[go: up one dir, main page]

WO2008085366A2 - Procédés et compositions destinés à tuer les cellules exprimant la cd59 humaine - Google Patents

Procédés et compositions destinés à tuer les cellules exprimant la cd59 humaine Download PDF

Info

Publication number
WO2008085366A2
WO2008085366A2 PCT/US2007/026005 US2007026005W WO2008085366A2 WO 2008085366 A2 WO2008085366 A2 WO 2008085366A2 US 2007026005 W US2007026005 W US 2007026005W WO 2008085366 A2 WO2008085366 A2 WO 2008085366A2
Authority
WO
WIPO (PCT)
Prior art keywords
ily
human
peg
intermedilysin
tissue
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.)
Ceased
Application number
PCT/US2007/026005
Other languages
English (en)
Other versions
WO2008085366A3 (fr
Inventor
Xuebin Qin
Weiguo Hu
Jose Alberto Halperin
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Harvard University
Original Assignee
Harvard University
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Harvard University filed Critical Harvard University
Publication of WO2008085366A2 publication Critical patent/WO2008085366A2/fr
Publication of WO2008085366A3 publication Critical patent/WO2008085366A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0034—Urogenital system, e.g. vagina, uterus, cervix, penis, scrotum, urethra, bladder; Personal lubricants

