000 description 1
- 108010063045 Lactoferrin Proteins 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 206010037742 Rabies Diseases 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- CSSYQJWUGATIHM-IKGCZBKSSA-N l-phenylalanyl-l-lysyl-l-cysteinyl-l-arginyl-l-arginyl-l-tryptophyl-l-glutaminyl-l-tryptophyl-l-arginyl-l-methionyl-l-lysyl-l-lysyl-l-leucylglycyl-l-alanyl-l-prolyl-l-seryl-l-isoleucyl-l-threonyl-l-cysteinyl-l-valyl-l-arginyl-l-arginyl-l-alanyl-l-phenylal Chemical compound C([C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 CSSYQJWUGATIHM-IKGCZBKSSA-N 0.000 description 1
- 229940078795 lactoferrin Drugs 0.000 description 1
- 235000021242 lactoferrin Nutrition 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000003641 microbiacidal effect Effects 0.000 description 1
- 238000000491 multivariate analysis Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 229910001868 water Inorganic materials 0.000 description 1
Classifications
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- 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
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/04—Antipruritics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/06—Antipsoriatics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/04—Centrally acting analgesics, e.g. opioids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/08—Antiallergic agents
-
- 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
- C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links 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
- A61K38/40—Transferrins, e.g. lactoferrins, ovotransferrins
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Immunology (AREA)
- Toxicology (AREA)
- Epidemiology (AREA)
- Dermatology (AREA)
- Nutrition Science (AREA)
- Pain & Pain Management (AREA)
- Oncology (AREA)
- Mycology (AREA)
- Communicable Diseases (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Neurosurgery (AREA)
- Neurology (AREA)
- Biomedical Technology (AREA)
- Rheumatology (AREA)
- Pulmonology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
Un péptido seleccionado de cualquiera de los péptidos **Fórmula**
Description
5
10
15
20
25
30
en forma de respuesta a la dosis. Los resultados se presentan como supervivencia bacteriana relativa (%) en comparación con el grupo de control ± EEM (n=15 heridas). Se estimó la significación estadística por la prueba de t de Student. *=p< 0,05, **=p <0,01, ***=p <0,001.
Figura 5. Efecto de dosis y respuesta del péptido 265 (A) sobre la colonización bacteriana de heridas infectadas en piel de cerdo. Las heridas infectadas con S. aureus en PBS/suero (50/50) y tratadas con el correspondiente péptido en H2O, a concentraciones de 0,1, 0,5 y 2 mg/ml demuestran una reducción significativa de los recuentos bacterianos en forma de respuesta a la dosis. Los resultados se presentan como supervivencia bacteriana relativa (%) en comparación con el grupo de control ± EEM (n= 10 heridas). Se estimó la significación estadística por la prueba de t de Student. *=p< 0,05, **=p <0,01, ***=p <0,001.
EJEMPLOS
Ejemplo 1. Cribado peptídico 1
Se diseñaron y ensayaron los péptidos derivados de lactoferrina mostrados en la Tabla 1. Se identificaron los péptidos activos.
Se diseñaron nuevas variantes peptídicas basándose en la actividad antiinflamatoria y antimicrobiana medida de péptidos que tienen secuencias similares a la SEQ ID NO:1. Además, se tuvieron en cuenta las consideraciones estructurales de las correspondientes secuencias de estos péptidos. En la práctica, esto significaba mantener y potenciar la helicidad de los péptidos. Se diseñaron nuevas variantes de péptidos aumentando la carga positiva y las regiones hidrófobas de los péptidos. Por tanto, se aumentó el carácter anfipático de los péptidos. (Figura. 2). Basándose en los nuevos diseños, se ordenaron los nuevos péptidos como una colección PEPscreen (Sigma) y se ensayaron tanto la actividad antiinflamatoria como antimicrobiana.
