CA2205771C - Improved delivery of disease modifiers - Google Patents
Improved delivery of disease modifiers Download PDFInfo
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- CA2205771C CA2205771C CA002205771A CA2205771A CA2205771C CA 2205771 C CA2205771 C CA 2205771C CA 002205771 A CA002205771 A CA 002205771A CA 2205771 A CA2205771 A CA 2205771A CA 2205771 C CA2205771 C CA 2205771C
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- hyaluronan
- hbm
- amino acids
- protein
- basic amino
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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
- A61K38/16—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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
Abstract
A pharmaceutical composition component comprising hyaluronan binding motif (HBM) interposed between a form of hyaluronan having a molecular weight (protein standard) less than 750,000 daltons and a disease modifier which comprises a peptide or protein.
Description
TITLE OF INVENTION
Improved Delivery of Disease Modifiers FIELD OF INVENTION
This invention relates to novel methods for delivery of disease modifiers using hyaluronan and to new compositions comprising the disease modifiers and hyaluronan.
BACKGROUND OF INVENTION
Hyaluronic acid is a large, complex oligosaccaride consisting of up to 50,000 pairs of the basic disaccharide glucuronic acid-Q(1-3) N-acetylglucos-amine Q(1-4). It is found in vivo as a major component of the extracellular matrix. Its tertiary structure is a random coil of about 50 nm in diameter.
Hyaluronic acid appears in nature in its sodium salt form. Hyaluronic acid and its pharmaceutically tolerable or acceptable salts (such as sodium hyaluronate) are referred to as Hyaluronan (HA).
Hyaluronan has the ability to bind a large amount of water, which in vivo makes it a viscous hydrated gel with viscoelastic properties. It is found in this form in the mammalian eye, both in the vitreous and in the extracellular matrix.
Hyaluronan (Hyaluronic Acid and pharmaceutically acceptable Salts Thereof) have been disclosed for use with medicine and/or therapeutic agents for the treatment of disease and/or conditions (see PCT Application, PCT/CA 90/00306, International Publication No. WO 91/04058).
Subsequent applications taught the combination of hyaluronic acid and pharmaceutically acceptable salts thereof for topical treatment and for accumulation (see PCT Application, PCT/CA 93/00061, International Publication No. WO 93/16732). It has been postulated that the medicine or therapeutic agent for example, an NSAID, appears to be associated with the hyaluronan as a clathrin (term is taken from clathrinida, an order of sponges which have an asconoid structure and lack a true dermal
Improved Delivery of Disease Modifiers FIELD OF INVENTION
This invention relates to novel methods for delivery of disease modifiers using hyaluronan and to new compositions comprising the disease modifiers and hyaluronan.
BACKGROUND OF INVENTION
Hyaluronic acid is a large, complex oligosaccaride consisting of up to 50,000 pairs of the basic disaccharide glucuronic acid-Q(1-3) N-acetylglucos-amine Q(1-4). It is found in vivo as a major component of the extracellular matrix. Its tertiary structure is a random coil of about 50 nm in diameter.
Hyaluronic acid appears in nature in its sodium salt form. Hyaluronic acid and its pharmaceutically tolerable or acceptable salts (such as sodium hyaluronate) are referred to as Hyaluronan (HA).
Hyaluronan has the ability to bind a large amount of water, which in vivo makes it a viscous hydrated gel with viscoelastic properties. It is found in this form in the mammalian eye, both in the vitreous and in the extracellular matrix.
Hyaluronan (Hyaluronic Acid and pharmaceutically acceptable Salts Thereof) have been disclosed for use with medicine and/or therapeutic agents for the treatment of disease and/or conditions (see PCT Application, PCT/CA 90/00306, International Publication No. WO 91/04058).
Subsequent applications taught the combination of hyaluronic acid and pharmaceutically acceptable salts thereof for topical treatment and for accumulation (see PCT Application, PCT/CA 93/00061, International Publication No. WO 93/16732). It has been postulated that the medicine or therapeutic agent for example, an NSAID, appears to be associated with the hyaluronan as a clathrin (term is taken from clathrinida, an order of sponges which have an asconoid structure and lack a true dermal
-2-membrane or cortex), or is associated with the hyaluronan in a patient to whom the combination is administered in association with the patient's serum albumin which serum albumin appears to bind to the hyaluronan.
It is possible to bind hyaluronan directly to a medicine or therapeutic agent. In this regard, see "Effects of Precipitates formed by insulin with hyaluronic acid and mucoid from vitreous humor i n depressing blood-sugar levels ", Science 1950; 111: 520-521 at 520; "Reaction of Cationic Groups of Chlorpromazine with Anionic Macromolecules:
Complexes with DNA, RNA, Hyaluronic Acid and Heparin ", Acta pharmacol. et toxicol 1974, 34, 27-32 at pages 30 to 31, and U.S. Patent 5,166,331.
PCT Application PCT/CA95/00477, International Publication No.
WO 96/06622, also owned by Hyal Pharmaceutical Corporation, teaches the modulation of cellular activity of tissue and cells expressing a high affinity cell-surface receptor for hyaluronic acid by the use of forms of hyaluronic acid. These cell surface receptors comprise adhesion molecule CD44 and adhesion molecule HARLEC (Hyaluronic Acid [Hyaluronan] Receptors Liver Endothelial Cells) and regulatory molecule RHAMM (Receptor for HA Mediated Motility), for binding hyaluronan. HARLEC is expressed (produced and put on the cell surface) in liver endothelial cells. The administration of an effective amount of a form of hyaluronic acid to bind with the cell-surface receptors modulates cellular activity of tissues and/or cells expressing such high affinity cell-surface receptors for hyaluronic acid (for example, an adhesion or regulatory molecule) in the human body.
One of the reasons why the hyaluronic acid is able to be used to transport the medicine and/or therapeutic agent is its selective binding to the cell-surface receptors through a Hyaluronan Binding Motif.
It is possible to bind hyaluronan directly to a medicine or therapeutic agent. In this regard, see "Effects of Precipitates formed by insulin with hyaluronic acid and mucoid from vitreous humor i n depressing blood-sugar levels ", Science 1950; 111: 520-521 at 520; "Reaction of Cationic Groups of Chlorpromazine with Anionic Macromolecules:
Complexes with DNA, RNA, Hyaluronic Acid and Heparin ", Acta pharmacol. et toxicol 1974, 34, 27-32 at pages 30 to 31, and U.S. Patent 5,166,331.
PCT Application PCT/CA95/00477, International Publication No.
WO 96/06622, also owned by Hyal Pharmaceutical Corporation, teaches the modulation of cellular activity of tissue and cells expressing a high affinity cell-surface receptor for hyaluronic acid by the use of forms of hyaluronic acid. These cell surface receptors comprise adhesion molecule CD44 and adhesion molecule HARLEC (Hyaluronic Acid [Hyaluronan] Receptors Liver Endothelial Cells) and regulatory molecule RHAMM (Receptor for HA Mediated Motility), for binding hyaluronan. HARLEC is expressed (produced and put on the cell surface) in liver endothelial cells. The administration of an effective amount of a form of hyaluronic acid to bind with the cell-surface receptors modulates cellular activity of tissues and/or cells expressing such high affinity cell-surface receptors for hyaluronic acid (for example, an adhesion or regulatory molecule) in the human body.
One of the reasons why the hyaluronic acid is able to be used to transport the medicine and/or therapeutic agent is its selective binding to the cell-surface receptors through a Hyaluronan Binding Motif.
-3-Hyaluronan Binding motif (HBM) has been identified and is identified BX7B. See the article entitled Identification of a common hyaluronan binding motif in the hyaluronan binding proteins RHAMM, CD44 and link protein; The EMBO Journal, Vol. 13, No. 2 (1994) pp. 286-296.
While disease modifiers such as Cytokines, peptides mimicking cytokines, and proteins mimicking cytokines for example, may be administered to humans with the subject matter of PCT/CA 90/00306 (International Publication Number WO 91/04058), we have developed an improved method of administration of these disease modifiers (cytokines, peptides mimicking cytokines and proteins mimicking cytokines and other proteins and peptides).
Hyaluronan interacts at sites of cell migration and proliferation via specific hyaluronan receptors (CD44, IZHAMM, CD38, TSG-6, and extra-cellular hyaluronan binding proteins (Versican, Aggrecan, Perlican, link-protein, GHAP) all being examples thereof). These receptors and binding proteins are upregulated at the cites of proliferation/migration. As a result, addition of exogenous hyaluronan targets to these sites for example, where injury has occurred.
It is therefore an object of this invention to provide a new method of delivery of disease modifiers to the human using hyaluronan.
It is a further and other object of the invention to provide new compositions for use in the new method of delivery.
These and other objects of the invention will be realized by those skilled in the art from the following summary of the invention and detailed description of embodiments thereof.
SUMMARY OF INVENTION
Hyaluronan binding motif (HBM) as previously stated has (have) been identified in all hyaluronan binding proteins and receptors and is
While disease modifiers such as Cytokines, peptides mimicking cytokines, and proteins mimicking cytokines for example, may be administered to humans with the subject matter of PCT/CA 90/00306 (International Publication Number WO 91/04058), we have developed an improved method of administration of these disease modifiers (cytokines, peptides mimicking cytokines and proteins mimicking cytokines and other proteins and peptides).
Hyaluronan interacts at sites of cell migration and proliferation via specific hyaluronan receptors (CD44, IZHAMM, CD38, TSG-6, and extra-cellular hyaluronan binding proteins (Versican, Aggrecan, Perlican, link-protein, GHAP) all being examples thereof). These receptors and binding proteins are upregulated at the cites of proliferation/migration. As a result, addition of exogenous hyaluronan targets to these sites for example, where injury has occurred.
It is therefore an object of this invention to provide a new method of delivery of disease modifiers to the human using hyaluronan.
It is a further and other object of the invention to provide new compositions for use in the new method of delivery.
These and other objects of the invention will be realized by those skilled in the art from the following summary of the invention and detailed description of embodiments thereof.
SUMMARY OF INVENTION
Hyaluronan binding motif (HBM) as previously stated has (have) been identified in all hyaluronan binding proteins and receptors and is
-4-strongly conserved amongst species. HBM is identified as BX7B and is known to persons skilled in the art. The affinity of the hyaluronan for the HBM is enhanced if flanking basic amino acids are added or if several internal basic amino acids are added at position 4,5. Furthermore, binding activity is enhanced if the intervening amino acids are hydrophobic and not acidic. Using the hyaluronan binding motif (HBM), it is now possible to bestow hyaluronan binding properties upon a protein or peptide that does not normally bind to hyaluronan. For example, the addition of the HBM to casein, a milk protein that does not bind to hyaluronan bestows binding activity on the protein.
Therefore, according to one aspect of the invention, I propose the use of the hyaluronan binding motif (HBM) to be interposed between a disease modifier (which may be a protein or peptide and which protein or peptide one skilled in the art would not consider under normal conditions to be capable of being bound effectively with hyaluronan) and hyaluronan.
By linking the components, I have found that the combination will target the disease modifier for example, proteins such as cytokines, peptides mimicking cytokines, and proteins mimicking cytokines to the sites of, for example, injury or disease. The hyaluronan by binding through HBM to the protein or peptide also protects the protein or peptide from attack from Proteases which appear in high numbers at the sites of injury. Further, the hyaluronan also protects protein/peptide from immune system recognition and attack and possible destruction.
While the disease modifier includes proteins and peptides (which may be considered drugs in the usual sense such as for example antibiotics), they also include any other disease modifier which could be chemically linked to the amino or carboxy terminus of the hyaluronan binding peptide. Such disease modifiers include smyonpford. cyclosporin
Therefore, according to one aspect of the invention, I propose the use of the hyaluronan binding motif (HBM) to be interposed between a disease modifier (which may be a protein or peptide and which protein or peptide one skilled in the art would not consider under normal conditions to be capable of being bound effectively with hyaluronan) and hyaluronan.
By linking the components, I have found that the combination will target the disease modifier for example, proteins such as cytokines, peptides mimicking cytokines, and proteins mimicking cytokines to the sites of, for example, injury or disease. The hyaluronan by binding through HBM to the protein or peptide also protects the protein or peptide from attack from Proteases which appear in high numbers at the sites of injury. Further, the hyaluronan also protects protein/peptide from immune system recognition and attack and possible destruction.