Definitions

  • This invention relates to the treatment of obesity and cancer, and the prevention of pregnancy.
  • compositions containing substantially pure ILY e.g., recombinant ILY
  • formulated e.g., a pharmaceutical formulation for contraceptive or topical administration.
  • the human CD59 gene and/or recombinase gene can be conditionally expressed, such that, for example, the gene is only expressed in a certain cell type (e.g., a certain cell type at a certain developmental stage) or tissue (e.g., neural tissue, hematopoietic tissue, skin, endothelial tissue, and muscular tissue).
  • a certain cell type e.g., a certain cell type at a certain developmental stage
  • tissue e.g., neural tissue, hematopoietic tissue, skin, endothelial tissue, and muscular tissue.
  • substantially pure is meant a preparation that is at least 50% by weight (dry weight) ILY.
  • composition comprising substantially pure ILY is meant to include any ratio of substantially pure intermedilysin to other compounds.
  • obesity is meant a condition in a human characterized by excessive weight due to stored fat. According to established standards, humans are "overweight” when they have a Body Mass Index (BMI) of greater than 25 and they are “obese” then they have a BMI of greater than 30.
  • BMI Body Mass Index
  • subject is meant a human or other mammal expressing a form of CD59 that sensitizes the expressing cell to ILY.
  • An ILY polypeptide can have at least 50%, 60%, 70%, 80%, 90%, 95%, or 99%, sequence identity to the ILY polypeptide having the sequence of BAE 16324, or a fragment having ILY activity (e.g., a fragment of the polypeptide having the sequence of BAEl 6324). Additionally, an ILY polypeptide can be encoded by a nucleic acid that hybridizes under high stringency conditions to a nucleic acid of ILY. ILY can be isolated from any Streptococcus intermedins strain (e.g., strains 1208-1 , UNS35, UNS46, and ATCC27335), or produced recombinantly.
  • Streptococcus intermedins strain e.g., strains 1208-1 , UNS35, UNS46, and ATCC27335
  • Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
  • hybridize is meant to form a double-stranded complex containing complementary paired nucleobase sequences, or portions thereof, under various conditions of stringency (see, e.g., Wahl. and Berger, Methods Enzymol. 152:399 (1987); Kimmel, Methods Enzymol. 152:507 (1987)).
  • hybridizes under high stringency conditions is meant under conditions of stringent salt concentration, stringent temperature, or in the presence of formamide.
  • stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and most preferably less than about 250 mM NaCl and 25 mM trisodium citrate.
  • Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and most preferably at least about 50% formamide.
  • Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, more preferably of at least about 37° C, and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred embodiment, hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS.
  • SDS sodium dodecyl sulfate
  • hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 ⁇ g/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 ⁇ g/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art. For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature.
  • stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.
  • Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, more preferably of at least about 42° C, and most preferably of at least about 68° C.
  • wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS.
  • wash steps will occur at 42° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS.
  • wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art.
  • Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180 (1977)); Grunstein and Hogness (Proc. Natl. Acad. Sci. USA 72:3961 (1975)); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York (2001)); Berger and Kimmel (Guide to Molecular Cloning Techniques, Academic Press, New York, (1987)); and Sambrook et al.
  • a Streptococcus intermedius ILY polypeptide By “having the activity of a Streptococcus intermedius ILY polypeptide” is meant a cholesterol-dependent cytolysing activity specific to cells expressing human CD59.
  • human CD59 or "hCD59” is meant a protein encoded by a nucleic acid sequence substantially similar to SEQ ID NO: 1 , or functional fragment thereof.
  • hCD59 is also meant a polypeptide with at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% percent sequence identity to the protein encoded by the cDNA sequence of SEQ ID NO: 1.
  • hCD59 is defined as a polypeptide encoded by a nucleic acid that hybridizes under high stringency conditions to the nucleic acid sequence of SEQ ID NO: 1.
  • Figure 2C is a western blot showing the isolation of ILY-binding proteins from human serum.
  • the lanes were loaded as follows: 1 : 0.8 ⁇ l of human serum, 2: 0.8 ⁇ l flow through from human serum loaded on the ILY-binding column, 3: 10 ⁇ g proteins from the eluted fraction of human serum loaded on ILY column, 4: 0.8 ⁇ l of mouse serum, 5: 0.8 ⁇ l flow through from mouse serum loaded on ILY-binding column, 6: 10 ⁇ g protein from the eluted fraction of mouse serum loaded on ILY column. Arrows indicate bands cut for protein sequencing.
  • Figure 2D is a table showing protein sequencing information.
  • Figure 3 A is a western blot showing further isolation of ILY-binding human IgG with protein G column. Lanes were loaded as follows: 1 : protein maker; 2: 10 ⁇ l of the fraction eluted from ILY column; 3: 20 ⁇ l of the eluted fraction after further purification with protein G column; 4: 20 ⁇ l of the flow through from further purification with protein G column. Samples used for lanes 2, 3, and 4 were equalized to the same volume by dialysis and concentration. Total original volume of the each sample of lane 2, lane 3, and lane 4 are equal.
  • Figure 3 B is a graph showing percent ILY mediated hemolysis with the inhibitory effect of human ILY-binding IgG.
  • the volumes of the eluted fraction from ILY column (crosses), the eluate from further purification with protein G column (rectangles), and the flow through from further purification with protein G column (triangles) were equalized by dialysis and concentration.
  • ILY concentration for the hemolytic assay is 1.6 x 10 "9 M. These data represent an experiment repeated three times.
  • Figure 3C is a graph showing percent ILY mediated hemolysis in the absence of an inhibitory effect of mouse ILY -binding IgG. The volumes of mouse and human serum loaded on the ILY-binding column were equal.
  • the total volume of the eluted fraction from mouse serum (rectangles), flow through from mouse serum (crosses), and the eluted fraction from human serum (triangles) were equalized by dialysis and concentration.
  • ILY concentration is 1.6 x 10 "9 M.
  • Figure 3D is a graph showing ILY binding to the Fab region of human ILY- binding. 200 ng ILY was used to coat each well.