Tabla 1. Lista de péptidos ensayados en el cribado 1
- Péptido
- Secuencia SEQ ID NO:
- Péptido 116
- SEQ ID NO: 4
- Péptido 126
- SEQ ID NO: 5
- Péptido 127
- SEQ ID NO: 6
- Péptido 130
- SEQ ID NO: 7
- Péptido 132
- SEQ ID NO: 8
- Péptido 150
- SEQ ID NO: 9
- Péptido 152
- SEQ ID NO: 10
- Péptido 153
- SEQ ID NO: 11
- Péptido 154
- SEQ ID NO: 12
- Péptido 155
-
imagen10 SEQ ID NO: 13
- Péptido 156
- SEQ ID NO: 14
- Péptido 157
- SEQ ID NO: 15
- Péptido 159
- SEQ ID NO: 16
Se midió la actividad antiinflamatoria como la inhibición de la producción en TNF- en células THP-1 estimuladas por LPS
Se mantuvo la estirpe celular THP-1 (TIB-202; ATCC, Manassas, VA, EE.UU.) correspondiente a monocitos humanos en RPMI 1640 (PAA Laboratories GmbH, Pasching, Austria) suplementado con suero fetal bovino al 10 % (FBS; PAA Laboratories GmbH, Pasching, Austria), piruvato de sodio 1 mM (Sigma-Aldrich, St. Louis, MO, EE.UU.) y HEPES 20 mM (PAA, Laboratories GmbH, Pasching, Austria).
Se ajustó la densidad celular a 106 células/ml y se añadieron 100 μI de la suspensión por pocillo a placas de cultivo celular de 96 pocillos (Sarstedt, Nümbrecht, Alemania). Se trataron las células con PMA 10 ng/ml (12-miristato 13acetato de forboI; Sigma-Aldrich, St. Louis, MO, EE.UU.) durante 48 horas para diferenciar los monocitos en células
11
- SEQ ID NO
- Péptido MMC99 μM en medio BHI al 1 %
- SEQ ID NO 4
- 116 10
- SEQ ID NO 5
- 126 10
- SEQ ID NO 9
- 150 10
- SEQ ID NO 10
- 152 10
- SEQ ID NO 11
- 153 10
- SEQ ID NO 12
- 154 10
- SEQ ID NO 13
- 155 10
- SEQ ID NO 14
- 156 20
- SEQ ID NO 15
- 157 20
- SEQ ID NO 16
- 159 20
Ejemplo 2. Cribado peptídico 2
Se sometieron las actividades de TNF- de los péptidos de esta primera ronda de cribado a análisis multivariante usando el software ProPHECY™ (Saromics, Lund, Suecia). Se computaron un gran número de descriptores para 5 cada péptido. Se correlacionaron entonces las actividades de TNF- con estos descriptores. Se crearon modelos de regresión separados para la clase peptídica. Además, se crearon también modelos globales que consideraban la clase peptídica. El análisis del modelo de regresión sugería varias variables que contribuían a una actividad de TNF mejorada. Se sugirieron nuevos péptidos para la segunda ronda de cribado por la clase peptídica, basados principalmente en la modulación de carga, anfipatía e hidrofobia. Basándose en los nuevos diseños, se ordenaron 10 aproximadamente 80 péptidos como una colección PEPscreen (Sigma) y se ensayaron tanto la actividad antiinflamatoria como antimicrobiana.
Tabla 4. Lista de péptidos ensayados en el cribado 2
- Péptido 224
- SEQ ID NO: 17
- Péptido 256
- SEQ ID NO: 18
- Péptido 257
- SEQ ID NO: 19
- Péptido 258
- SEQ ID NO: 20
- Péptido 259
- SEQ ID NO: 21
- Péptido 260
- SEQ ID NO: 22
- Péptido 261
- SEQ ID NO: 23
- Péptido 262
- SEQ ID NO: 24
- Péptido 263
- SEQ ID NO: 25
- Péptido 264
- SEQ ID NO: 26
- Péptido 265
- SEQ ID NO: 27
- Péptido 266
- SEQ ID NO: 28
- Péptido 268
- SEQ ID NO: 29
- Péptido 269
- SEQ ID NO: 30
- Péptido 270
- SEQ ID NO: 31
13
- Péptido 271
- SEQ ID NO: 32
- Péptido 272
-
imagen12 SEQ ID NO: 33
- Péptido 273
- SEQ ID NO: 34
- Péptido 276
- SEQ ID NO: 35
- Péptido 282
- SEQ ID NO: 36
Se midió la actividad antiinflamatoria como inhibición de la producción de TNF- en células THP-1 estimuladas por LPS
Se mantuvo la estirpe celular THP-1 (TIB-202; ATCC, Manassas, VA, EE.UU.) correspondiente a monocitos
5 humanos, en RPMI 1640 (PAA Laboratories GmbH, Pasching, Austria) suplementado con suero fetal bovino al 10 % (FBS; PAA Laboratories GmbH, Pasching, Austria), piruvato de sodio 1 mM (Sigma-Aldrich, St. Louis, MO, EE.UU.) y HEPES 20 mM (PAA, Laboratories GmbH, Pasching, Austria).