While the disease modifier includes proteins and peptides (which may be considered drugs in the usual sense such as for example antibiotics), they also include any other disease modifier which could be chemically linked to the amino or carboxy terminus of the hyaluronan binding peptide. Such disease modifiers include smyonpford. cyclosporin
-5-and other therapeutic peptides such as cytokine peptides, or cell adhesion peptides but are not limited thereto. They also include the following:
Drug Use Route Protein Hormones Follicle Stimulating Hormone (FSH) Leutinizing Hormone amenorrhea intravenous (LH) Prolactin chronic renal insufficiency Human Growth human growth intramuscular Hormone (GH) hormone deficiency parenteral in children Adrenocorticotropin hypercalcemia intravenous Hormone (ACTH) and inflamation analogues (eg. diagnosis of adrenal Leuprolide Acetate) insufficiency Vasopressin, Lypressindiabetes insipidus intranasal Desmopressin parenteral injection Oxytocin (OT) lactation intranasal, intravenous postpartum bleedingparenteral induction of labour Gonadotropin Releasinginfertility, suppressionintranasal Hormone (GnRH) of ovulation, prostateinjection and breast tumours Gonadorelin diagnosis of subcutaneous, hypothalemic - pituitaryintravenous; intranasal - gonadal dysfunction amenorrhoea infertility Corticotropin Releasing Hormone (CRH) Thyrotropin Releasinglactation transdermal, oral Hormone (TRH) Leutinizing Hormone cryptorchidism intranasal Releasing Hormone endometriosis (LHRH) Melanocyte - depression oral Stimulating Hormone Tardive dyskinesia Inhibiting Factor (MIF) Melanocyte - transdermal Stimulating Hormone Releasing Factor (MSH)
Drug Use Route Protein Hormones Follicle Stimulating Hormone (FSH) Leutinizing Hormone amenorrhea intravenous (LH) Prolactin chronic renal insufficiency Human Growth human growth intramuscular Hormone (GH) hormone deficiency parenteral in children Adrenocorticotropin hypercalcemia intravenous Hormone (ACTH) and inflamation analogues (eg. diagnosis of adrenal Leuprolide Acetate) insufficiency Vasopressin, Lypressindiabetes insipidus intranasal Desmopressin parenteral injection Oxytocin (OT) lactation intranasal, intravenous postpartum bleedingparenteral induction of labour Gonadotropin Releasinginfertility, suppressionintranasal Hormone (GnRH) of ovulation, prostateinjection and breast tumours Gonadorelin diagnosis of subcutaneous, hypothalemic - pituitaryintravenous; intranasal - gonadal dysfunction amenorrhoea infertility Corticotropin Releasing Hormone (CRH) Thyrotropin Releasinglactation transdermal, oral Hormone (TRH) Leutinizing Hormone cryptorchidism intranasal Releasing Hormone endometriosis (LHRH) Melanocyte - depression oral Stimulating Hormone Tardive dyskinesia Inhibiting Factor (MIF) Melanocyte - transdermal Stimulating Hormone Releasing Factor (MSH)
-6-Growth Hormone transdermal Releasing Hormone (GHRH) Somatostatin acromegaly, GI intranasal tumours, intravenous gastric ulcers Corticotrophin diagnostic agent to intravenous investigate adrenocortical insufficiency Tetracosactide diagnostic agent to intramuscular investigate intravenous adrenocortical insufficiency Octreotide Acetategastrointestinal subcutaneous endocrine tumours acromegaly Parathyroid Hormoneosteoporosis subcutaneous (PTH) Thyroid Stimulatingdiagnosis of thyroid injection Hormone (TSH) / disease Thyroid Releasing Hormone (TRH) Insulin diabetes mellitus intravenous transdermal Glucagon hypocalcemia parenteral intravenous intramuscular Cholecystokinin chronic pancreatitis,intranasal, transdermal appetite, postoperativeintravenous paralytic ileus Gastrin Secretin a1 - Antitrypsin congenital a1 - parenteral antitrypsin deficiency Trypsin GI disorders debridement of wounds topical oedema and inflammation associated with infection or trauma oral Pepsin digestive aid in pepsinoral deficiency gastric hypochlorhydria dyspepsia GI disorders Neurotensin (NT) gastric juice secretionintravenous _7_ Calcitonin (CT) Paget's disease of intranasal, bone osteoporosis subcutaneous, hypercalcemia intramuscular gastric secretion intravenous intractable pain oral, parenteral, transdermal Human Chorionic cryptorchidism intravenous, parenteral Gonadotropin (hcG)induction of ovulation Relaxin facilitates birth scleroderma Therapeutic Cytokines Interferons IFNa-2a Kaposi's sarcoma parenteral IFNa-2b genital warts Kaposi's sarcoma parenteral AIDS-related hairy cell leukemia IFNa-n3 parenteral IFN(3-1b Multiple Sclerosis (relapsing, remitting type) IFN~y 1b chronic granulomatous parenteral disease Tumour Necrosis Factor TNFa, TNF~i Interleukins protection against the parenteral effects of radiation and chemotherapeutic agents renal cell carcinoma IL.-1, IL.-3, IL-4, IL-5, IL-6, IL-7, IL-8 Hemotopoietic Proteins Erythropoietin (EPO) dialysis anemia parenteral chemotherapy-induced anemia chemotherapy-induced neutropenia _8-Granuloycyte - Colony bone marrow transplant parenteral Stimulating Factor (G-CSF) Granulocyte bone marrow transplant parenteral Macrophage - Colony Stim. Fac. (GM-CSF) Macrophage-Colony Stimulating Factor (M-CSF) Growth Factors Epidermal Growth corneal and cateract topical Factor (EGF) surgeries Transforming Growth Factor a(TGFa) Platelet-derived growth diabetic and decubitus factor (PDGF) ulcers wound healing Transforming Growth Factor~3 (TGF(3) Fibroblast Growth neuropathic ulcers Factor - basic FGF pressure sores -acidic FGF
Insulin-like Growthnutritional Factor 1 (IGFl) support/metabolisn and type II diabetes osteoporosis Insulin-like Growth Factor2 (IGF2) Nerve Growth Factorperipheral neuropathies Skeletal Growth Factor Cardiovascular Therapeutic Proteins Proteins of the Blood Coagulation Pathway Factor VIII Hemophilia A parenteral Factor IX
Factor VII/VIIa Factor XII
Tissue Factor (Factor III) Protein C
Antithrombin III
Fibrinolytic Therapeutic Proteins Tissue Plasminogenacute myocardial parenteral Activator infarction Streptokinase and thromboembolism injection Anisoylated Streptokinase Fibrolase myocardial infarction Urokinase and Single-Chain Urokinase parenteral Kidney Plasminogen Activator Angiotensin-Converting Enzyme Inhibitors Captopril hypertension oral Enalapril heart failure Vaccines Active Immunization injection or oral Hepatitis B virus surface antigens Influenza virus surface antigens Plasmodium surface antigens Mycobacterium surface antigens Schistosoma surface antigens Herpes simplex virus surface antigens Trypanosoma surface antigens Streptococcus surface antigens Epstein - Barr virus surface antigens HTLV III virus surface antigens Therapeutic and Diagnostic Antibodies Murine Native &
Radioimmuno-conjugate Antibodies &
Fragments - Muromonab-CD3 heart, kidney & liver parenteral transplant rejection - Staumonab pendetide detection, staging, and follow-up of colorectal and ovarian cancers Murine Chemoimmuno-conjugate Antibodies Murine Immunotoxin Antibodies & Fragments Nonmurine Polyclonal Antibodies Human Antibodies &
Fragments Murine/Human Monoclonal Antibodies & Fragments Opioids (3-Endorphin cancer pain intravenous childbirth pain intrathecal narcotic abstinence Dermorphin syndrome Dynorphins Enkephalins Other Proteins Cylosporine immunosuppression intravenous in allogenic transplants Delta Sleep-inducingInsomnia intravenous peptide (DSIP) Bestatin cancer therapy oral Bacitracin bacterial infection topical Gramicidin bacterial infection topical Terprotide hypertension parenteral Serum Thymide Factorimmune deficiencies intravenous (FTS) Crude Thymosin autoimmune disordersparenteral collagen vascular disease intramuscular chemotherapy intravenous rheumatoid arthritisintravenous Angiogenin induces formation of blood vessels Albumin & Plasma blood volume Proteins replacement Atrial Natriuretic fluid and electrolyte Factor homoeostasis regulation of blood pressure Benin arterial pressure control Superoxide Dismutaseinflammation rheumatoid arthritis protection against radiation therapy Glucocerebrosidase Gaucher's Disease (Algucerase) rh DNase Cystic Fibrosis Aprotinin haemorrhage associatedintravenous with raised plasma concentrations of plasmin Protamine neutralize effect intravenous of heparin Asparaginase induction of remissionparenteral in acute lymphoblastic leukemia rhBMP-2 bone and cartilage repair Flab) fragment Digoxin overdose injection Other disease modifiers may also be used with this invention.
These may include:
Anti microbial Peptides Gramicidin and Related Peptides (eg. Gramicidin S, Tyrocidines, Gratisin) ~3-lactam and (3-lactam like Antibiotics Sulfazecin Type (eg. Sulfazecin, Isosulfazecin) Carbapenem Type Cephabacin Type (eg. Chitinovorin, Cephabacin) Norcardicin Type (eg. Formacidin) Lactivicin Type (eg. Lactivicin) Glycopeptide Antibiotics of the Vancomycin Group Vancomycin Type (eg. Vancomycin, Orienticin, Eremomycin) Actinoidin Type (eg. Actinoidin, Avoparcin, Chloropolysporin) Ristocetin Type (eg. Ristocetin, Actaplanin) Teicoplanin Type (eg. Teicoplanin, Ardacin, Kibdelin, Parvodicin) N-methylated Peptides and Peptolides Linear Peptides (eg. Stenothricin) Cyclic Peptides (eg. Ilamycins, cyclosporins) Diketopiperazines (eg. Gliotoxin) True Depsipeptides (eg. Enniatins, Beauvericin) Actinomycins Sideromycins (eg. Grisein, Albomycin, Desferrioxamine B) - use iron chelation therapy for removal of excessive iron resulting from genetic defects (primary hemochromatosis, anemias) or repeated blood transfusions Phleomycin - Like Antibiotics (eg. Bleomycin) - use - squamous cell carcinomas, Hodgkin's lymphomas, testis tumour Protease Inhibitors Inhibitors Against Endopeptidases Serine and Cysteine Proteinase Inhibitors Leupeptin - use - fertilization, inflammation, chemical carcinogenesis, burns, pancreatitis, muscular dystrophy, antoimmune diseases Antipain - use - fertilization, inflammation, chemical carcinogenesis, muscular dystrophy Chymostatin - use - fertilization, inflammation Elastatinal - use - inflammation Ac-Leu-Argal Lystatin Poststatin Aspartic Proteinase Inhibitors Pepstatin - use - inflammation, hypertension, ascites and pleural fluid Pepstanone Hydroxypepstatin Metal Proteinase Inhibitors Phosphoramidon - use - inflammation Steffimycins B & D
Inhibitors Against Exopeptidases Aminopeptidases Inhibitors Amastatin Actinonin - use - immunopotentiation, analgesia Arphamenines A & B - use - immunopotentiation, analgesia, autoimmune diseases Bestatin - use - immunopotentiation, analgesia, hypertension, malignant diseases, muscular dystrophy Ebelactones A & B - use - immunopotentiation Formestin Probestin Prostatin Leuhistin Dipeptidylamino Peptidase Inhibitors Ac-Leu-Argal Antipain Leupeptin Diprotins A & B - use - immunopotentiation Octastatins A & B
Carboxy Peptidase Inhibitors (S)-a-Benzylmalic acid - use - immunopotentiation Histargin - use - immunopotentiation, hypertension Dipeptidylcarboxy Peptidase Inhibitors EDDS
Foroxymithine - use - immunopotentiation, hypertension Histargin Inhibitors Against Plasma-Membrane-Located-Enzymes Forphenicine - use - immunopotentiation, muscular dystrophy, malignant diseases Forphenicinol - use - immunopotentiation, muscular dystrophy, malignant diseases Esterastin - use - immunopotentiation, inflammation, auto immune diseases Ebelactone A & B
Thus, I have provided a new composition comprising hyaluronan, hyaluronan binding motif (HBM), for example, found in a hyaluronan binding protein or receptor, and disease modifier (including a drug and/or therapeutic agent) which can be chemically linked or bound by the HBM
(eg. at the amino or carboxy terminus of the hyaluronan binding peptide) to the disease modifier. Thus, the hyaluronan indirectly binds through the hyaluronan binding motif (HBM) to the disease modifier such as a drug which is a protein such that the hyaluronan binding motif (HBM) is interposed between the hyaluronan and the disease modifier.