  • Figure 4B is a graph showing percent lysis of the indicated cell type in response to the indicated amount of ILY.
  • Human and ThCD59 RBC mouse RBCs are more sensitive to ILY -mediated lysis than WT erythrocytes.
  • Figure 4D is a graph showing percent reduction in hematocrit at the indicated dosage of ILY.
  • ILY treatment induces dose-dependent intravascular hemolysis in ThCD59 RBC mice. *P ⁇ 0.001 versus mice injected with ILY (30 ng per g body weight).
  • Figure 4E is a photograph and graph showing free hemoglobin in the indicated cells. ILY treatment (45 ng/g body weight) induces dose-dependent intravascular hemolysis in ThCD59 RBC mice. *P ⁇ 0.001 versus WT injected with ILY. Hb, hemoglobin.
  • Figure 4F is a photograph and graph showing gross hemoglobinuria as a function of time after ILY injection.
  • WT and ThCD59 RBC mice were injected with ILY (45 ng/g body weight).
  • Urine was collected at various time points after injection (5 h after ILY injection and again after 1 d, 2 d, 3 d and 4 d).
  • Gross hemoglobinuria is shown in the left panel; hemoglobin levels in the urine (1 to 4 dilution in PBS) are shown in the right panel.
  • Results shown in 4B, 4D, 4E and 4F are mean ⁇ s.e.m. *P ⁇ 0.001 versus WT at day 1.
  • Figure 5A is a graph showing lysis as a function of ILY concentration in the indicated species. Hemolytic activity in human erythrocytes after addition of serial dilutions of ILY plus diluted sera (1 :8 in PBS) from various species. Results represent mean from three independent experiments with different batches of purchased sera.
  • Figure 5D is a graph showing percent lysis as a function of dilution of the indicated samples. Hemolytic activity in human erythrocytes after addition of ILY (1.6 x 10 ⁇ 9 M) plus serially diluted human serum, eluate or flow-through from the G protein column.
  • Figure 5E is a graph showing percent lysis as a function of dilution of the indicated samples. Hemolytic activity in human erythrocytes after addition of ILY (1.6 x 10 ⁇ 9 M) plus serially diluted mouse serum, eluate or flow-through from the ILY affinity column. Values shown in Figures 5B, 5D, and 5E are mean ⁇ s.e.m. from three independent experiments per group. **P ⁇ 0.001, *P ⁇ 0.05 versus flow- through fraction.
  • Figure 6A is a graph showing percent survival mice treated with the indicated amount of ILY-binding IgG.
  • Human ILY-binding IgG rescued ILY-induced acute death in ThCD59 RBC mice, but bovine ILY-binding IgG did not.
  • Mice were injected intravenously with ILY-neutralizing human IgG and then injected with ILY (283.5 ng per g body weight, three times the lethal dose (LD i O o)) after 15 min.
  • Figure 7B is a graph showing circulating endothelial cells counts in the indicated sample. * P ⁇ 0.01 versus WT/ILY or T/ILY + Ab. Values shown are mean ⁇ s.e.m.
  • Figure 7D and 7E are photomicrographs of endothelial tissue. Ultrastructural features of endothelial damage in hepatic vessels. Note the typical chromatin condensation, irregular luminal membranes with numerous processes and blebs (grey arrows) and enlarged vacuoles in the dead endothelial cells (Figure 7D), and the absence of continuous basement membrane (inside the black circle) and widening of the intercellular spaces (star) ( Figure 7E). EC, endothelium; V, vacuole. Black arrow points to basement membrane.
  • DIC features included intra-alveolar edema (black arrow), perivascular inflammation (dark gray, right facing arrow) with neutrophil infiltration (white arrow), thrombosis (light gray, right facing arrows) and neutrophil margination (Left facing, gray arrow).
  • Left column shows 2Ox magnification.
  • Right column shows a 6Ox magnification of the area outlined by the black rectangle in the image at left. Scale bar, 40 ⁇ m.
  • Figure 8B is a graph showing concentration of TAT in the indicated samples. ILY resulted in increased TAT in ThCD59 END mice but not in WT or ThCD59 END mice pretreated with ILY-neutralizing human IgG.
  • Figure 8C is a graph showing platelet counts in the indicated samples. Decreased platelet counts in ThCDS ⁇ 0 mice but not in WT or ThCD59 END mice pretreated with ILY-neutralizing human IgG.
  • WT/ILY WT treated with PBS plus ILY; T/ILY: ThCD59 RBC treated with PBS plus ILY; T/ILY + Ab: ThCD59 RBC treated with ILY-neutralizing human IgG plus ILY.
  • Values shown in 8B and 8C are mean ⁇ s.e.m. *P ⁇ 0.01 versus WT/ILY or T/ILY + Ab.
  • Figure 9A is a graph showing number of events as a function of fluorescence. FACS analysis was performed using the anti-hCD59 antibody, Brie 229, to examine
  • Figure 9B is graph showing percent lysis as a function of dilution. hCD59
  • Figure 9C is a photomicrograph of a northern blot showing RNA concentration.
  • Northern blot analysis shows that hCD59 mRNA is present in the spleen and bone marrow only. Both 28S and 18S are shown as RNA loading controls.
  • Figure 9D is a photomicrograph of a northern blot showing RNA
  • hCD59 mRNAs were detected in ThCD59 mice.
  • Figure 9E is a series of fluorescent photomicrographs showing hCD59 protein. HCD59 protein was detected (gray arrows) in small size vessels and glomeruli of
  • ThCD59 but not of WT mice (immunofluorescence staining with anti-hCD59 antibody).
  • Lower panels are phase-contrast controls for the corresponding upper panels.
  • the area inside the black circle is a renal glomerulus and the black arrows point to renal tubules.
  • KG Kidney glomeruli;
  • KV Kidney vein;
  • Figure 1OA and 1OB are a graphs showing hematocrit in the indicated samples.
  • ILY in tissue by double immunofluorescence colocalizationof macrophage and recombinant ILY in(l 1 A) brain, (HB) liver and (HC) lung.
  • ILY ILY injection.
  • PBS PBS injection. Macrophages were labeled by anti-
  • CD68 and FITC-conjugated secondary antibodies were labeled by anti-His tag and Rhodamine-conjugated secondary antibodies.
  • the invention features compositions and methods for selectively killing CD59 expressing cells in a subject by administering ILY. These compositions and methods are useful for treating conditions associated with cells expressing CD59, including proliferative diseases, obesity, and obesity related syndromes. These compositions can be used in combination with anti-hCD59 antibody, such that in desired locations, the lytic activity of ILY is suppressed by the antibody, and in other locations, where antibody is not applied, ILY lytic activity is permitted. This invention also features methods and compositions useful for preventing pregnancy.
  • the invention embodies local administration of ILY to the tissue of interest in a subject.
  • the ILY can be formulated, for example, for topical, percutaneous, surgical, or transdermal administration.
  • ILY may, for example, be formulated for sustained release.
  • the methods and compositions of the invention are useful for treating obesity and obesity related syndromes.