Se ajustó la densidad celular a 106 células/ml y se añadieron 100 μI de la suspensión por pocillo a placas de cultivo celular de 96 pocillos (Sarstedt, Nümbrecht, Alemania). Se trataron las células con PMA 10 ng/ml (12-miristato 1310 acetato de forboI; Sigma-Aldrich, St. Louis, MO, EE.UU.) durante 48 horas para diferenciar los monocitos en células similares a macrófagos. Después de ello, se estimularon las células mediante la adición de lipopolisacárido 0,1 ng/ml (LPS; serotipo de E. coli O55:B5; Sigma-Aldrich, St. Louis, MO, EE.UU.) al medio especificado anteriormente excepto por contener FBS termoinactivado al 5 %. 30 minutos después de la adición de LPS, se añadieron péptidos (40 μM, 10 μM y 4 μM) por triplicado. Después de 6 horas de incubación, se recogieron los sobrenadantes celulares,
15 se centrifugaron y se mantuvieron congelados a -20 ºC hasta analizar el contenido de TNF- por ELISA (R&D Systems, Minneapolis, MN, EE.UU.). Se presentan los resultados como secreción relativa media (%), fijándose el nivel de TNF- estimulado sin péptido en 100 % y fijándose la secreción basal en 0 % (Tabla 5).
Tabla 5. Efectos antiinflamatorios de los péptidos ensayados en el cribado 2
- SEQ ID NO
- Péptido TNF- a péptido 40 μM TNF- a péptido 10 μM TNF- a péptido 4 μM
- SEQ ID NO 17
- 224 113,4 % nd nd
- SEQ ID NO 18
- 256 100,5 % nd nd
- SEQ ID NO 19
- 257 62,8 % 109,1 % 114,5 %
- SEQ ID NO 20
- 258 39,8 % 115,4 % 110,4 %
- SEQ ID NO 21
- 259 33,0 % 108,5 % 108,4 %
- SEQ ID NO 22
- 260 5,9 % 74,0 % 99,3 %
- SEQ ID NO 23
- 261 24,5 % 56,2 % 86,0 %
- SEQ ID NO 24
- 262 12,1 % 45,3 % 79,6 %
- SEQ ID NO 25
- 263 61,4 % 100,1 % 93,6 %
- SEQ ID NO 26
- 264 87,2 % nd nd
- SEQ ID NO 27
- 265 13,8 % 31,1 % 87,0 %
- SEQ ID NO 28
- 266 101,1 % nd nd
- SEQ ID NO 29
- 268 74,4 % 129,7 % 114,5 %
- SEQ ID NO 30
- 269 66,2 % 113,8 % 115,7 %
- SEQ ID NO 31
- 270 23,1 % 71,5 % 96,2 %
- SEQ ID NO 32
- 271 24,5 % 74,4 % 101,7 %
- SEQ ID NO 33
- 272 102,8 % nd nd
14
- SEQ ID NO
- Péptido TNF- a péptido 40 μM TNF- a péptido 10 μM TNF- a péptido 4 μM
- SEQ ID NO 34
- 273 52,7 % 78,6 % 86,2 %
- SEQ ID NO 35
- 276 38,8 % 135,3 % 111,0 %
- SEQ ID NO 36
- 282 114,8 % nd nd
nd= no realizado
Se midió la actividad antimicrobiana como el efecto bactericida sobre S. aureus usando la concentración microbicida mínima, MMC99, ensayo)
Se transfirieron S. aureus (nº 1800; CCUG, Gotemburgo, Suecia) cultivadas en placas de agar sanguíneo [agar
5 Columbia (Oxoid, Basingstoke, RU) suplementado con sangre de caballo desfibrinada al 5 % (National Veterinary Institute (SVA), Uppsala, Suecia)] a caldo de infusión de cerebro y corazón (BHI al 3,7 %; Difco, BD Diagnostics, Franklin Lakes, NJ, EE.UU.) y se incubaron en un agitador a 250 rpm +37º C durante una noche. Se diluyó después de ello el cultivo 1:10 con caldo BHI reciente y se incubó durante 2 horas adicionales para alcanzar el crecimiento en fase logarítmica. Se sedimentaron las bacterias y se suspendieron en medio BHI al 1 % (caldo BHI diluido 100 veces