The new composition (or compound) may be used and administered in manners previously described for example, intravenously, interarterially, interperitoneally, intrapleurally, into the skin, applied topically onto the skin for penetration into the skin, to the oral mucosa, rectally or by direct injection into a tumour, abscess, or similar disease focus or put on a patch to be secured to the skin of the patient or administered via an enema.
Many forms of hyaluronan may be suitable although those preferred are those discussed hereinafter:
One form of hyaluronic acid and/or pharmaceutically acceptable salts thereof (for example sodium salt) suitable for use with my invention is an amount having the following specifications/characteristics:
TESTS SPECIFICATIONS RESULTS
pH 5.0 to 7.0 at 25 degrees C. 6.0 Specific Gravity 0.990 to 1.010 at 25 degrees C. 1.004 Intrinsic Viscosity 4.5 to 11.0 dL/g. 7.07 Molecular Weight 178,000 to 562,000 daltons (protein 319,378 daltons standard) Sodium Hyaluronate 9.0 to 11.0 mg/mL. Positive 9.9 mg/ML
Assay and Identification Positive Another such amount may comprise:
TESTS SPECIFICATIONS
1. Description White or cream odourless powder 2. Identification (IR Spectrum)Conforms to Ref. Std.
Spectrum 3. pH (1% solution) 5.0 to 7.0 4. Loss on Drying NMT 10%
5. Residue on Ignition 15.0% to 19.0%
6. Protein Content NMT 0.1%
Insulin-like Growthnutritional Factor 1 (IGFl) support/metabolisn and type II diabetes osteoporosis Insulin-like Growth Factor2 (IGF2) Nerve Growth Factorperipheral neuropathies Skeletal Growth Factor Cardiovascular Therapeutic Proteins Proteins of the Blood Coagulation Pathway Factor VIII Hemophilia A parenteral Factor IX
Factor VII/VIIa Factor XII
Tissue Factor (Factor III) Protein C
Antithrombin III
Fibrinolytic Therapeutic Proteins Tissue Plasminogenacute myocardial parenteral Activator infarction Streptokinase and thromboembolism injection Anisoylated Streptokinase Fibrolase myocardial infarction Urokinase and Single-Chain Urokinase parenteral Kidney Plasminogen Activator Angiotensin-Converting Enzyme Inhibitors Captopril hypertension oral Enalapril heart failure Vaccines Active Immunization injection or oral Hepatitis B virus surface antigens Influenza virus surface antigens Plasmodium surface antigens Mycobacterium surface antigens Schistosoma surface antigens Herpes simplex virus surface antigens Trypanosoma surface antigens Streptococcus surface antigens Epstein - Barr virus surface antigens HTLV III virus surface antigens Therapeutic and Diagnostic Antibodies Murine Native &
Radioimmuno-conjugate Antibodies &
Fragments - Muromonab-CD3 heart, kidney & liver parenteral transplant rejection - Staumonab pendetide detection, staging, and follow-up of colorectal and ovarian cancers Murine Chemoimmuno-conjugate Antibodies Murine Immunotoxin Antibodies & Fragments Nonmurine Polyclonal Antibodies Human Antibodies &
Fragments Murine/Human Monoclonal Antibodies & Fragments Opioids (3-Endorphin cancer pain intravenous childbirth pain intrathecal narcotic abstinence Dermorphin syndrome Dynorphins Enkephalins Other Proteins Cylosporine immunosuppression intravenous in allogenic transplants Delta Sleep-inducingInsomnia intravenous peptide (DSIP) Bestatin cancer therapy oral Bacitracin bacterial infection topical Gramicidin bacterial infection topical Terprotide hypertension parenteral Serum Thymide Factorimmune deficiencies intravenous (FTS) Crude Thymosin autoimmune disordersparenteral collagen vascular disease intramuscular chemotherapy intravenous rheumatoid arthritisintravenous Angiogenin induces formation of blood vessels Albumin & Plasma blood volume Proteins replacement Atrial Natriuretic fluid and electrolyte Factor homoeostasis regulation of blood pressure Benin arterial pressure control Superoxide Dismutaseinflammation rheumatoid arthritis protection against radiation therapy Glucocerebrosidase Gaucher's Disease (Algucerase) rh DNase Cystic Fibrosis Aprotinin haemorrhage associatedintravenous with raised plasma concentrations of plasmin Protamine neutralize effect intravenous of heparin Asparaginase induction of remissionparenteral in acute lymphoblastic leukemia rhBMP-2 bone and cartilage repair Flab) fragment Digoxin overdose injection Other disease modifiers may also be used with this invention.
These may include:
Anti microbial Peptides Gramicidin and Related Peptides (eg. Gramicidin S, Tyrocidines, Gratisin) ~3-lactam and (3-lactam like Antibiotics Sulfazecin Type (eg. Sulfazecin, Isosulfazecin) Carbapenem Type Cephabacin Type (eg. Chitinovorin, Cephabacin) Norcardicin Type (eg. Formacidin) Lactivicin Type (eg. Lactivicin) Glycopeptide Antibiotics of the Vancomycin Group Vancomycin Type (eg. Vancomycin, Orienticin, Eremomycin) Actinoidin Type (eg. Actinoidin, Avoparcin, Chloropolysporin) Ristocetin Type (eg. Ristocetin, Actaplanin) Teicoplanin Type (eg. Teicoplanin, Ardacin, Kibdelin, Parvodicin) N-methylated Peptides and Peptolides Linear Peptides (eg. Stenothricin) Cyclic Peptides (eg. Ilamycins, cyclosporins) Diketopiperazines (eg. Gliotoxin) True Depsipeptides (eg. Enniatins, Beauvericin) Actinomycins Sideromycins (eg. Grisein, Albomycin, Desferrioxamine B) - use iron chelation therapy for removal of excessive iron resulting from genetic defects (primary hemochromatosis, anemias) or repeated blood transfusions Phleomycin - Like Antibiotics (eg. Bleomycin) - use - squamous cell carcinomas, Hodgkin's lymphomas, testis tumour Protease Inhibitors Inhibitors Against Endopeptidases Serine and Cysteine Proteinase Inhibitors Leupeptin - use - fertilization, inflammation, chemical carcinogenesis, burns, pancreatitis, muscular dystrophy, antoimmune diseases Antipain - use - fertilization, inflammation, chemical carcinogenesis, muscular dystrophy Chymostatin - use - fertilization, inflammation Elastatinal - use - inflammation Ac-Leu-Argal Lystatin Poststatin Aspartic Proteinase Inhibitors Pepstatin - use - inflammation, hypertension, ascites and pleural fluid Pepstanone Hydroxypepstatin Metal Proteinase Inhibitors Phosphoramidon - use - inflammation Steffimycins B & D
Inhibitors Against Exopeptidases Aminopeptidases Inhibitors Amastatin Actinonin - use - immunopotentiation, analgesia Arphamenines A & B - use - immunopotentiation, analgesia, autoimmune diseases Bestatin - use - immunopotentiation, analgesia, hypertension, malignant diseases, muscular dystrophy Ebelactones A & B - use - immunopotentiation Formestin Probestin Prostatin Leuhistin Dipeptidylamino Peptidase Inhibitors Ac-Leu-Argal Antipain Leupeptin Diprotins A & B - use - immunopotentiation Octastatins A & B
Carboxy Peptidase Inhibitors (S)-a-Benzylmalic acid - use - immunopotentiation Histargin - use - immunopotentiation, hypertension Dipeptidylcarboxy Peptidase Inhibitors EDDS
Foroxymithine - use - immunopotentiation, hypertension Histargin Inhibitors Against Plasma-Membrane-Located-Enzymes Forphenicine - use - immunopotentiation, muscular dystrophy, malignant diseases Forphenicinol - use - immunopotentiation, muscular dystrophy, malignant diseases Esterastin - use - immunopotentiation, inflammation, auto immune diseases Ebelactone A & B
Thus, I have provided a new composition comprising hyaluronan, hyaluronan binding motif (HBM), for example, found in a hyaluronan binding protein or receptor, and disease modifier (including a drug and/or therapeutic agent) which can be chemically linked or bound by the HBM
(eg. at the amino or carboxy terminus of the hyaluronan binding peptide) to the disease modifier. Thus, the hyaluronan indirectly binds through the hyaluronan binding motif (HBM) to the disease modifier such as a drug which is a protein such that the hyaluronan binding motif (HBM) is interposed between the hyaluronan and the disease modifier.
The new composition (or compound) may be used and administered in manners previously described for example, intravenously, interarterially, interperitoneally, intrapleurally, into the skin, applied topically onto the skin for penetration into the skin, to the oral mucosa, rectally or by direct injection into a tumour, abscess, or similar disease focus or put on a patch to be secured to the skin of the patient or administered via an enema.
Many forms of hyaluronan may be suitable although those preferred are those discussed hereinafter:
One form of hyaluronic acid and/or pharmaceutically acceptable salts thereof (for example sodium salt) suitable for use with my invention is an amount having the following specifications/characteristics:
TESTS SPECIFICATIONS RESULTS
pH 5.0 to 7.0 at 25 degrees C. 6.0 Specific Gravity 0.990 to 1.010 at 25 degrees C. 1.004 Intrinsic Viscosity 4.5 to 11.0 dL/g. 7.07 Molecular Weight 178,000 to 562,000 daltons (protein 319,378 daltons standard) Sodium Hyaluronate 9.0 to 11.0 mg/mL. Positive 9.9 mg/ML
Assay and Identification Positive Another such amount may comprise:
TESTS SPECIFICATIONS
1. Description White or cream odourless powder 2. Identification (IR Spectrum)Conforms to Ref. Std.
Spectrum 3. pH (1% solution) 5.0 to 7.0 4. Loss on Drying NMT 10%
5. Residue on Ignition 15.0% to 19.0%
6. Protein Content NMT 0.1%
7. Heavy Metals NMT 20 ppm
8. Arsenic NMT 2 ppm
9. Residual Solvents a) Formaldehyde NMT 100 ppm b) Acetone NMT 0.1%
c) Ethanol NMT 2.0%
c) Ethanol NMT 2.0%
10. Sodium Hyaluronate Assay 97.0 to 102.0%
(dried basis)
(dried basis)
11. Intrinsic Viscosity 10.0 to 14.5 dL/g
12. Molecular Weight 500,000 to 800,000 daltons
13. Total Aerobic Microbial NMT 50 microorganisms/g Count (USP 23)
14. Escherichia coli (USP 23) Absent
15. Yeasts and Moulds (USP 23) NMT 50 microorganisms/g
16. Bacterial Endotoxins (LAL) NMT 0.07 EU/mg (USP 23) Another such amount is available from Hyal Pharmaceuticals Limited and comes in a 15 ml of Sodium hyaluronate 20mg/ml vial (300mg/vial - Lot 2F3). The sodium hyaluronate amount is a 2% solution with a mean average molecular weight of about 225,000. The amount also contains water q.s. which is triple distilled and sterile in accordance with the U.S.P. for injection formulations.The vials of hyaluronic acid and/or salts thereof may be carried in a Type 1 borosilicate glass vial closed by a butyl stopper which does not react with the contents of the vial.