  • diseases include depression, type 2 diabetes, dyslipidemia, respiratory complications, sleep apnea, hypertension, gall bladder disease, heart disease (e.g., coronary artery disease), and osteoarthritis.
  • compositions of this invention are also useful for reducing undesired adipose tissue.
  • ILY is administered to the subject at the site of undesired
  • CD59-expressing adipose cells CD59-expressing adipose cells.
  • ILY can be administered directly to an hCD59-expressing neoplasia, or systemically to a subject having a neoplasia characterized by the cell surface expression of hCD59.
  • Therapy may be performed alone or in conjunction with another therapy (e.g., surgery, radiation therapy, chemotherapy, immunotherapy, anti-angiogenesis therapy, or gene therapy).
  • the duration of the therapy depends on the type of disease or disorder being treated, the age and condition of the patient, the stage and type of the patient's disease, and how the patient responds to the treatment.
  • the methods and compositions of the invention are more effective than other methods and compositions.
  • “more effective” is meant that a method, composition, or kit exhibits greater efficacy, is less toxic, safer, more convenient, better tolerated, or less expensive, or provides more treatment satisfaction than another method, composition, or kit with which it is being compared.
  • Cancers include, without limitation, leukemias (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblasts leukemia, acute promyelocyte leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease, non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymph
  • the invention also features topical administration of ILY to treat skin-related disorders.
  • Inflammatory skin-related disorders result in the damage of healthy skin tissue by an inflammatory process.
  • Examples of inflammatory skin-related disorders include scleroderma, systemic lupus erythematosus, and inflammatory dermatoses.
  • Inflammatory dermatoses include, for example, psoriasis, atopic dermatitis, nonspecific dermatitis, lamellar ichthyosis, epidemlolytic hyperkeratosis, premalignant keratosis, acne, and seborrheic dermatitis, pityriasis roseas, acute febrile neutrophilic dermatosis, eczema (e.g., histotic eczema, dyshidrotic eczema, vesicular palmoplanar eczema), balanitis circumscripta plasmacellularis, balanoposthitis, Behcet disease, erythema annulare centrifugum, erythema dyschromicum perstans, erythema multiforme, granuloma annulare, lichen nitidus, lichen planus, lichen sclerosus et atrophicus, lichen simplex chronic
  • the invention is also useful for treating intrinsic aging (i.e., chronological aging) as well as extrinsic aging (i.e., resulting from environmental conditions).
  • Examples of such conditions include wrinkles (e.g., fine and coarse wrinkles), brown spots, dyspigmentation, laxity, yellow hue, telangiectasia, leathery appearance, lentigines, guttate hypomelanosis, solar keratoses, seborrhoeic keratoses, ephelides, actinic lentigo, and cutaneous malignancies.
  • ILY can be administered topically in combination with, for example, a neutralizing anti-ILY antibody.
  • the neutralizing anti-ILY antibody is administered portions of the skin where ILY-mediated cell death is undesired.
  • ILY can be delivered to the skin in a topical formulation.
  • Topical formulations include, without limitation, creams, lotions, gels, sticks, ointments, sprays, foams, patches, aerosols, wound dressings, and drops.
  • the formulations can be administered, for example, using a metered dose spray applicator, a micro-needle, iontophoresis, ultrasound penetration enhancement, electroporation, nano/micro-injection, sponge, or by applying and spreading the formulation by hand. Any conventional pharmacologically and cosmetically acceptable vehicles may be used.
  • compounds may be administered in liposomal formulations that allow the biologically active compounds to enter the skin. Such liposomal formulations are described in, for example, U.S.
  • Examples of other appropriate vehicles are described in U.S. Patent No. 4,877,805 and EP Publication No. 0586106A1.
  • Suitable vehicles of the invention may also include mineral oil, petrolatum, polydecene, stearic acid, isopropyl myristate, polyoxyl 40 stearate, stearyl alcohol, or vegetable oil.
  • the formulations can include various conventional colorants, fragrances, thickeners (e.g., xanthan gum), preservatives, humectants, emollients (e.g., hydrocarbon oils, waxes, or silicones), demulcents, emulsifying excipients, dispersants, penetration enhancers, plasticizing agents, preservatives, stabilizers, demulsifiers, wetting agents, emulsifiers, moisturizers, astringents, deodorants, and the like can be added to provide additional benefits and improve the feel and/or appearance of the topical preparation.
  • emollients e.g., hydrocarbon oils, waxes, or silicones
  • demulcents emulsifying excipients
  • dispersants e.g., hydrocarbon oils, waxes, or silicones
  • demulcents e.g., hydrocarbon oils, waxes, or silicones
  • demulcents emulsifying excip
  • the topical formulations of the invention will typically have a pH of between 5.5 and 8.5 and include from about 0.000001% to 10% (w/v), desirably 0.001% to 0.1% (w/v), of the compounds of the invention.
  • the formulations of the invention can also contain one or more antioxidants.
  • Useful antioxidants include, without limitation, thiols (e.g., aurothioglucose, dihydrolipoic acid, propylthiouracil, thioredoxin, glutathione, cysteine, cystine, cystamine, thiodipropionic acid), sulphoximines (e.g., buthionine-sulphoximines, homo-cysteine-sulphoximine, buthionine-sulphones, and penta-, hexa- and heptathionine-sulphoximine), metal chelators (e.g, ⁇ -hydroxy-fatty acids, palmitic acid, phytic acid, lactoferrin, citric acid, lactic acid, and malic acid, humic acid, bile acid, bile extracts, bilirubin, biliverdin, EDTA, EGTA, and DTPA),
  • Antioxidants that may be incorporated into the formulations of the invention include natural antioxidants prepared from plant extracts, such as extracts from aloe vera; avocado; chamomile; echinacea; ginko biloba; ginseng; green tea; heather; jojoba; lavender; lemon grass; licorice; mallow; oats; peppermint; St. John's wort; willow; wintergreen; wheat wild yam extract; marine extracts; and mixtures thereof.
  • plant extracts such as extracts from aloe vera; avocado; chamomile; echinacea; ginko biloba; ginseng; green tea; heather; jojoba; lavender; lemon grass; licorice; mallow; oats; peppermint; St. John's wort; willow; wintergreen; wheat wild yam extract; marine extracts; and mixtures thereof.
  • the total amount of antioxidant included in the formulations can be from 0.001% to 3% by weight, preferably 0.01% to 1% by weight, in particular 0.05% to 0.5% by weight, based on the total weight of the formulation.
  • Formulations of the invention can further include one or more emulsifying excipients.