10 en agua ultrapura) hasta una concentración de 107 bacterias/ml estimada midiendo la densidad óptica a 600 nm.
Se diluyeron en serie los péptidos en etapas de dos veces desde 400 μM hasta 0,78 μM en medio BHI al 1 % o bien en fluido de herida simulado termoinactivado al 50 % [SWF, que contiene 1 parte de peptona al 0,1 % (Oxoid, Basingstoke, RU) en solución salina y 1 parte de suero fetal bovino, diluido 2 veces en agua ultrapura].
Se incubaron después de ello los péptidos (100 μl) con bacterias (5 μl a 107 bact./ml) durante 2 horas a +37 ºC. Se
15 pusieron gotas (5 μl) de la suspensión en placas de agar sanguíneo. Se incubaron las placas de agar sanguíneo durante una noche a 37 ºC. Se registraron los valores de MMC99, concretamente la concentración de péptido mínima necesaria para conseguir un 99 % de reducción de las bacterias viables (Tabla 6). Se confirmó la concentración de la suspensión bacteriana usada en el ensayo por los recuentos viables en placas de agar sanguíneo.
Tabla 6. Efectos antibacterianos de los péptidos ensayados en el cribado 2
- SEQ ID NO
- Péptido MMC99 μM en medio BHI al 1 % MMC99 μM en SWF al 50 %
- SEQ ID NO 17
- 224 12,5 400
- SEQ ID NO 18
- 256 12,5 200
- SEQ ID NO 19
- 257 6,25 200
- SEQ ID NO 20
- 258 6,25 12,5
- SEQ ID NO 21
- 259 6,25 25
- SEQ ID NO 22
- 260 6,25 25
- SEQ ID NO 23
- 261 12,5 12,5
- SEQ ID NO 24
- 262 6,25 6,25
- SEQ ID NO 25
- 263 6,25 100
- SEQ ID NO 27
- 265 6,25 12,5
- SEQ ID NO 29
- 268 6,25 200
- SEQ ID NO 30
- 269 6,25 200
- SEQ ID NO 31
- 270 6,25 12,5
- SEQ ID NO 32
- 271 6,25 6,25
- SEQ ID NO 34
- 273 6,25 12,5
- SEQ ID NO 35
- 276 6,25 12,5
- SEQ ID NO 36
- 282 12,5 200
20 nd= no realizado
15
Claims (1)
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imagen1
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP11152213A EP2481751A1 (en) | 2011-01-26 | 2011-01-26 | Human lactoferrin derived peptides |
| EP11152213 | 2011-01-26 | ||
| PCT/EP2012/051112 WO2012101157A1 (en) | 2011-01-26 | 2012-01-25 | Human lactoferrin derived peptides and there use |
Publications (1)
| Publication Number | Publication Date |
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| ES2633353T3 true ES2633353T3 (es) | 2017-09-20 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| ES12700715.1T Active ES2645959T3 (es) | 2011-01-26 | 2012-01-25 | Péptidos basados en lactoferrina humana que tienen actividad antiinflamatoria |
| ES12701866.1T Active ES2633353T3 (es) | 2011-01-26 | 2012-01-25 | Péptidos derivados de lactoferrina humana y su uso |
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| Application Number | Title | Priority Date | Filing Date |