The amount of hyaluronic acid and/or salts thereof (for example sodium salt) may also comprise the following characteristics:
a purified, substantially pyrogen-free amount of hyaluronic acid obtained from a natural source having at least one characteristic selected from the group (and preferably all characteristics) consisting of the following:
i) a molecular weight within the range of 150,000-225,000;
ii) less than about 1.25% sulphated mucopoly-saccharides on a total weight basis;
iii) less than about 0.6% protein on a total weight basis;
iv) less than about 150 ppm iron on a total weight basis;
v ) less than about 15 ppm lead on a total weight basis;
vi) less than 0.0025% glucosamine;
vii) less than 0.025% glucuronic acid;
viii) less than 0.025% N-acetylglucosamine;
ix) less than 0.0025% amino acids;
x) a UV extinction coefficient at 257 nm of less than about 0.275;
xi) a UV extinction coefficient at 280 nm of less than about 0.25; and xii) a pH within the range of 7.3-7.9. Preferably, the hyaluronic acid is mixed with sterile water and the amount of hyaluronic acid has a mean average molecular weight within the range of 150,000-225,000 daltons (protein standard). More preferably, the amount of hyaluronic acid comprises at least one characteristic selected from the
The amount of hyaluronic acid and/or salts thereof (for example sodium salt) may also comprise the following characteristics:
a purified, substantially pyrogen-free amount of hyaluronic acid obtained from a natural source having at least one characteristic selected from the group (and preferably all characteristics) consisting of the following:
i) a molecular weight within the range of 150,000-225,000;
ii) less than about 1.25% sulphated mucopoly-saccharides on a total weight basis;
iii) less than about 0.6% protein on a total weight basis;
iv) less than about 150 ppm iron on a total weight basis;
v ) less than about 15 ppm lead on a total weight basis;
vi) less than 0.0025% glucosamine;
vii) less than 0.025% glucuronic acid;
viii) less than 0.025% N-acetylglucosamine;
ix) less than 0.0025% amino acids;
x) a UV extinction coefficient at 257 nm of less than about 0.275;
xi) a UV extinction coefficient at 280 nm of less than about 0.25; and xii) a pH within the range of 7.3-7.9. Preferably, the hyaluronic acid is mixed with sterile water and the amount of hyaluronic acid has a mean average molecular weight within the range of 150,000-225,000 daltons (protein standard). More preferably, the amount of hyaluronic acid comprises at least one characteristic selected from the
-17-group (and preferably all characteristics) consisting of the following characteristics:
i) less than about 1% sulphated mucopolysaccharides on a total weight basis;
ii) less than about 0.4% protein on a total weight basis;
iii) less than about 100 ppm iron on a total weight basis;
iv) less than about 10 ppm lead on a total weight basis;
v ) less than 0.00166% glucosamine;
vi) less than 0.0166% glucuronic acid;
vii) less than 0.0166% N-acetylglucosamine;
viii) less than 0.00166% amino acids;
x) a UV extinction coefficient at 257 nm of less than about 0.23;
xi) a UV extinction coefficient at 280 nm of less than 0.19;
and xii) a pH within the range of 7.5-7.7 Applicants may also use sodium hyaluronate produced and supplied by LifeCoreTM Biomedical, Inc., having the following specifications:
Characteristics Specification Appearance White to cream colored particles Odor No perceptible odor Viscosity Average < 750,000 Daltons Molecular Weight UV/Vis Scan, 190-820nm Matches reference scan OD, 260nm < 0.25 OD units Hyaluronidase Sensitivity Positive response
i) less than about 1% sulphated mucopolysaccharides on a total weight basis;
ii) less than about 0.4% protein on a total weight basis;
iii) less than about 100 ppm iron on a total weight basis;
iv) less than about 10 ppm lead on a total weight basis;
v ) less than 0.00166% glucosamine;
vi) less than 0.0166% glucuronic acid;
vii) less than 0.0166% N-acetylglucosamine;
viii) less than 0.00166% amino acids;
x) a UV extinction coefficient at 257 nm of less than about 0.23;
xi) a UV extinction coefficient at 280 nm of less than 0.19;
and xii) a pH within the range of 7.5-7.7 Applicants may also use sodium hyaluronate produced and supplied by LifeCoreTM Biomedical, Inc., having the following specifications:
Characteristics Specification Appearance White to cream colored particles Odor No perceptible odor Viscosity Average < 750,000 Daltons Molecular Weight UV/Vis Scan, 190-820nm Matches reference scan OD, 260nm < 0.25 OD units Hyaluronidase Sensitivity Positive response
-18-IR Scan Matches reference pH, l0mg/g solution 6.2 - 7.8 Water 8% maximum Protein < 0.3 mcg/mg NaHy Acetate < 10.0 mcg/mg NaHy Heavy Metals, maximum ppm As Cd Cr Co Cu Fe Pb Hg N i 2.0 5.0 5.0 10.0 10.0 25.0 10.0 10.0 5.0 Microbial Bioburden None observed Endotoxin < 0.07EU/mg NaHy Biological Safety Testing Passes Rabbit Ocular Toxicity Test Another amount of sodium hyaluronate proposed to be used is sold under the name Hyaluronan HA-M5070 Skymart Enterprises, by Inc.
having the following specifications:
Specifications' Test Results Lot No. HG1004 pH 6.12 Condroitin Sulfate not detected Protein 0.05%
Heavy Metals Not more than 20 ppm Arsenic Not more than 2 ppm Loss on Drying 2.07%
Residue on Ignition 16.69%
Intrinsic Viscosity 12.75 dl/s (XW: 679,000) Nitrogen 3.14%
Assay 104.1 Microbiological Counts 80/g
having the following specifications:
Specifications' Test Results Lot No. HG1004 pH 6.12 Condroitin Sulfate not detected Protein 0.05%
Heavy Metals Not more than 20 ppm Arsenic Not more than 2 ppm Loss on Drying 2.07%
Residue on Ignition 16.69%
Intrinsic Viscosity 12.75 dl/s (XW: 679,000) Nitrogen 3.14%
Assay 104.1 Microbiological Counts 80/g
-19-E. coli Negative Mold and Yeast Not more than 50/g Other forms of hyaluronic acid and/or its salts may be chosen from other suppliers and those described in prior art documents provided they are suitable.
The following references teach hyaluronic acid, sources thereof, and processes for the manufacture and recovery thereof which may be suitable.
As there is no toxicity of the form of hyaluronic acide, the form of hyaluronic acid may be administered in doses in excess of l2mg/kg of body weight, for example, in excess of 1000mg/70kg person and even up to amounts of 3000mg/70kg person without adverse toxic effects. Lower amounts may include 10-50mg of hyaluronan. Exemplary amounts of Hyaluronan used may be 3-l0mg of hyaluronan (HA)/kg of body weight of the patient wherein the molecular weight (protein standard) is less than 750,000 daltons.
Many forms of hyaluronan may be suitable for use herein although those preferred are those discussed hereinafter. Particularly, molecular weights of forms of hyaluronan between about 150,000 daltons and about 750,000 daltons (protein standard) in sterile water prepared having a viscosity for intravenous administration are suitable.
One specific form of pharmaceutical grade is a 1% sterile sodium hyaluronate solution (50 ml vials) provided by Hyal Pharmaceutical Corporation which has the following characteristics:
Tests Svecifications 1. Container Description 150 mL Flint glass vial with a red or gray rubber stopper and an aluminum seal, 20 mm m size.
The following references teach hyaluronic acid, sources thereof, and processes for the manufacture and recovery thereof which may be suitable.
As there is no toxicity of the form of hyaluronic acide, the form of hyaluronic acid may be administered in doses in excess of l2mg/kg of body weight, for example, in excess of 1000mg/70kg person and even up to amounts of 3000mg/70kg person without adverse toxic effects. Lower amounts may include 10-50mg of hyaluronan. Exemplary amounts of Hyaluronan used may be 3-l0mg of hyaluronan (HA)/kg of body weight of the patient wherein the molecular weight (protein standard) is less than 750,000 daltons.
Many forms of hyaluronan may be suitable for use herein although those preferred are those discussed hereinafter. Particularly, molecular weights of forms of hyaluronan between about 150,000 daltons and about 750,000 daltons (protein standard) in sterile water prepared having a viscosity for intravenous administration are suitable.
One specific form of pharmaceutical grade is a 1% sterile sodium hyaluronate solution (50 ml vials) provided by Hyal Pharmaceutical Corporation which has the following characteristics:
Tests Svecifications 1. Container Description 150 mL Flint glass vial with a red or gray rubber stopper and an aluminum seal, 20 mm m size.
-20-2. Product Description A clear, colourless, odourless, transparent, slightly viscous liquid.
3. Fill Volume 50.0 to 52.0 mL
4. pH 5.0 to 7.0 at 25 degrees C.
5. Specific Gravity 0.990 to 1.010 at 25 degrees C.
6. Intrinsic Viscosity 4.5 to 11.0 dL/g 7. Molecular Weight 178,000 to 562,000 daltons 8. Sodium Hyaluronate Assay 9.0 to 11.0 mg/mL. Positive and Identification 9. Particulate Matter No visible Particulate Matter 10. Sterility Meets Requirements for Sterility, 11. Bacterial Endotoxins Meets Requirements for Bacterial (LAL) Endotoxins, USP 23.
This pharmaceutical grade % sterile solution of hyaluronan 1 may be made from granules/powder having the following characteristics:
Tests Specifications 1. Description White or cream-coloured granules or powder, odourless 2. Identification (IR Spectrum)Must conform with the Reference Standard Spectrum.
3. pH (1% Solution) Between 5.0 and 7.0 at 25 degrees C.
4. Loss on Drying NMT 10.0% at 102 degrees C.
for 6 hours.
5. Residue on Ignition Between 15.0 and 19.0%
6. Protein Content NMT 0.10%
3. Fill Volume 50.0 to 52.0 mL
4. pH 5.0 to 7.0 at 25 degrees C.
5. Specific Gravity 0.990 to 1.010 at 25 degrees C.
6. Intrinsic Viscosity 4.5 to 11.0 dL/g 7. Molecular Weight 178,000 to 562,000 daltons 8. Sodium Hyaluronate Assay 9.0 to 11.0 mg/mL. Positive and Identification 9. Particulate Matter No visible Particulate Matter 10. Sterility Meets Requirements for Sterility, 11. Bacterial Endotoxins Meets Requirements for Bacterial (LAL) Endotoxins, USP 23.
This pharmaceutical grade % sterile solution of hyaluronan 1 may be made from granules/powder having the following characteristics:
Tests Specifications 1. Description White or cream-coloured granules or powder, odourless 2. Identification (IR Spectrum)Must conform with the Reference Standard Spectrum.
3. pH (1% Solution) Between 5.0 and 7.0 at 25 degrees C.
4. Loss on Drying NMT 10.0% at 102 degrees C.
for 6 hours.
5. Residue on Ignition Between 15.0 and 19.0%
6. Protein Content NMT 0.10%
-21-7. Heavy Metals NMT 20 ppm (as per USP 23 p.
1727).
8. Arsenic NMT 2 ppm (as per USP 23, p.
1724).
9. Residual Solvents a) Acetone: NMT 0.1%
b) Ethanol: NMT 2.0%
c) Formaldehyde: NMT 100 ppm 10. Sodium Hyaluronate Assay 97.0 to 102.0% (dried basis) 11. Intrinsic Viscosity Between 10.0 to 14.5 deciliters per gram.
12. Molecular Weight Between 500,000 to 800,000 daltons (calculated using the Laurent Formula) (based Ori iritrlnciS vISCOSIty).
13. Total Aerobic Microbial Count NMT 50 microorganism/gram (as per USP 23, p. 1684).
14. Test for Escherichia coli Escherichia coli is absent (as per USP 23, p. 1685).
15. Yeasts & Molds NMT 50 microorganisms/gram (as per USP 23, p. 1686).
16. Endotoxins (LAL) NMT 0.07 EU/mg (as per USP
23, p. 1696).
A topical grade of hyaluronan may, in certain circumstances be suitable and may be made from the following granules/powder which have the following characteristics:
Tests Svecifications 1. Description White or cream-coloured granules or powder, odourless
1727).
8. Arsenic NMT 2 ppm (as per USP 23, p.
1724).