  • Emulsifying excipients that may be used in the formulations of the invention include, without limitation, compounds belonging to the following classes: polyethoxylated fatty acids, PEG-fatty acid diesters, PEG-fatty acid mono-ester and di-ester mixtures, polyethylene glycol glycerol fatty acid esters, alcohol-oil transesterification products, polyglycerized fatty acids, propylene glycol fatty acid esters, mixtures of propylene glycol esters and glycerol esters, mono- and diglycerides, sterol and sterol derivatives, polyethylene glycol sorbitan fatty acid esters, polyethylene glycol alkyl ethers, sugar esters, polyethylene glycol alkyl phenols, polyoxyethylene-polyoxypropylene block copolymers, sorbitan fatty acid esters, lower alcohol fatty acid esters, ionic surfactants, to
  • Polyethoxylated fatty acids may be used as excipients for the formulations of the invention.
  • Examples of commercially available polyethoxylated fatty acid monoester surfactants include: PEG 4-100 monolaurate (Crodet L series, Croda), PEG 4-100 monooleate (Crodet O series, Croda), PEG 4-100 monostearate (Crodet S series, Croda, and Myrj Series, Atlas/ICI), PEG 400 distearate (Cithrol 4DS series, Croda), PEG 100, 200, or 300 monolaurate (Cithrol ML series, Croda), PEG 100, 200, or 300 monooleate (Cithrol MO series, Croda), PEG 400 dioleate (Cithrol 4DO series, Croda), PEG 400-1000 monostearate (Cithrol MS series, Croda), PEG-I stearate (Nikkol MYS-IEX, Nikko, and
  • Formulations of the invention may include one or more of the polyethoxylated fatty acids above.
  • Polyethylene glycol fatty acid diesters may be used as excipients for the formulations of the invention.
  • Examples of commercially available polyethylene glycol fatty acid diesters include: PEG-4 dilaurate (Mapeg® 200 DL, PPG), PEG-4 dioleate (Mapeg® 200 DO, PPG), PEG-4 distearate (Kessco® 200 DS, Stepan), PEG- 6 dilaurate (Kessco® PEG 300 DL, Stepan), PEG-6 dioleate (Kessco® PEG 300 DO, Stepan), PEG-6 distearate (Kessco® PEG 300 DS, Stepan), PEG-8 dilaurate (Mapeg® 400 DL, PPG), PEG-8 dioleate (Mapeg® 400 DO 5 PPG), PEG-8 distearate (Mapeg® 400 DS, PPG), PEG
  • PEG-fatty acid mono- and di-ester mixtures may be used as excipients for the formulations of the invention.
  • Examples of commercially available PEG-fatty acid mono- and di-ester mixtures include: PEG 4-150 mono, dilaurate (Kessco® PEG 200- 6000 mono, Dilaurate, Stepan), PEG 4-150 mono, dioleate (Kessco® PEG 200-6000 mono, Dioleate, Stepan), and PEG 4-150 mono, distearate (Kessco® 200-6000 mono, Distearate, Stepan).
  • Formulations of the invention may include one or more of the PEG-fatty acid mono- and di-ester mixtures above.
  • Propylene glycol fatty acid esters may be used as excipients for the formulations of the invention.
  • Examples of commercially available propylene glycol fatty acid esters include: propylene glycol monocaprylate (Capryol 90, Gattefosse), propylene glycol monolaurate (Lauroglycol 90, Gattefosse), propylene glycol oleate (Lutrol OP2000, BASF), propylene glycol myristate (Mirpyl), propylene glycol monostearate (LIPO PGMS, Lipo Chem.), propylene glycol hydroxystearate, propylene glycol ricinoleate (PROPYMULS, Henkel), propylene glycol isostearate, propylene glycol monooleate (Myverol P-06, Eastman), propylene glycol dicaprylate dicaprate (Captex® 200, ABITEC), propylene glycol dioctanoate (Captex® 800, ABITEC
  • Formulations of the invention may include one or more of the mono- and diglycerides above.
  • Sterol and sterol derivatives may be used as excipients for the formulations of the invention.
  • Examples of commercially available sterol and sterol derivatives include: cholesterol, sitosterol, lanosterol, PEG-24 cholesterol ether (Solulan C-24, Amerchol), PEG-30 cholestanol (Phytosterol GENEROL series, Henkel), PEG-25 phytosterol (Nikkol BPSH-25, Nikko), PEG-5 soyasterol (Nikkol BPS-5, Nikko), PEG-IO soyasterol (Nikkol BPS-IO, Nikko), PEG-20 soyasterol (Nikkol BPS-20, Nikko), and PEG-30 soyasterol (Nikkol BPS-30, Nikko).
  • Formulations of the invention may include one or more of the sterol and sterol derivatives above.
  • Typical counterions are provided above. It will be appreciated by one skilled in the art, however, that any bioacceptable counterion may be used.
  • the fatty acids are shown as sodium salts, other cation counterions can also be used, such as, for example, alkali metal cations or ammonium.
  • Formulations of the invention may include one or more of the ionic surfactants above.
  • Ointments, pastes, jellies, liquids, foams, gels and creams may, for example, be formulated with an aqueous or oil base with the addition of suitable thickening and/or gelling agents.
  • Lotions may be formulated with an aqueous base or an oil base and will in general also contain one or more emulsifying agents, coloring agents, stabilizing agents, suspending agents, thickening agents or surfactants, such as a nonionic surfactant, for example, a polyoxyethylene higher alcohol ether or polyethylene glycol.
  • Additives that may be used in pharmaceutical compositions containing ILY may include excipients (such as starch, dextrin, mannitol, cyclodextrin and traganth) binding agents, fillers, colorants (such as beta-carotin), lubricants, isotonic agents (such as sodium chloride or glucose), disintegrants, antioxidants (such as ascorbic acid, erythorbic acid, or a salt or ester thereof), or wetting agents. Additional additives utilized in pharmaceutical compositions can be found, for example, in Remington: The Science and Practice of Pharmacy (Remington the Science and Practice of Pharmacy), 21 st ed., (2005), edited by University of the Sciences in Philadelphia.
  • tissue-specific cell ablation transgenic mouse models one expressing hCD59 only on erythrocytes (ThCDS ⁇ 0 ) and the other only on endothelial cells ⁇ ThCD5 ⁇ NO ).
  • ThCD ⁇ 80 the transgene was under the control of the alpha hemoglobin promoter and for ThCD5 ⁇ ND under the control of the ICAM-2 promoter (see experimental data set forth below).
  • the invention features tissue specific expression of hCD59 by placing the hCD59 under other tissue specific promoters.
  • An exemplary nucleic acid sequence encoding hCD59 is set forth in Table 1. Such promoters include, for example, the those set forth in Table 2.
  • hCD59 can also be expressed in a cell-type or tissue specific manner through the use of a recombinase activation system.
  • a recombinase activation system for example the Cre- recombinase or FLP recombinase systems
  • Cre- recombinase or FLP recombinase systems contain a recombinase under the control of a tissue specific or cell-type specific promoter (see, for example, Buch, Nat Methods 2:419 (2005) and Saito et al., Nat Biotechnol 19:746 (2001)).
  • this recombinase modifies the hCD59 transgene in the cellular genome causing hCD59 to be functionally expressed (for example, as described below).
  • a transgenic Cre-recombinase expressing animal e.g., mouse