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| ES12700715.1T Active ES2645959T3 (es) | 2011-01-26 | 2012-01-25 | Péptidos basados en lactoferrina humana que tienen actividad antiinflamatoria |
Country Status (13)
| Country | Link |
|---|---|
| US (2) | US9132165B2 (es) |
| EP (3) | EP2481751A1 (es) |
| JP (2) | JP6158712B2 (es) |
| KR (2) | KR101949210B1 (es) |
| CN (2) | CN103476792B (es) |
| AU (2) | AU2012210564B2 (es) |
| BR (2) | BR112013018365A2 (es) |
| CA (2) | CA2825246A1 (es) |
| ES (2) | ES2645959T3 (es) |
| MX (2) | MX341020B (es) |
| PL (1) | PL2668205T3 (es) |
| RU (2) | RU2593757C2 (es) |
| WO (2) | WO2012101156A2 (es) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| EP2481751A1 (en) * | 2011-01-26 | 2012-08-01 | PharmaSurgics in Sweden AB | Human lactoferrin derived peptides |
| GB201401877D0 (en) * | 2014-02-04 | 2014-03-19 | Univ Tromsoe | Peptides |
| CN104888202A (zh) * | 2014-05-22 | 2015-09-09 | 闫牧乔 | 一种治疗扁桃体炎的药物 |
| IT201800002457A1 (it) * | 2018-02-07 | 2019-08-07 | Neilos S R L | Composizione per la prevenzione e il trattamento delle affezioni delle vie respiratorie |
| RU2681216C1 (ru) * | 2018-09-05 | 2019-03-05 | Федеральное государственное бюджетное образовательное учреждение высшего образования "Курский государственный медицинский университет" Министерства здравоохранения Российской Федерации | Способ стимуляции регенерации инфицированных ран кожи и мягких тканей с применением пептида Gly-His-Lys-D-Ala |
| RU2681310C1 (ru) * | 2018-09-05 | 2019-03-06 | Федеральное государственное бюджетное образовательное учреждение высшего образования "Курский государственный медицинский университет" Министерства здравоохранения Российской Федерации | Способ первичной хирургической обработки инфицированных ран кожи и мягких тканей с использованием пептида Gly-His-Lys-D-Ala |
| WO2020096672A1 (en) | 2018-11-09 | 2020-05-14 | The Wistar Institute Of Anatomy And Biology | Use of lactoferrin for generating myeloid-derived suppressor cells |
| KR102032945B1 (ko) * | 2018-12-03 | 2019-10-16 | 순천대학교 산학협력단 | 항염증 활성을 나타내는 펩타이드 및 이를 유효성분으로 포함하는 항염증용 조성물 |
| NL2024839B1 (en) * | 2020-02-05 | 2021-09-13 | Cbmr Scient Nanoscience B V | Antimicrobial peptide for treatment and controlling skin disorders relating to microbial infection |
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| JP2818056B2 (ja) | 1990-09-07 | 1998-10-30 | 森永乳業株式会社 | 抗菌性ペプチドおよび抗菌剤 |
| EP0629347B1 (en) | 1992-01-23 | 1997-07-02 | Morinaga Milk Industry Co., Ltd. | Antibacterial agent and treatment of article therewith |
| JP3347819B2 (ja) | 1993-06-28 | 2002-11-20 | 森永乳業株式会社 | 抗菌性ペプチドの精製方法 |
| US5595756A (en) * | 1993-12-22 | 1997-01-21 | Inex Pharmaceuticals Corporation | Liposomal compositions for enhanced retention of bioactive agents |
| JP3529423B2 (ja) | 1994-04-01 | 2004-05-24 | 森永乳業株式会社 | 抗菌性ペプチドの製造方法 |
| JP3812957B2 (ja) | 1994-08-02 | 2006-08-23 | 森永乳業株式会社 | 角膜損傷治療剤 |