9. Residual Solvents a) Acetone: NMT 0.1%
b) Ethanol: NMT 2.0%
c) Formaldehyde: NMT 100 ppm 10. Sodium Hyaluronate Assay 97.0 to 102.0% (dried basis) 11. Intrinsic Viscosity Between 10.0 to 14.5 deciliters per gram.
12. Molecular Weight Between 500,000 to 800,000 daltons (calculated using the Laurent Formula) (based Ori iritrlnciS vISCOSIty).
13. Total Aerobic Microbial Count NMT 50 microorganism/gram (as per USP 23, p. 1684).
14. Test for Escherichia coli Escherichia coli is absent (as per USP 23, p. 1685).
15. Yeasts & Molds NMT 50 microorganisms/gram (as per USP 23, p. 1686).
16. Endotoxins (LAL) NMT 0.07 EU/mg (as per USP
23, p. 1696).
A topical grade of hyaluronan may, in certain circumstances be suitable and may be made from the following granules/powder which have the following characteristics:
Tests Svecifications 1. Description White or cream-coloured granules or powder, odourless
-22-2. Identification (IR Spectrum) Must conform to the Reference Standard Spectrum.
3. pH (1% Solution) Between 6.0 and 8.0 at degrees C.
4. Loss on Drying NMT 10.0% at 102 degrees C. for 6 hours.
5. Residue on Ignition Between 15.0 and 19.0%
6. Protein Content NMT 0.40%
7. Heavy Metals NMT 20 ppm (as per USP
3. pH (1% Solution) Between 6.0 and 8.0 at degrees C.
4. Loss on Drying NMT 10.0% at 102 degrees C. for 6 hours.
5. Residue on Ignition Between 15.0 and 19.0%
6. Protein Content NMT 0.40%
7. Heavy Metals NMT 20 ppm (as per USP
23 p.
1727).
8. Arsenic NMT 2 ppm (as per USP
23, p.
1724).
9. Residual Solvents a) Acetone: NMT 0.1%
b) Ethanol: NMT 2.0%
c) Formaldehyde: NMT 100 ppm 10. Sodium Hyaluronate Assay 97.0 to 102.0% (dried basis) 11. Intrinsic Viscosity Between 11.5 to 14.5 deciliters per gram.
12. Molecular Weight Between 600,000 to 800,000 daltons (calculated using the Laurent (based Ori lritririSiC
Formula) V1SCOSity).
13. Total Aerobic Microbial CountNMT 100 microorganism/gram (as per USP 23, p. 1684).
14. Test for Staphylococcus aureusStaphylococcus aureus is absent (as per USP 23, p. 1684).
15. Test for Pseudomonas aeruginosaPseudomonas aeruginosa is absent (as per USP 23, p. 1684).
16. Yeasts & Molds NMT 200 CFU/gram (as per USP 23, p. 1686).
This topical grade may then be sterilized.
Other forms of hyaluronic acid and/or its salts may be chosen from other suppliers, for example those described in prior art documents disclosing forms of hyaluronic acid having lower molecular weights between about 150,000 daltons and 750,000 daltons being prepared as for example, 1-2%
solutions in sterile water for intravenous administration.
The following references teach hyaluronic acid, sources thereof and processes of the manufacture and recovery thereof.
Canadian Letters Patent 1,205,031 (which refers to United States Patent 4,141,973 as prior art) refers to hyaluronic acid fractions having average molecular weights of from 50,000 to 100,000; 250,000 to 350,000; and 500,000 to 730,000 and discusses processes of their manufacture Where high molecular weight hyaluronic acid (or salts or other forms thereof) is used, it must, prior to use, be diluted to permit administration and ensure no intramuscular coagulation. Recently, it has been found that large molecular weight hyaluronic acid having a molecular weight exceeding about 1,000,000 daltons self-aggregates and thus, does not interact very well with HA
receptors. Thus, the larger molecular weight hyaluronic acid should be avoided.
Briefly, the methods for linking or combining the hyaluronan targeting sequence onto other proteins using the hyaluronan binding motif (HBM), may be the methods outlined as follows or any other suitable methods as would be understood by persons skilled in the art:
A first method is recombinant technology which involves linking the HBM sequence to a DNA sequence encoding a therapeutic protein.
The whole recombinant DNA is then translated by bacteria to make an
1727).
8. Arsenic NMT 2 ppm (as per USP
23, p.
1724).
9. Residual Solvents a) Acetone: NMT 0.1%
b) Ethanol: NMT 2.0%
c) Formaldehyde: NMT 100 ppm 10. Sodium Hyaluronate Assay 97.0 to 102.0% (dried basis) 11. Intrinsic Viscosity Between 11.5 to 14.5 deciliters per gram.
12. Molecular Weight Between 600,000 to 800,000 daltons (calculated using the Laurent (based Ori lritririSiC
Formula) V1SCOSity).
13. Total Aerobic Microbial CountNMT 100 microorganism/gram (as per USP 23, p. 1684).
14. Test for Staphylococcus aureusStaphylococcus aureus is absent (as per USP 23, p. 1684).
15. Test for Pseudomonas aeruginosaPseudomonas aeruginosa is absent (as per USP 23, p. 1684).
16. Yeasts & Molds NMT 200 CFU/gram (as per USP 23, p. 1686).
This topical grade may then be sterilized.
Other forms of hyaluronic acid and/or its salts may be chosen from other suppliers, for example those described in prior art documents disclosing forms of hyaluronic acid having lower molecular weights between about 150,000 daltons and 750,000 daltons being prepared as for example, 1-2%
solutions in sterile water for intravenous administration.
The following references teach hyaluronic acid, sources thereof and processes of the manufacture and recovery thereof.
Canadian Letters Patent 1,205,031 (which refers to United States Patent 4,141,973 as prior art) refers to hyaluronic acid fractions having average molecular weights of from 50,000 to 100,000; 250,000 to 350,000; and 500,000 to 730,000 and discusses processes of their manufacture Where high molecular weight hyaluronic acid (or salts or other forms thereof) is used, it must, prior to use, be diluted to permit administration and ensure no intramuscular coagulation. Recently, it has been found that large molecular weight hyaluronic acid having a molecular weight exceeding about 1,000,000 daltons self-aggregates and thus, does not interact very well with HA
receptors. Thus, the larger molecular weight hyaluronic acid should be avoided.
Briefly, the methods for linking or combining the hyaluronan targeting sequence onto other proteins using the hyaluronan binding motif (HBM), may be the methods outlined as follows or any other suitable methods as would be understood by persons skilled in the art:
A first method is recombinant technology which involves linking the HBM sequence to a DNA sequence encoding a therapeutic protein.
The whole recombinant DNA is then translated by bacteria to make an
-24- . , artificial protein that is used for therapy. In this regard, see "Identification of Two Hyaluronan-binding Domains in the Hyaluronan Receptor", RHAMM, The Journal of Biological Chemistry, April 25, 1993, Vol. 268, No.l2, pp. 8617 to 8623 previously discussed herein. In this report, the scientists (including me) have disclosed:
"In the course of preparing RHAMM cDNAs that were defective in binding HA to be used for genetic studies, we have identified the carboxyl terminus as the HA binding region of RHAMM.
We sought to identify the precise motifs that contained hyaluronan binding activity within this region.
In this report we demonstrate that the RHAMM cDNA fusion protein retains its ability to bind to HA in two types of binding assays including a new transblot assay using bio-tinylated HA
(20) and HA-Sepharose affinity chromatography. We have defined the HA-binding domains) on RHAMM as a 35-amino-acid region located near the carboxyl terminus of RHAMM. We show that two motifs within this region, containing 11 and 10 amino acids, respectively, represent the HA binding motifs of RHAMM. Neither of these motifs nor the entire 35-amino-acid region containing these motifs bears any amino acid sequence homology to other characterized hyaluronan-binding proteins."
In the Experimental Procedures, the scientists disclosed the following:
"Construction of Recombinant RHAMM-containing Oligonucleotides Encoding HA-Binding Peptides--PCR was used to incorporate the HA binding regions (peptideaa401-411 and peptideaa423-432 respectively)into a cDNA encoding the NH2 terminus of RHAMM that was prepared as a 0.71-kb fragment (aa
"In the course of preparing RHAMM cDNAs that were defective in binding HA to be used for genetic studies, we have identified the carboxyl terminus as the HA binding region of RHAMM.
We sought to identify the precise motifs that contained hyaluronan binding activity within this region.
In this report we demonstrate that the RHAMM cDNA fusion protein retains its ability to bind to HA in two types of binding assays including a new transblot assay using bio-tinylated HA
(20) and HA-Sepharose affinity chromatography. We have defined the HA-binding domains) on RHAMM as a 35-amino-acid region located near the carboxyl terminus of RHAMM. We show that two motifs within this region, containing 11 and 10 amino acids, respectively, represent the HA binding motifs of RHAMM. Neither of these motifs nor the entire 35-amino-acid region containing these motifs bears any amino acid sequence homology to other characterized hyaluronan-binding proteins."
In the Experimental Procedures, the scientists disclosed the following:
"Construction of Recombinant RHAMM-containing Oligonucleotides Encoding HA-Binding Peptides--PCR was used to incorporate the HA binding regions (peptideaa401-411 and peptideaa423-432 respectively)into a cDNA encoding the NH2 terminus of RHAMM that was prepared as a 0.71-kb fragment (aa
-25-1-238, see above and Fig. 2). The fusion protein product of this fragment did not have the ability to bind HA (Fig. 7). The procedure was carried out by making two PCR primers (5'TAG
AAT GAA TTC TTT CAA TTT CAC AAC ATG TTT GAT TTT
TTG TTT AAG ATC TTC TAT TTC and 5'TAG AAT GAA TTC
TTT CCT TTT AAC AAG CTG AGA TCG CAG TTT AAG ATC
TTC TAT TTC) which contained both a region mimicking the oligonucleotides encoding either peptideaa401-411 or peptideaa423-432 (creating an EcoRI site at the end of each primer) and a region mimicking 18 base pairs of the 3' end cDNA
of the 0.71-kb insert. Recombinant cDNA was obtained with a PCR reaction by using either of these two primers together with a primer that mimicked the 5' end of the RHAMM cDNA
(nucleotide 1-22) (creating a B a m HI site) with the same conditions described in the construction of RHAMM cDNA.
Both PCR products were digested with EcoRI and BamHI and purified in 1% agarose gel electrophoresis. Recombinant cDNAs were then inserted into pGEX-2T and transformed into HB101 as above. The correct insertion of the recombinant cDNAs was confirmed by restriction endonuclease digestion of the selected clones and by sizing of the insert with agarose gel electrophoresis.
The major findings of this paper are that a critical interaction of hyaluronan with the RHAMM receptor can be localized to a region of 35 amino acids (aa 400-434) near the carboxyl terminus of this protein. This region contains sequences that exhibit clusters of basic amino acids. Peptides mimicking these sequences contain HA binding activity, and
AAT GAA TTC TTT CAA TTT CAC AAC ATG TTT GAT TTT
TTG TTT AAG ATC TTC TAT TTC and 5'TAG AAT GAA TTC
TTT CCT TTT AAC AAG CTG AGA TCG CAG TTT AAG ATC
TTC TAT TTC) which contained both a region mimicking the oligonucleotides encoding either peptideaa401-411 or peptideaa423-432 (creating an EcoRI site at the end of each primer) and a region mimicking 18 base pairs of the 3' end cDNA
of the 0.71-kb insert. Recombinant cDNA was obtained with a PCR reaction by using either of these two primers together with a primer that mimicked the 5' end of the RHAMM cDNA
(nucleotide 1-22) (creating a B a m HI site) with the same conditions described in the construction of RHAMM cDNA.
Both PCR products were digested with EcoRI and BamHI and purified in 1% agarose gel electrophoresis. Recombinant cDNAs were then inserted into pGEX-2T and transformed into HB101 as above. The correct insertion of the recombinant cDNAs was confirmed by restriction endonuclease digestion of the selected clones and by sizing of the insert with agarose gel electrophoresis.