  • Cre-recombinase in that tissue will remove the STOP codon and allow the expression of hCD59 protein in the membrane of the specific cells selected.
  • Examples of animals suitable for tissue specific expression of Cre recombinase and FLP recombinase are set forth in Table 2.
  • CD59 is also highly expressed in various cancer cells such as prostate, breast and gastric adenomas and intestinal-type gastric carcinomas, and B-cell lymphoma. Higher expression levels of hCD59 in neoplastic cells has been correlated with cellular resistance to certain chemotherapeutic drugs.
  • ILY specifically binds to human CD59 and lyses human cells that express human CD59 on the cell surface. We found that humans develop specific immunity to protect cells from ILY-mediated cell lysis.
  • RBC of HCD59RBC transgenic mice makes the hCD59RBC +/ ⁇ mRBCs hyper-sensitive to ILY-mediated lysis. This lysis is comparable to the level of ILY-mediated lysis of human RBC (Fig. IA). WT mRBC are resistant to ILY-mediated lysis.
  • ILY in vivo study, we administrated different doses of ILY by tail vein injection. With three different doses of ILY (95, 47, 30 ng/g body weight), the percentages of ILY-induced cell death o ⁇ hCD59RBC transgenic mice were 100% (15/15 animals), 50% (8/16 animals) and 0% (0/6 animals), respectively. Based on this data, we consider the dose of 95 ng ILY/g body weight as the lethal dose (LDioo) in our in vivo experiments.
  • LDioo lethal dose
  • Inhibitors in human serum neutralize the lytic effect of ILY.
  • SI (1) is part of the normal human oral micro flora, (2) can cause liver and brain abscesses, and (3) secretes ILY that specifically lyses human cells due to the presence of hCD59 in their surface
  • humans may develop some immune defenses that protect against ILY -mediated cell lysis and pathogenic SI infection.
  • hemolytic assays with human RBC. Human RBC were incubated with different concentrations of ILY and a 1 to 8 dilution of serum in PBS from different species.
  • Fig. 2A shows that human serum, and not the serum from other 11 animal species, significantly blocked ILY-mediated human RBC lysis.
  • ILY-binding human IgG In order to determine the in vivo effect of ILY-binding human IgG, we first treated hCD59RBC v/' with an intravenous (IV; injection of different doses of the ILY- binding human IgG. Fifteen minutes later, we injected these mice with 285 ng ILY/g body weight (three times lethal dose (LD) of ILY in untreated hCD59RBC ⁇ /' mice). We found that the survival percentage of hCD59RBC +/ ⁇ pre-treated with 1 and 0.75 ⁇ g ILY-binding human IgG/g body weight was 89% (8/9) and 62.5% (5/8), respectively. This result confirms that the ILY-binding human IgG blocks ILY function in vivo and suggests that an anti-ILY antibody may be useful for the treatment of SI infectious disease.
  • IV intravenous
  • the methods of the invention are useful for tissue specific cell ablation in animal models of development and disease.
  • the below example demonstrates several working embodiments of tissue specific cell ablation using expression of hCD59 and ILY.
  • Transgenic mice express human CD59 on erythrocytes and endothelia
  • CD59 is a membrane-bound complement regulator that inhibits formation of the membrane attack complex (Qin Immunity 18:217-227 (2003)).
  • ThCD59 RBC mice expressing human CD59 in erythrocytes (Kooyman et al. Science 269:89-92 (1995)); human CD59 expression on erythrocytes in these mice was associated with increased resistance to membrane attack complex-mediated lysis, and expression of human CD59 mRNA was specific to hematopoietic organs (Figs. 9A- C).
  • ThCD59 END mice expressing human CD59 on endothelia; human CD59 mRNA and protein was expressed only in blood vessels in these mice (Figs. 9D and 9E).
  • ThCD59 RBC mice Pretreatment of ThCD59 RBC mice with ILY-neutralizing human IgG prevented death after administration of a lethal dose of ILY, but pretreatment with ILY-binding bovine IgG did not have this preventive effect (Fig. 6A). ILY-mediated intravascular hemolysis in ThCD59 RBC mice was also prevented by human IgG but not by bovine IgG (Fig. 6B).
  • ThCD59 RBC mice markedly decreased NO levels and increased platelet activation, as shown by increased plasma P selectin (sP-selectin) — an effect prevented by pretreatment with ILY-neutralizing human IgG (Figs. 6C and 6D).
  • ThCD59 RBC and ThCD59 END mice for the prototypic development of a new model of rapid conditional targeted cell ablation that takes advantage of ILY-mediated lysis exclusively on cells carrying human CD59.
  • Transgenic mice carrying human CD59 helped confirm in vivo that human CD59 is the only receptor for ILY.
  • This ILY-mediated cell ablation system may be useful for cellular function and/or tissue regeneration studies not only in mice but also in intermediate-sized and large animals transgenically expressing human CD59 in the cells or organs of interest.
  • the erythrocytic and endothelial ablation models established here provide a new tool for studying the pathogenesis of and potential treatments for human diseases characterized by hemolytic anemia or endothelial damage.
  • ILY mediates intravascular hemolysis, a common manifestation of several human diseases, including paroxysmal nocturnal hemoglobinuria, sickle cell disease, autoimmune anemias, thalassemias, transfusion reactions and infection-induced anemias.
  • the intravascular hemolysis caused by these conditions results in common clinical manifestations such as abdominal pain, dysphagia, erectile dysfunction, endothelial dysfunction, pulmonary hypertension, renal failure and platelet activation with thrombosis.
  • ILY -mediated targeted cell ablation not only in mice but also in other animal species is made possible by specific features that we have documented here: (i) ILY does not damage cells from any animal species other than humans, (ii) ILY shows high affinity and specificity for human CD59 in vivo, allowing the ablation of specific cell populations by targeted expression of transgenic human CD59, and (iii) ILY-neutralizing antibodies are absent in species other than humans, allowing the use of ILY in transgenic animals.
  • ILY-neutralizing antibodies are present only in humans is still unclear.
  • humans are routinely exposed to infection with Streptococcus intermedins and ILY and have developed specific antibodies against functional domain(s) of ILY — in particular, domain 4, which serves as the binding site for human CD59.
  • domain 4 which serves as the binding site for human CD59.
  • the presence of non-neutralizing ILY antibodies in other species may be explained by ILY belonging to the CDC family of toxins, which contributes to the pathogenesis of a variety of human and animal diseases caused by Gram-positive bacteria.
  • the 40-80% sequence similarity between ILY and more than 20 members of the CDC family is probably responsible for the antibody cross-reactivity.
