WO2012040295A2 - Therapeutic applications of smad7 - Google Patents
Therapeutic applications of smad7 Download PDFInfo
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- WO2012040295A2 WO2012040295A2 PCT/US2011/052499 US2011052499W WO2012040295A2 WO 2012040295 A2 WO2012040295 A2 WO 2012040295A2 US 2011052499 W US2011052499 W US 2011052499W WO 2012040295 A2 WO2012040295 A2 WO 2012040295A2
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- 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
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
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- A61K38/00—Medicinal preparations containing peptides
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- 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
- A61K38/162—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from virus
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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
- A61K38/18—Growth factors; Growth regulators
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- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/06—Antipsoriatics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
- C07K14/4703—Inhibitors; Suppressors
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/09—Fusion polypeptide containing a localisation/targetting motif containing a nuclear localisation signal
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16033—Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory
Definitions
- the present invention relates general to the fields of oncology and cancer therapies, inflammatory diseases and chronic wound healing. More specifically, the invention relates to methods and compositions for the treatment of inflammatory diseases, chronic wound healing/ulceration, and side effects resulting from chemotherapy and radiation therapy, as well as overexposure to radiation in military/industrial/safety/rescue personnel.
- side effects can include oral mucositis, gut mucositis and bone marrow failure.
- Smad7 mothers against decapentaplegic-7) protein compositions to prevent or treat the indications described above is provided.
- Inflammation is a protective attempt by the organism to remove the injurious stimuli and to initiate the healing process. Without inflammation, wounds and infections would never heal. Similarly, progressive destruction of the tissue would compromise the survival of the organism.
- chronic inflammation can also lead to a host of diseases, such as hay fever, atherosclerosis, rheumatoid arthritis, psoriasis and even cancer (e.g., gall bladder carcinoma), and acute inflammation may cause injury through overresponse to an acute stimulus. It is for that reason that inflammation is normally closely regulated by the body.
- inflammation can be classified as either acute or chronic.
- Acute inflammation is the initial response of the body to harmful stimuli and is achieved by the increased movement of plasma and leukocytes (especially granulocytes) from the blood into the injured tissues.
- a cascade of biochemical events propagates and matures the inflammatory response, involving the local vascular system, the immune system, and various cells within the injured tissue.
- Prolonged inflammation known as chronic inflammation, leads to a progressive shift in the type of cells present at the site of inflammation and is characterized by simultaneous destruction and healing of the tissue from the inflammatory process.
- Acute inflammation begins within seconds to minutes following the injury of tissues.
- the damage may be purely physical, or it may involve the activation of an immune response.
- Three main processes occur: (a) increased blood flow due to dilation of blood vessels (arterioles) supplying the region; (b) increased permeability of the capillaries, allowing fluid and blood proteins to move into the interstitial spaces; and (c) migration of neutrophils (and perhaps a few macrophages) out of the venules and into interstitial spaces
- One particular type of inflammatory injury is that stemming from the use of therapeutic agents, many of which are intended to produce inflammation in a diseased tissue or organ, but which unfortunately are rarely able to specifically target those regions. For example, over 80% of oral cancer patients are treated with radiation therapy and at least 75% of these individuals will develop oral mucositis. Oral mucositis also occurs in patients undergoing a hematopoietic stem cell transplant and in other cancer patients requiring radiotherapy and/or chemotherapy, and is often the most severe complication of radiotherapy. Severe oral mucositis is extremely painful and impairs oral intake. Subjects suffering from oral mucositis often require long-term pain medications to alleviate the symptoms of this condition.
- a method for treating an inflammatory condition and/or tissue damage conditions in a subject comprising providing to the subject a therapeutically effective amount of a Smad7 (mothers against decapentaplegic homolog 7).
- the Smad7 may be provided as a protein, an expression vector encoding a Smad7 protein, such as a viral or non-viral vector.
- the composition may comprise a Smad7 protein fused to a protein transduction domain (PTD), and said Smad7 protein fusion may further comprises a regulatory domain that prevents Smad7 function in the absence of an activator, and the method further comprises provision of said activator.
- the Smad7 may be provided in a formulation on a patch, in a gelatinous composition, in a microsphere, in a microbead or combination thereof.
- the patch may comprise a biodegradable patch, such as an alginate polymer.
- the Smad7 may be provided local to an affected region or systemically.