The major findings of this paper are that a critical interaction of hyaluronan with the RHAMM receptor can be localized to a region of 35 amino acids (aa 400-434) near the carboxyl terminus of this protein. This region contains sequences that exhibit clusters of basic amino acids. Peptides mimicking these sequences contain HA binding activity, and
-26-furthermore, these peptides confer HA binding activity to an NH2-terminal fragment of RHAMM that does not bind to HA.
Collectively, these results indicate that these sequences represent two critical HA binding motifs of RHAMM.
In the article Identification of a common Hyaluronan binding motif in the hyaluronan binding proteins RHAMM, CD44 and link protein , The EMBO Journal, Vol. 13, no. 2, PP. 286-296, 1994, the authors disclosed that:
We have previously identified two hyaluronan (HA) binding domains in the HA receptor, RHAMM, that occur near the carboxyl-terminus of this protein. We show here that these two HA binding domains are the only HA binding regions in RHAMM, and that they contribute approximately equally to the HA binding ability of this receptor. Mutation of domain II using recombinant polypeptides of RHAMM demonstrates that K423 and 8431, spaced seven amino acids apart, are critical for HA
binding activity. Domain I contains two sets of two basic amino acids, each spaced seven residues apart, and mutation of these basic amino acids reduced their binding to HA-Sepharose. These results predict that two basic amino acids flanking a seven amino acid stretch [hereafter called B(X~)B] are minimally required for HA binding activity. To assess whether this motif predicts HA
binding in the intact RHAMM protein, we mutated all basic amino acids in domains I and II that form part of these motifs using site-directed mutagenesis and prepared fusion protein from the mutated cDNA. The altered RHAMM protein did not bind HA, confirming that the basic amino acids and their spacing are critical for binding. A specific requirement for arginine or lysine residues was identified since mutation of K430, 8431 and
Collectively, these results indicate that these sequences represent two critical HA binding motifs of RHAMM.
In the article Identification of a common Hyaluronan binding motif in the hyaluronan binding proteins RHAMM, CD44 and link protein , The EMBO Journal, Vol. 13, no. 2, PP. 286-296, 1994, the authors disclosed that:
We have previously identified two hyaluronan (HA) binding domains in the HA receptor, RHAMM, that occur near the carboxyl-terminus of this protein. We show here that these two HA binding domains are the only HA binding regions in RHAMM, and that they contribute approximately equally to the HA binding ability of this receptor. Mutation of domain II using recombinant polypeptides of RHAMM demonstrates that K423 and 8431, spaced seven amino acids apart, are critical for HA
binding activity. Domain I contains two sets of two basic amino acids, each spaced seven residues apart, and mutation of these basic amino acids reduced their binding to HA-Sepharose. These results predict that two basic amino acids flanking a seven amino acid stretch [hereafter called B(X~)B] are minimally required for HA binding activity. To assess whether this motif predicts HA
binding in the intact RHAMM protein, we mutated all basic amino acids in domains I and II that form part of these motifs using site-directed mutagenesis and prepared fusion protein from the mutated cDNA. The altered RHAMM protein did not bind HA, confirming that the basic amino acids and their spacing are critical for binding. A specific requirement for arginine or lysine residues was identified since mutation of K430, 8431 and
-27-K432 to histidine residues abolished binding. Clustering of basic amino acids either within or at either end of the motif enhanced HA binding activity while the occurrence of acidic residues between the basic amino acids reduced binding. The B(X7) B
motif, in which B is either R or K and X7 contains no acidic residues and at least one basic amino acid, was found in all HA
binding proteins molecularly characterized to date.
Recombinant techniques were used to generate chimeric proteins containing either the B(X7)B motifs present in CD44 or link protein, with the amino-terminus of RHAMM (Amino acids 1-238) that does not bind HA. All chimeric proteins containing the motif bound HA in transblot analyses. Site-directed mutations of these motifs in CD44 sequences abolished HA binding. Collectively, these results predict that the motif of B(X7)B as a minimal binding requirement for HA in RHAMM, CD44 and link protein, and occurs in all HA binding proteins described to date.
The protein -HBM may then be combined with hyaluronan and will become bound thereto.
A second method is to prepare the HA targeting sequence by synthesis (a standard procedure as would be understood by persons skilled in the art) and then link to a protein via carbodiimide linkage. This is a chemical method for linking carboxyl and amino groups together. Such a procedure is described in Spontaneous Glycosylation of Glycosaminoglycan Substrates of Adherent Fibroblasts, Cell, May 1979, Vol.
17, 109-115 by E.A. Turley and S. Roth. At page 114, the following experimental procedure is found:
motif, in which B is either R or K and X7 contains no acidic residues and at least one basic amino acid, was found in all HA
binding proteins molecularly characterized to date.
Recombinant techniques were used to generate chimeric proteins containing either the B(X7)B motifs present in CD44 or link protein, with the amino-terminus of RHAMM (Amino acids 1-238) that does not bind HA. All chimeric proteins containing the motif bound HA in transblot analyses. Site-directed mutations of these motifs in CD44 sequences abolished HA binding. Collectively, these results predict that the motif of B(X7)B as a minimal binding requirement for HA in RHAMM, CD44 and link protein, and occurs in all HA binding proteins described to date.
The protein -HBM may then be combined with hyaluronan and will become bound thereto.
A second method is to prepare the HA targeting sequence by synthesis (a standard procedure as would be understood by persons skilled in the art) and then link to a protein via carbodiimide linkage. This is a chemical method for linking carboxyl and amino groups together. Such a procedure is described in Spontaneous Glycosylation of Glycosaminoglycan Substrates of Adherent Fibroblasts, Cell, May 1979, Vol.
17, 109-115 by E.A. Turley and S. Roth. At page 114, the following experimental procedure is found:
-28-"Derivatization and Characterization of Glycosaminoglycan Dishes"
The glycosaminoglycans, chondroitin-6-sulfate (Type C, Sigma;
molecular weight 50,000), hyaluronic acid (bovine vitreous humor, Sigma;
molecular weight 1,000,000) and polygalacturonic acid (Sigma; molecular weight 20,000-400,000), were covalently linked to 35 x lOmm polystyrene tissue culture dishes (Falcon Plastics) by a modification of the procedure of Keener, McDermott and Sheppard (1971). Each dish was treated for 1 hr with 1 ml of concentrated sulfuric acid at 37°C, washed extensively with water and then treated with 1 ml of aqueous ammonium hydroxide (30%, v/v) at room temperature for 24 hr. The resulting polysulfonamide dishes were incubated with 1 ml of an aqueous solution of 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (25 mg/ml) and either chondroitin sulfate (CS dishes), hyaluronic acid (HA dishes) or polygalacturonic acid (PGA dishes) all at 5 mg/ml for 48 hr in a humidified atmosphere a t 37°C. Dishes were then boiled in 8 M urea and 10% sodium dodecylsulfate (SDS-urea) for 20 min to remove noncovalently bound material. Dishes were pulverized, and hydrolysis of bound sugars was achieved with 1.5 ml of 90% formic acid (v/v) at 105°C for 6-12 hr. Hydrolysates were lyophilized and the residue was dissolved in 500 ~,1 water. This solution was assayed for uronic acid (Dische, 194' in the case of HA and PGA
dishes, or counted in a Searle~ Mark III liquid scintillation counter in the case of CS dishes derivatized with 3H-chondroitin sulfate (3nmole per plate; spec. act. 1.5 x 108 cpm/mg). Aliquots of all samples were analyzed by high voltage electro-phoresis (50 V/an for 45 min) on Whatman~
3MM paper impregnated with 1% borate (w/v). For standards, D-U-14C-glucuronic acid (8 nmole, spec. act. 76mCi/nmole; Amerisham-Searle), hyaluronic acid, chondroitin sulfate and polygalacturonic acid were a 11 hydrolyzed under identical conditions. The unlabeled hexosamine
The glycosaminoglycans, chondroitin-6-sulfate (Type C, Sigma;
molecular weight 50,000), hyaluronic acid (bovine vitreous humor, Sigma;
molecular weight 1,000,000) and polygalacturonic acid (Sigma; molecular weight 20,000-400,000), were covalently linked to 35 x lOmm polystyrene tissue culture dishes (Falcon Plastics) by a modification of the procedure of Keener, McDermott and Sheppard (1971). Each dish was treated for 1 hr with 1 ml of concentrated sulfuric acid at 37°C, washed extensively with water and then treated with 1 ml of aqueous ammonium hydroxide (30%, v/v) at room temperature for 24 hr. The resulting polysulfonamide dishes were incubated with 1 ml of an aqueous solution of 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (25 mg/ml) and either chondroitin sulfate (CS dishes), hyaluronic acid (HA dishes) or polygalacturonic acid (PGA dishes) all at 5 mg/ml for 48 hr in a humidified atmosphere a t 37°C. Dishes were then boiled in 8 M urea and 10% sodium dodecylsulfate (SDS-urea) for 20 min to remove noncovalently bound material. Dishes were pulverized, and hydrolysis of bound sugars was achieved with 1.5 ml of 90% formic acid (v/v) at 105°C for 6-12 hr. Hydrolysates were lyophilized and the residue was dissolved in 500 ~,1 water. This solution was assayed for uronic acid (Dische, 194' in the case of HA and PGA
dishes, or counted in a Searle~ Mark III liquid scintillation counter in the case of CS dishes derivatized with 3H-chondroitin sulfate (3nmole per plate; spec. act. 1.5 x 108 cpm/mg). Aliquots of all samples were analyzed by high voltage electro-phoresis (50 V/an for 45 min) on Whatman~
3MM paper impregnated with 1% borate (w/v). For standards, D-U-14C-glucuronic acid (8 nmole, spec. act. 76mCi/nmole; Amerisham-Searle), hyaluronic acid, chondroitin sulfate and polygalacturonic acid were a 11 hydrolyzed under identical conditions. The unlabeled hexosamine
-29-standards were visualized on paper using periodate-benzidine (Smith, 1969)."
Once again the protein-HBM is then combined with hyaluronan.
A third method is simply to synthesize the therapeutic peptide and HA targeting sequence (HBM) together by standard peptide synthesis known to persons skilled in the art. This could be done for example, if the disease modifier (therapeutic agent) were a peptide of for example, 10-20 amino acids. This procedure would make economic sense rather than using bacteria to make it as a small protein. The product is then combined with hyaluronan. The new compounds formed by interposing the hyaluronan binding motif (HBM) between the disease modifier (e.g. drug) and hyaluronan, may be administered in the usual manner as one administers the hyaluronan or disease modifier either together or individually.
Because toxicity may be associated with the disease modifier, toxicity will have to be considered in the amounts bound through the HBM to the hyaluronan. However because of the improvement in delivery by my invention, less of the disease modifier may be required (than would be used normally to treat the disease or condition) and therefore toxicity concerns are less. Further where dosages of the hyaluronan exceed 200 mg per person (for example a 70 kg person), side effects attributed to the drug modifier may be reduced such as gastrointestinal distress, neurological abnormalities, depression, etc.
The invention may thus be used to bind hyaluronan through HBM
to a protein or peptide. For example the protein tissue inhibitors of metalloproteinases (TIMPS) which break down collagen can be made recombinantly. HA binding motif (HBM) may be added to TIMPS in one of the many known manners and the product can be combined with
Once again the protein-HBM is then combined with hyaluronan.
A third method is simply to synthesize the therapeutic peptide and HA targeting sequence (HBM) together by standard peptide synthesis known to persons skilled in the art. This could be done for example, if the disease modifier (therapeutic agent) were a peptide of for example, 10-20 amino acids. This procedure would make economic sense rather than using bacteria to make it as a small protein. The product is then combined with hyaluronan. The new compounds formed by interposing the hyaluronan binding motif (HBM) between the disease modifier (e.g. drug) and hyaluronan, may be administered in the usual manner as one administers the hyaluronan or disease modifier either together or individually.
Because toxicity may be associated with the disease modifier, toxicity will have to be considered in the amounts bound through the HBM to the hyaluronan. However because of the improvement in delivery by my invention, less of the disease modifier may be required (than would be used normally to treat the disease or condition) and therefore toxicity concerns are less. Further where dosages of the hyaluronan exceed 200 mg per person (for example a 70 kg person), side effects attributed to the drug modifier may be reduced such as gastrointestinal distress, neurological abnormalities, depression, etc.