  • CDC binding antibodies may bind to ILY but not to ILY domain 4, which is critical for the binding of ILY to human CD59 and for lytic activity.
  • the ILY-mediated cell ablation model has other attractive features.
  • the effective cell ablation occurs very rapidly — within seconds after ILY administration.
  • diphtheria toxin-mediated cell damage in Hbegf transgenic mice is effective within a time frame of days.
  • ILY-mediated rapid cell ablation may help define primary pathophysiological changes without confounding effects due to adaptive responses of other cells.
  • ILY-mediated cell ablation is specifically confined to cells carrying human CD59. ILY lyses only human cells, although cells from other species also carry homologous CD59.
  • ILY-mediated cell ablation is highly efficient and potent. At doses as low as 30 ng per g body weight and 4.5 ng per g body weight, ILY induces massive intravascular hemolysis in ThCD59 RBC mice and severe endothelial damage in ThCD59 END mice, respectively, without any evidence of off-target effects in wild-type mice or in transgenic mice pretreated with neutralizing antibodies. Fourth, ILY- mediated cell ablation can be achieved by several routes of administration: intravenous, intraperitoneal and intramuscular administration all produce comparable results (Figs.
  • ILY can pass through the endothelial barrier.
  • ILY injection ILY is engulfed by resident macrophages in organs such as brain, liver and lung, as shown by double immunofluorescence assay (Figs. 1 IA- 11 C).
  • Figs. 1 IA- 11 C a double immunofluorescence assay
  • recombinant ILY can be easily procured from prokaryotic expression systems.
  • ILY-mediated cell ablation may be the method of choice for ablation of nondividing cells such as erythrocytes.
  • the ILY-mediated in vivo specific cell ablation method reported here represents an alternative model applicable to studies of cell function, tissue regeneration and differentiation and will be useful for investigating the pathogenesis of and potential therapies for prevalent human ailments such as hemolytic anemia and cardiovascular diseases.
  • Hemolytic assay We performed hemolytic assays as described previously (Giddings et al. Nat. Struct. MoI. Biol. 1 1 :1173-1178 (2004)). To evaluate a possible protective effect against ILY-mediated hemolysis of red blood cells, we added serially diluted ILY to human erythrocytes preincubated with human serum at a 1 :8 dilution in PBS (ComTech). We tested sera from other species (Valley Biomedical) for ILY inhibitory effects by a hemolytic assay. Before the hemolytic assay, the eluate and flow-through fractions were dialyzed and concentrated to volumes equal to the volume of serum that was loaded on the column.
  • ILY LD 50 in ThCD59 RBC mice We randomly assigned eight mice per group (four males and four females) to one of five groups and recorded the percentage of mice surviving or dying within 6 h after intravenous (i.v.) ILY injection.
  • Hematocrit, plasma hemoglobin and hemoglobinuria measurement We measured hematocrit, plasma hemoglobin and hemoglobinuria as described previously (Qin Immunity 18:217-227 (2003)). Measurement of nitric oxide products nitrite and nitrate. We measured the concentration of the NO products nitrite and nitrate 1 h after ILY treatment, using a
  • Nitric Oxide Quantitation Kit (ActiveMotif). sP-selectin measurement.
  • mice blood samples by venipuncture from the inferior vena cava with a syringe containing 10 mM EDTA and centrifuged samples (2,00Og, 20 min) 1 h after ILY treatment.
  • Evans blue dye (Sigma-Aldrich) in PBS into the tail vein 2 h after ILY treatment. We killed the mice 10 min after Evans blue dye injection and then fixed them by perfusion with 4% phosphate-buffered formalin (pH 7). Next, we harvested mouse aortas, opened them along the long axis and evaluated blue-stained areas by light microscopy. We quantified Evans blue staining areas using Image ProPlus 6.0. von Willebrand Factor (vWF) measurement. vWF is a biomarker for endothelial damage. We measured mouse plasma vWF by ELISA as described previously (Denis et al. Proc. Natl. Acad. Sci. USA 95:9524-9529 (1998)).
  • mice pretreated mice with either PBS or ILY-neutralizing human IgG (1 ⁇ g per g body weight) for 15 min, injected them with ILY (3 ng per g body weight) and then measured plasma vWF 3 h after ILY injection. Electron microscopy.
  • TAT a stable complex formed by the reaction of thrombin with its major inhibitor anti thrombin III, represents a sensitive marker for the activation of intravascular coagulation,.
  • Platelet count We counted platelets as described previously (Qin Immunity 18:217-227 (2003)).
  • ThCD59 transgenic mice We generated ThCD59 mice expressing hCD59 in erythrocytes in C57BL/BJ background with the construct described previously (Saito et al. Nat. Biotechnol. 19:746-750 (2001)).
  • the transgenic vector consisted of the human alpha hemoglobin locus control region, alpha hemoglobin gene promoter, and hCD59 cDNA (from 5' to 3').
  • Non-transgenic C57BL/6J mice were WT controls.
  • ThCD59 transgenic mice We generated ThCD59 mice expressing hCD59 in endothelial cells with a transgenic vector by established methods.
  • the vector consisted of the human ICAM-II gene promoter and hCD59 cDNA (from 5' to 3').
  • Northern Blot Analysis We isolated total RNA from multiple tissues of
  • ThCD 59 and ThCD 59 mice using TRIZOL reagent Invitrogen
  • FACS analysis We incubated erythrocytes from ThCD59 with Brie 229 for 30 min at room temperature, washed three times with 3% BSA/PBS buffer, and incubated for 30 min with a FITC-conjugated goat anti-mouse secondary antibody. We then washed the cells in PBS three times before analysis of fluorescence intensity by FACScan (Becton Dickinson).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Epidemiology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Urology & Nephrology (AREA)
  • Engineering & Computer Science (AREA)
  • Endocrinology (AREA)
  • Zoology (AREA)
  • Rheumatology (AREA)
  • Diabetes (AREA)
  • Dermatology (AREA)
  • Biomedical Technology (AREA)
  • Toxicology (AREA)
  • Pathology (AREA)
  • Gynecology & Obstetrics (AREA)
  • Reproductive Health (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne des compositions et des procédés qui font appel à l'intermedilysine pour traiter les états associés à l'expression de la CD59 humaine. L'invention porte également sur des procédés et des compositions qui font appel à l'intermedilysine afin de prévenir une grossesse. L'invention se rapporte en outre à des procédés qui permettent de tuer les cellules exprimant la CD59 chez un animal non humain.
PCT/US2007/026005 2006-12-26 2007-12-21 Procédés et compositions destinés à tuer les cellules exprimant la cd59 humaine Ceased WO2008085366A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US87714706P 2006-12-26 2006-12-26
US60/877,147 2006-12-26