| JPH08143468A (ja) | 1994-11-17 | 1996-06-04 | Morinaga Milk Ind Co Ltd | 抗潰瘍剤 |
| WO1998006425A1 (en) | 1996-08-12 | 1998-02-19 | A+ Science Invest Ab | Treatment and prevention of infections, inflammations and/or tumours with lactoferrin and/or lactoferricin |
| US6696545B1 (en) * | 1997-04-11 | 2004-02-24 | Sangstat Medical Corporation | Cytomodulating lipophilic peptides for modulating immune system activity and inhibiting inflammation |
| SE9804614A0 (en) * | 1998-07-06 | 2000-01-07 | A+ Science Invest Ab | New peptides and use thereof |
| US6399570B1 (en) * | 1999-02-05 | 2002-06-04 | Agennix, Inc. | Antimicrobial/endotoxin neutralizing polypeptide |
| MXPA01011606A (es) * | 1999-05-14 | 2003-09-10 | Univ California | Terapia anti-inflamatoria para infeccion mediada por inflamacion. |
| JP2003521483A (ja) | 1999-11-11 | 2003-07-15 | エイエム ファーマ ビーブイ | ヒトラクトフェリンの第１カチオンクラスターの抗菌活性 |
| RU2165769C1 (ru) * | 2000-07-13 | 2001-04-27 | Якубовская Раиса Ивановна | Антибактериальный, антиоксидантный, иммуномодулирующий и антиканцерогенный препарат и способ его применения |
| SE0102067D0 (sv) | 2001-06-11 | 2001-06-11 | A & Science Invest Ab | Prevention of neovascularization of intervertebral discs and/or of tissues with local inflammation |
| WO2006047744A2 (en) | 2004-10-26 | 2006-05-04 | Agennix Incorporated | Compositions of lactoferrin related peptides and uses thereof |
| AU2007272272B2 (en) * | 2006-07-10 | 2012-04-12 | Pba3 Biomed Gmbh | Antimicrobial peptides |
| WO2008096816A1 (ja) * | 2007-02-09 | 2008-08-14 | Genomidea Inc. | 血管新生誘導剤及びそれに用いられるポリペプチド |
| WO2008096814A1 (ja) * | 2007-02-09 | 2008-08-14 | Genomidea Inc. | 新規ポリペプチド及びそれを有効成分として含有する抗菌剤 |
| EP2050461A1 (en) | 2007-10-19 | 2009-04-22 | PharmaSurgics in Sweden AB | Peptides based on the sequence of human lactoferrin and their use |
| EP2060586A1 (en) * | 2007-11-14 | 2009-05-20 | PharmaSurgics in Sweden AB | New synthetic arginine substituted peptides and their use |
| KR101350251B1 (ko) * | 2008-05-23 | 2014-01-14 | 다이이찌 산쿄 가부시키가이샤 | 목적 펩타이드의 혈장내 반감기 연장 작용을 가진 펩타이드 |
| RU2501566C2 (ru) * | 2009-01-13 | 2013-12-20 | Пергамум АБ | Новые фармацевтические композиции |
| EP2481751A1 (en) * | 2011-01-26 | 2012-08-01 | PharmaSurgics in Sweden AB | Human lactoferrin derived peptides |
| KR101341210B1 (ko) * | 2013-01-24 | 2013-12-12 | 재단법인 지능형 바이오 시스템 설계 및 합성 연구단 | 신규한 항균 펩타이드 및 이의 용도 |
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- 2012-01-25 KR KR1020137021855A patent/KR101949210B1/ko not_active Expired - Fee Related
- 2012-01-25 ES ES12700715.1T patent/ES2645959T3/es active Active
- 2012-01-25 EP EP12701866.1A patent/EP2668205B1/en not_active Not-in-force
- 2012-01-25 WO PCT/EP2012/051111 patent/WO2012101156A2/en not_active Ceased
- 2012-01-25 MX MX2013007995A patent/MX341020B/es active IP Right Grant
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