The invention may thus be used to bind hyaluronan through HBM
to a protein or peptide. For example the protein tissue inhibitors of metalloproteinases (TIMPS) which break down collagen can be made recombinantly. HA binding motif (HBM) may be added to TIMPS in one of the many known manners and the product can be combined with
-30-hyaluronan to form the dosage amount to be administered to a patient.
The amounts of TIMPS and HA are chosen in amounts suitable for use to treat a patient in need of treatment. When administered to the patients (for example by injection), the HA bound TIMPS goes to the site of injury (the pathological tissue site which expresses a surplus of HA receptors) for treatment of the injury. The invention can be used for administration of any protein disease modifier.
Thus the invention can be used to target disease modifiers which are proteins such as recombinant proteins or peptides such as TIMPS, enzymes, collagenese, cytokines, growth facts, therapeutic proteins (such as antibiotics which may be proteins).
Figures 1, 2, 3 and 4 are provided illustrating methods of binding proteins to hyaluronan binding motifs.
Figure 1 relates to the competition and direct binding assays of synthetic peptides corresponding to positive charge clusters in RHAMM protein. Panel A, transblotted RHAMM fusion protein was stained with a 1:3,000 dilution of biotin-labeled HA that had been preincubated for 1 h with 3 mg/ml bovine serum albumin (BSA) (lane 1), 3 mg/ml peptideaa401-411 (lane 2), or 3 mg/ml peptideaa423-432 (lane 3). Both peptides significantly reduced the binding of HA to RHAMM fusion protein. Panel B, HA-Sepharose affinity gel was prepared according to the manufacturer's instruction/ RHAMM peptides (peptideaa401-411 peptideaa423-432; randomized peptideaa401-411 (LKQKKVKKHIV); randomized peptideaa423-432 (QSKRLKKRVL);
peptideaa125-145 and peptideaa269-288 20) were applied to HA-Sepharose.
Unbound peptides were removed by washing the gel with PBS containing 0.15 M NaCI. The amounts of peptides applied and the unbound peptide removed from the gel were determined by measuring their OD value. The results
The amounts of TIMPS and HA are chosen in amounts suitable for use to treat a patient in need of treatment. When administered to the patients (for example by injection), the HA bound TIMPS goes to the site of injury (the pathological tissue site which expresses a surplus of HA receptors) for treatment of the injury. The invention can be used for administration of any protein disease modifier.
Thus the invention can be used to target disease modifiers which are proteins such as recombinant proteins or peptides such as TIMPS, enzymes, collagenese, cytokines, growth facts, therapeutic proteins (such as antibiotics which may be proteins).
Figures 1, 2, 3 and 4 are provided illustrating methods of binding proteins to hyaluronan binding motifs.
Figure 1 relates to the competition and direct binding assays of synthetic peptides corresponding to positive charge clusters in RHAMM protein. Panel A, transblotted RHAMM fusion protein was stained with a 1:3,000 dilution of biotin-labeled HA that had been preincubated for 1 h with 3 mg/ml bovine serum albumin (BSA) (lane 1), 3 mg/ml peptideaa401-411 (lane 2), or 3 mg/ml peptideaa423-432 (lane 3). Both peptides significantly reduced the binding of HA to RHAMM fusion protein. Panel B, HA-Sepharose affinity gel was prepared according to the manufacturer's instruction/ RHAMM peptides (peptideaa401-411 peptideaa423-432; randomized peptideaa401-411 (LKQKKVKKHIV); randomized peptideaa423-432 (QSKRLKKRVL);
peptideaa125-145 and peptideaa269-288 20) were applied to HA-Sepharose.
Unbound peptides were removed by washing the gel with PBS containing 0.15 M NaCI. The amounts of peptides applied and the unbound peptide removed from the gel were determined by measuring their OD value. The results
-31-indicated that peptideaa401-411 and peptideaa423-432 bound in highest amounts to HA-Sepharose gel.
Figure 2 relates to construction of recombinant RHAMM containing HA
binding domains. Panel A, cDNA encoding peptideaa401-411 (~) and peptideaa423-432 (.) were, respectively, aligned by PCR to a cDNA encoding RHAMM NH2-terminal polypeptideaal-238 that did not have the ability to bind HA. This was carried out as described under "Experimental Procedures."
Both PCR products were digested with EcoRI and BamHI and purified with agarose gel electrophoresis. The cDNAs were inserted into pGEX-2T opened with BamHI and EcoRI, which were cloning sites that were followed by stop codons, and transformed into HB101. The correct inserts were confirmed by restriction endonuclease digestion of the selected clones and were expressed as glutathione S-transferase-RHAMM fusion proteins. Panel B, bacterial cell lysates containing the glutathione S-transferase-RHAMM fusion proteins were fractionated on SDS-PAGE, transblotted onto nitrocellulose membrances, and visualized with either polyclonal antibody to peptideaa125-145 (lanes 1-3) or biotin-labeled HA (lanes 4-6). The glutathione S-transferase fusion non-recombinant polypeptideaal-238 was used as a control (lanes 1 and 4). The linkage of either peptideaa401-411 (lanes 2 and 5) or peptideaa423-432 (lanes 3 and 6) to the NH2-terminal RHAMM polypeptideaal-238 created HA-binding domains (lanes 5 and 6) although their antibody binding properties remained the same (lanes 2 and 3).
Figure 3 relates to the deletion and mutation of HA binding domains in RHAMM. (A) The HA binding domain II (aa423-432) was completely deleted and the HA binding domain I (aa 401-411) was partially deleted. The remaining domain (aa 401-411) was altered by mutating K405 and K409 to E.
(B) The PCR product (Figure 2A, lane 2) was ligated into the plasmid-containing fragment (5.3 kb in lane 3) and transformed into E.coli HB101. The
Figure 2 relates to construction of recombinant RHAMM containing HA
binding domains. Panel A, cDNA encoding peptideaa401-411 (~) and peptideaa423-432 (.) were, respectively, aligned by PCR to a cDNA encoding RHAMM NH2-terminal polypeptideaal-238 that did not have the ability to bind HA. This was carried out as described under "Experimental Procedures."
Both PCR products were digested with EcoRI and BamHI and purified with agarose gel electrophoresis. The cDNAs were inserted into pGEX-2T opened with BamHI and EcoRI, which were cloning sites that were followed by stop codons, and transformed into HB101. The correct inserts were confirmed by restriction endonuclease digestion of the selected clones and were expressed as glutathione S-transferase-RHAMM fusion proteins. Panel B, bacterial cell lysates containing the glutathione S-transferase-RHAMM fusion proteins were fractionated on SDS-PAGE, transblotted onto nitrocellulose membrances, and visualized with either polyclonal antibody to peptideaa125-145 (lanes 1-3) or biotin-labeled HA (lanes 4-6). The glutathione S-transferase fusion non-recombinant polypeptideaal-238 was used as a control (lanes 1 and 4). The linkage of either peptideaa401-411 (lanes 2 and 5) or peptideaa423-432 (lanes 3 and 6) to the NH2-terminal RHAMM polypeptideaal-238 created HA-binding domains (lanes 5 and 6) although their antibody binding properties remained the same (lanes 2 and 3).
Figure 3 relates to the deletion and mutation of HA binding domains in RHAMM. (A) The HA binding domain II (aa423-432) was completely deleted and the HA binding domain I (aa 401-411) was partially deleted. The remaining domain (aa 401-411) was altered by mutating K405 and K409 to E.
(B) The PCR product (Figure 2A, lane 2) was ligated into the plasmid-containing fragment (5.3 kb in lane 3) and transformed into E.coli HB101. The
-32-clone containing the correct insert (lane 4) was used to prepare RHAMM
fusion protein. (C) Cell lysates containing the complete fusion proteins (lanes 2 and 5), deleted fusion protein (lanes 1 and 4) and HB101 lysate (lanes 3 and 6) were prepared by sonication, then separated by SDS-PAGE and immunoblotted. RHAMM protein was visualized using anti-RHAMM
antibody (lanes 1-3) or biotin-labelled HA (lanes 4-6). The results show that after deletion of domains I and II, RHAMM lost its ability to bind to biotin-labelled HA (lane 4). The bacterial lysate contains an HA binding protein of <26 kDa that is not related to RHAMM (lanes 4-6).
Figure 4 relates to the strategy for defining the critical basic amino acids that determine the HA binding properties of domain II. To investigate the basic amino acids required in domain II for HA binding, six independent mutations were carried out and a RHAMM fusion peptide was genesuted recombining domain II with the amino-terminus (aa 1-238) of RHAMM using a recombinant technique. (A) The primers used to generate the altered cDNAs. (B) The resultant amino acid sequences. Highlighted amino acids indicate mutations. Six cDNAs, each containing site-directed mutations) were generated in the PCRs diagrammed in panels A and B, using RHAMM
cDNA1-~20 as the template DNA and containing the oligonucleotides encoding as 423-432 of RHAMM with different mutations. PCR products from the six primers containing the mutated nucleotides were doubly digested with BamHI+EcoRI, ligated into pGEX-2T and transformed into HB101. Selected clones were confirmed to contain correct inserts by double digestion with BamHI+EcoRI and electrophoresis on agarose gels. Fusion proteins were prepared from clones and analyzed in Western blots with either anti-RHAMM antibody to visualize RHAMM protein (C) or biotin-labelled HA (D) to assay HA binding activity. The results show that the HA binding ability of mutations I-VI (panel D) was reduced to 0, 67, 38, 21, 2 and 40%, of
fusion protein. (C) Cell lysates containing the complete fusion proteins (lanes 2 and 5), deleted fusion protein (lanes 1 and 4) and HB101 lysate (lanes 3 and 6) were prepared by sonication, then separated by SDS-PAGE and immunoblotted. RHAMM protein was visualized using anti-RHAMM
antibody (lanes 1-3) or biotin-labelled HA (lanes 4-6). The results show that after deletion of domains I and II, RHAMM lost its ability to bind to biotin-labelled HA (lane 4). The bacterial lysate contains an HA binding protein of <26 kDa that is not related to RHAMM (lanes 4-6).
Figure 4 relates to the strategy for defining the critical basic amino acids that determine the HA binding properties of domain II. To investigate the basic amino acids required in domain II for HA binding, six independent mutations were carried out and a RHAMM fusion peptide was genesuted recombining domain II with the amino-terminus (aa 1-238) of RHAMM using a recombinant technique. (A) The primers used to generate the altered cDNAs. (B) The resultant amino acid sequences. Highlighted amino acids indicate mutations. Six cDNAs, each containing site-directed mutations) were generated in the PCRs diagrammed in panels A and B, using RHAMM
cDNA1-~20 as the template DNA and containing the oligonucleotides encoding as 423-432 of RHAMM with different mutations. PCR products from the six primers containing the mutated nucleotides were doubly digested with BamHI+EcoRI, ligated into pGEX-2T and transformed into HB101. Selected clones were confirmed to contain correct inserts by double digestion with BamHI+EcoRI and electrophoresis on agarose gels. Fusion proteins were prepared from clones and analyzed in Western blots with either anti-RHAMM antibody to visualize RHAMM protein (C) or biotin-labelled HA (D) to assay HA binding activity. The results show that the HA binding ability of mutations I-VI (panel D) was reduced to 0, 67, 38, 21, 2 and 40%, of
-33-ability of mutations I-VI (panel D) was reduced to 0, 67, 38, 21, 2 and 40%, of the control (lane 1), respectively. Lane 1, control; lane 2, mutation I; lane 3, mutation II; lane 4, mutation III; lane 5, mutation IV; lane 6, mutation V;
lane 7, mutation VI.
The amounts of disease modifiers and form of hyaluronan may be those previously used even band together through HBM. Because of the beneficial effects of the form of hyaluronan taking the disease modifier to the pathological tissue (having excess HA receptors) in need of treatment, less of the disease modifier than would normally be expected to be used may be useful to treat and resolve the condition/disease affecting the pathological tissue. The amounts of the forms of hyaluronan may be those amounts specified in W091 /04058 -- at least about l0mg of the form of hyaluronan i n each dosage amount to in excess of 1000-1500mg of the form of hyaluronan i n each dosage amount administered to a patient.