Publications (2)

Publication Number Publication Date
WO2008085366A2 true WO2008085366A2 (fr) 2008-07-17
WO2008085366A3 WO2008085366A3 (fr) 2009-05-14

Family

ID=39609187

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2007/026005 Ceased WO2008085366A2 (fr) 2006-12-26 2007-12-21 Procédés et compositions destinés à tuer les cellules exprimant la cd59 humaine

Country Status (1)

Country Link
WO (1) WO2008085366A2 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2142566A4 (fr) * 2007-03-30 2010-06-30 Harvard College Procedes et compositions pour le traitement de maladies proliferatives
EP2467492A4 (fr) * 2009-08-18 2013-01-09 Harvard College Méthodes et compositions pour le traitement de maladies prolifératives et pathogènes

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7094750B2 (en) * 2000-05-09 2006-08-22 Greenville Hospital System Therapeutic pore-forming peptides

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2142566A4 (fr) * 2007-03-30 2010-06-30 Harvard College Procedes et compositions pour le traitement de maladies proliferatives
EP2467492A4 (fr) * 2009-08-18 2013-01-09 Harvard College Méthodes et compositions pour le traitement de maladies prolifératives et pathogènes
US9163086B2 (en) 2009-08-18 2015-10-20 President And Fellows Of Harvard College Methods and compositions for the treatment of proliferative and pathogenic diseases

Also Published As

Publication number Publication date
WO2008085366A3 (fr) 2009-05-14

Similar Documents

Publication Publication Date Title
Yabluchanskiy et al. CORM-3, a carbon monoxide-releasing molecule, alters the inflammatory response and reduces brain damage in a rat model of hemorrhagic stroke
Arteel et al. Chronic enteral ethanol treatment causes hypoxia in rat liver tissuein vivo
DE3854536T2 (de) Interzellulare Adhäsions-Moleküle und deren Bindungsliganden.
Yuan et al. The iron hypothesis of atherosclerosis and its clinical impact
Poltera Pathology of human African trypanosomiasis with reference to experimental African trypanosomiasis and infections of the central nervous system
AU2006275891B2 (en) SCF extract containing cardiac glycoside
Van Beneden et al. Comparison of trichostatin A and valproic acid treatment regimens in a mouse model of kidney fibrosis
Morita et al. The role of complement in the pathogenesis of tubulointerstitial lesions in rat mesangial proliferative glomerulonephritis.
AU2017245395A1 (en) Methods and reagents for creating monoclonal antibodies
Gautier et al. Neutrophils contribute to intracerebral haemorrhages after treatment with recombinant tissue plasminogen activator following cerebral ischaemia
DE68923675T2 (de) Interzellulare Adhäsions-Moleküle und deren Bindungsliganden.
Soma et al. Immunoglobulin γ3-heavy-chain deposition disease: report of a case and relationship with hypocomplementemia
Speeg et al. Effect of the nonimmunosuppressive cyclosporin analog SDZ PSC-833 on colchicine and doxorubicin biliary secretion by the rat in vivo
Thamilarasan et al. Mn porphyrins as a novel treatment targeting sickle cell NOXs to reverse and prevent acute vaso-occlusion in vivo
van Alem et al. Liposomal delivery improves the efficacy of prednisolone to attenuate renal inflammation in a mouse model of acute renal allograft rejection
Ono et al. Sclerosing effect of OC-108, a novel agent for hemorrhoids, is associated with granulomatous inflammation induced by aluminum
WO2008085366A2 (fr) Procédés et compositions destinés à tuer les cellules exprimant la cd59 humaine
Wang et al. A rationally designed CD19 monoclonal antibody-triptolide conjugate for the treatment of systemic lupus erythematosus
Morioka et al. Histone mediates glomerular deposition of small size DNA anti-DNA complex
Andriole Acceleration of the inflammatory response of the renal medulla by water diuresis.
DE69935603T2 (de) Inhibitoren von autoantikörpern
Figueiredo et al. Antinociceptive activity and toxicology of the lectin from Canavalia boliviana seeds in mice
Selye On the protective action of testosterone against the kidney damaging effect of sublimate
Kamalpour et al. Injection adipolysis: mechanisms, agents, and future directions
WO2011047309A2 (fr) Traitement de tissu ischémique

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07867857

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 07867857

Country of ref document: EP

Kind code of ref document: A2