As many changes could be made to the examples without departing from the scope of the invention, it is intended that all material herein be interpreted as illustrative of the invention and not in a limiting sense.
lane 7, mutation VI.
The amounts of disease modifiers and form of hyaluronan may be those previously used even band together through HBM. Because of the beneficial effects of the form of hyaluronan taking the disease modifier to the pathological tissue (having excess HA receptors) in need of treatment, less of the disease modifier than would normally be expected to be used may be useful to treat and resolve the condition/disease affecting the pathological tissue. The amounts of the forms of hyaluronan may be those amounts specified in W091 /04058 -- at least about l0mg of the form of hyaluronan i n each dosage amount to in excess of 1000-1500mg of the form of hyaluronan i n each dosage amount administered to a patient.
As many changes could be made to the examples without departing from the scope of the invention, it is intended that all material herein be interpreted as illustrative of the invention and not in a limiting sense.
Claims (34)
PROPERTY OR PRIVILEGE IS BEING CLAIMED ARE AS FOLLOWS:
1. A pharmaceutical composition component comprising a hyaluronan binding motif (HBM) interposed between a form of hyaluronan and a disease modifier wherein the form of hyaluronan has a molecular weight less than 750,000 daltons (protein standard) and is selected from the group consisting of hyaluronan, pharmaceutically acceptable salts thereof and combinations thereof and wherein the disease modifier comprises a peptide or protein.
2. The pharmaceutical composition component of claim 1 wherein flanking basic amino acids are added to the HBM to enhance the affinity of the form of hyaluronan for the HBM.
3. The pharmaceutical composition component of claim 1 wherein several internal basic amino acids are added at position 4,5 to the HBM to enhance the affinity of the form of hyaluronan for the HBM.
4. The pharmaceutical composition component of claim 2 wherein the flanking basic amino acids are hydrophobic.
5. The pharmaceutical composition component of claim 3 wherein the internal basic amino acids are hydrophobic.
6. The pharmaceutical composition component of claim 1, 2, 3, 4 or 5 wherein the disease modifier is selected from the group consisting of a cytokine, a peptide mimicking a cytokine, and a protein mimicking a cytokine.
7. The pharmaceutical composition component of claim 1, 2, 3, 4, 5 or 6 wherein the form of hyaluronan is sodium hyaluronate.
8. A pharmaceutical composition comprising an effective amount of the pharmaceutical composition component of claim 1, 2, 3, 4, 5, 6 or 7 together with suitable pharmaceutical excipients.
9. The pharmaceutical composition of claim 8 wherein the amount of the form of hyaluronan exceeds 10mg.
10. The pharmaceutical composition of claim 9 wherein the amount of the form of hyaluronan is less than 3000mg.
11. A use of (i) a hyaluronan binding motif (HBM), (ii) a form of hyaluronic acid having a molecular weight less than 750,000 daltons (protein standard) and selected from the group consisting of hyaluronan, pharmaceutically acceptable salts thereof, and combinations thereof, and (iii) a disease modifier comprising a peptide or protein;
in the manufacture of a pharmaceutical composition component for treating a disease and/or condition of a patient wherein the HBM is interposed between the form of hyaluronan and the disease modifier.
in the manufacture of a pharmaceutical composition component for treating a disease and/or condition of a patient wherein the HBM is interposed between the form of hyaluronan and the disease modifier.
12. The use of claim 11 wherein flanking basic amino acids are added to the HBM to enhance the affinity of the form of hyaluronan for the HBM.
13. The use of claim 11 wherein several internal basic amino acids are added at position 4,5 to the HBM to enhance the affinity of the form of hyaluronan for the HBM.
14. The use of claim 12 wherein the flanking basic amino acids are hydrophobic.
15. The use of claim 13 wherein the internal basic amino acids are hydrophobic.
16. The use of claim 11, 12, 13, 14 or 15 wherein the disease modifier is selected from the group consisting of a cytokine, a peptide mimicking a cytokine, and a protein mimicking a cytokine.
17. The use of claim 11, 12, 13, 14, 15 or 16 wherein the form of hyaluronan is sodium hyaluronate.
18. A use of (i) a hyaluronan binding motif (HBM), (ii) a form of hyaluronic acid having a molecular weight less than 750,000 daltons (protein standard) and selected from the group consisting of hyaluronan, pharmaceutically acceptable salts thereof, and combinations thereof, and (iii) a disease modifier comprising a peptide or protein;
in the manufacture of a pharmaceutical composition component for protecting a disease modifier from attack from proteases wherein an effective amount of the disease modifier is bound through the HBM to the form of hyaluronan which is in an effective amount to protect the disease modifier from attack from proteases.
in the manufacture of a pharmaceutical composition component for protecting a disease modifier from attack from proteases wherein an effective amount of the disease modifier is bound through the HBM to the form of hyaluronan which is in an effective amount to protect the disease modifier from attack from proteases.
19. The use of claim 18 wherein flanking basic amino acids are added to the HBM to enhance the affinity of the form of hyaluronan for the HBM.
20. The use of claim 18 wherein several internal basic amino acids are added at position 4,5 to the HBM to enhance the affinity of the form of hyaluronan for the HBM.
21. The use of claim 19 wherein the flanking basic amino acids are hydrophobic.
22. The use of claim 20 wherein the internal basic amino acids are hydrophobic.
23. The use of claim 18, 19, 20, 21 or 22 wherein the disease modifier is selected from the group consisting of a cytokine, a peptide mimicking a cytokine, and a protein mimicking a cytokine.
24. The use of claim 18, 19, 20, 21, 22 or 23 wherein the form of hyaluronan is sodium hyaluronate.
25. A use of (i) a hyaluronan binding motif (HBM), (ii) a form of hyaluronic acid having a molecular weight less than 750,000 daltons (protein standard) and selected from the group consisting of hyaluronan, pharmaceutically acceptable salts thereof, and combinations thereof, and (iii) a disease modifier comprising a peptide or protein;
in the manufacture of a pharmaceutical composition component for protecting a disease modifier from recognition and attack from the patient's immune system wherein an effective amount of the disease modifier is bound through the HBM to the form of hyaluronan which is in an effective amount to protect the disease modifier from attack from proteases.
in the manufacture of a pharmaceutical composition component for protecting a disease modifier from recognition and attack from the patient's immune system wherein an effective amount of the disease modifier is bound through the HBM to the form of hyaluronan which is in an effective amount to protect the disease modifier from attack from proteases.
26. The use of claim 25 wherein flanking basic amino acids are added to the HBM to enhance the affinity of the form of hyaluronan for the HBM.
27. The use of claim 25 wherein several internal basic amino acids are added at position 4,5 to the HBM to enhance the affinity of the form of hyaluronan for the HBM.
28. The use of claim 26 wherein the flanking basic amino acids are hydrophobic.
29. The use of claim 27 wherein the internal basic amino acids are hydrophobic.
30. The use of claim 25, 26, 27, 28 or 29 wherein the disease modifier is selected from the group consisting of a cytokine, a peptide mimicking a cytokine, and a protein mimicking a cytokine.
31. The use of claim 25, 26, 27, 28, 29 or 30 wherein the form of hyaluronan is sodium hyaluronate.
32. A use of an effective amount of the pharmaceutical composition component of claim 1, 2, 3, 4, 5, 6 or 7 together with suitable pharmaceutical excipients in the manufacture of a pharmaceutical composition for treating a disease and/or condition of a patient.
33. The use of claim 32 wherein the amount of the form of hyaluronan exceeds 10 mg.
34. The use of claim 33 wherein the amount of the form of hyaluronan exceeds 3,000 mg.
Priority Applications (6)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA002205771A CA2205771C (en) | 1997-05-22 | 1997-05-22 | Improved delivery of disease modifiers |
| JP54972998A JP2001526666A (en) | 1997-05-22 | 1998-05-11 | Improved release of disease modulators |
| PCT/CA1998/000448 WO1998052590A2 (en) | 1997-05-22 | 1998-05-11 | Improved delivery of disease modifiers |
| NZ501270A NZ501270A (en) | 1997-05-22 | 1998-05-11 | Pharmaceutical composition comprising hyaluronan (Mr less than 750,00) and a disease modifier (cytokine, a peptide mimicking a cytokine or a protein mimicking a cytokine) to enhance delivery of disease modifiers |
| AU73286/98A AU740631B2 (en) | 1997-05-22 | 1998-05-11 | Improved delivery of disease modifiers |
| EP98920426A EP0998297A2 (en) | 1997-05-22 | 1998-05-11 | Improved delivery of disease modifiers |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA002205771A CA2205771C (en) | 1997-05-22 | 1997-05-22 | Improved delivery of disease modifiers |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2205771A1 CA2205771A1 (en) | 1998-11-22 |
| CA2205771C true CA2205771C (en) | 2002-05-14 |
Family
ID=4160694
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002205771A Expired - Fee Related CA2205771C (en) | 1997-05-22 | 1997-05-22 | Improved delivery of disease modifiers |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP0998297A2 (en) |
| JP (1) | JP2001526666A (en) |
| AU (1) | AU740631B2 (en) |
| CA (1) | CA2205771C (en) |
| NZ (1) | NZ501270A (en) |
| WO (1) | WO1998052590A2 (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6875753B1 (en) | 1996-03-14 | 2005-04-05 | The Governors Of The University Of Alberta | Methods for cell mobilization using in vivo treatment with hyaluronan (HA) |
| US6960651B2 (en) | 1999-06-29 | 2005-11-01 | Millennium Pharmaceuticals, Inc. | TANGO 332 polypeptides |
| US20040037834A1 (en) * | 2000-04-20 | 2004-02-26 | Woloski B. Michael R. | Rhamm peptide conjugates |
| AU2004242091C1 (en) * | 2003-05-07 | 2009-12-24 | La Jolla Institute For Molecular Medicine | Administration of hyaluronic acid to enhance the function of transplanted stem cells |
| DE102011122236A1 (en) * | 2011-12-09 | 2013-06-13 | Maria Clementine Martin Klosterfrau Vertriebsgesellschaft Mbh | Composition useful e.g. in topical treatment of inflammatory diseases of skin and/or mucous membrane, comprises fusafungine and its derivate, and acidic glycosaminoglycan or its derivatives |
| FR3002452B1 (en) * | 2013-02-28 | 2016-02-12 | Dermaconcept Jmc | TOPIC ANTIMICROBIAL DERMATOLOGICAL COMPOSITION |
| WO2017053807A2 (en) | 2015-09-23 | 2017-03-30 | Genentech, Inc. | Optimized variants of anti-vegf antibodies |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA1340994C (en) * | 1989-09-21 | 2000-05-16 | Rudolf Edgar Dr. Falk | Treatment of conditions and disease |
| GB9207949D0 (en) * | 1992-04-09 | 1992-05-27 | Univ Manitoba | Molecular cloning of a novel hyaluronan receptor that mediates tumor cell motility |
| EP0778776A1 (en) * | 1994-08-30 | 1997-06-18 | Hyal Pharmaceutical Corporation | Hyaluronic acid and derivatives for modulation of cellular activity |
-
1997
- 1997-05-22 CA CA002205771A patent/CA2205771C/en not_active Expired - Fee Related
-
1998
- 1998-05-11 JP JP54972998A patent/JP2001526666A/en active Pending
- 1998-05-11 AU AU73286/98A patent/AU740631B2/en not_active Ceased
- 1998-05-11 WO PCT/CA1998/000448 patent/WO1998052590A2/en not_active Application Discontinuation
- 1998-05-11 EP EP98920426A patent/EP0998297A2/en not_active Withdrawn
- 1998-05-11 NZ NZ501270A patent/NZ501270A/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| WO1998052590A3 (en) | 1999-04-22 |
| AU7328698A (en) | 1998-12-11 |
| CA2205771A1 (en) | 1998-11-22 |
| WO1998052590A2 (en) | 1998-11-26 |
| AU740631B2 (en) | 2001-11-08 |
| JP2001526666A (en) | 2001-12-18 |
| NZ501270A (en) | 2001-08-31 |
| EP0998297A2 (en) | 2000-05-10 |
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