- the method may further comprise administering a second mucositis therapy to said subject, such as viscous 2% lidocaine, baking soda solution, saline solution, BAX solution (lidocaine, diphenhyramine, sorbitol and Mylanta), beta carotene, tocopherol, laser irradiation, silver- nitrate, misoprostol, leucovorin, systemic keratinocyte growth factor, pentoxifylline, allopurinol, systemic sucralfate, chlorhexidine gluconate or cryotherapy.
- the composition may comprise a detectable marker.
- the inflammatory condition is selected from mucositis, psoriasis, an autoimmune disease, chronic wound, trauma, chemotherapy, radiotherapy or cytokine therapy.
- the method may further comprise administering said composition to said subject a second time.
- a method for treating or preventing oral mucositis in a subject undergoing radiation therapy and/or chemotherapy comprising providing to the subject a therapeutically effective amount of a Smad7 (mothers against decapentaplegic homolog 7).
- the Smad7 may be provided as a protein, an expression vector encoding a Smad7 protein, such as a viral or non-viral vector.
- the composition may comprise a Smad7 protein fused to a protein transduction domain (PTD), and said Smad7 protein fusion may further comprises a regulatory domain that prevents Smad7 function in the absence of an activator, and the method further comprises provision of said activator.
- PTD protein transduction domain
- the Smad7 may be provided in a formulation on a patch, in a gelatinous composition, in a microsphere, in a microbead or combination thereof.
- the patch may comprise a biodegradable patch, such as an alginate polymer.
- the Smad7 may be provided local to an affected region or systemically.
- the method may further comprise administering a second mucositis therapy to said subject, such as viscous 2% lidocaine, baking soda solution, saline solution, BAX solution (lidocaine, diphenhyramine, sorbitol and Mylanta), beta carotene, tocopherol, laser irradiation, silver- nitrate, misoprostol, leucovorin, systemic keratinocyte growth factor, pentoxifylline, allopurinol, systemic sucralfate, chlorhexidine gluconate or cryotherapy.
- a second mucositis therapy such as viscous 2% lidocaine, baking soda solution, saline solution, BAX solution (lidocaine, diphenhyramine, sorbitol and Mylanta), beta carotene, tocopherol, laser irradiation, silver- nitrate, misoprostol, leucovorin, systemic keratinocyte
- the subject may comprises a subject having a transplant, a subject having cancer or a subject having a condition requiring radiation therapy, such where the subject has cancer and the cancer is selected from the group consisting of oral cancer, colon cancer, breast cancer, head and neck cancer, pancreatic cancer and other cancers treated with upper body radiation or repeated cycles of chemotherapy.
- the subject may have undergone upper-body radiation.
- the method may further comprise administering said composition to said subject a second time.
- kits for treating oral mucositis in a subject comprising (a) a Smad7 agent composition; and (b) a delivery system.
- the agent may comprises Smad7 protein, optionally fused to a protein transduction domain (PTD), and/or optionally fused to a regulatory domain that prevents Smad7 function in the absence of an activator, and said kit further comprises said activator.
- the agent may be a Smad7 expression vector, such as a viral or a non-viral expression vector.
- the delivery system may comprise a gel, salve or patch delivery system.
- the composition may be lyophilized.
- the kit may further comprise a pharmaceutically acceptable buffer, solvent or diluent.
- compositions and methods herein concern, induction of Smad7 (mothers against decapentaplegic homolog 7) genes or protein levels or introduction of a Smad7 composition to a subject to treat a subject having a condition disclosed herein.
- Other embodiments concern over-expression of Smad7 (mothers against decapentaplegic homolog 7) genes and/or induction of Smad7 protein levels in a subject in need of such a treatment.
- Some embodiments concern using compositions of recombinant forms of Smad7 for expression of Smad7 in a subject.
- Other embodiments herein concern treating or preventing oral mucositis in a subject having undergone or that will undergo radiation therapy or chemotherapy.
- these subjects are undergoing treatment for cancer including, but not limited to, oral cancer, head and neck cancer, and other cancers treated with repeated cycles of chemotherapy including, but not limited to, colon cancer, breast cancer, pancreatic cancer, and other cancers.
- FIG. 1 Resistance to radiation-induced oral mucositis in Smad7 transgenic mice. Dotted lines in H&E highlight the epithelial-stromal boundary. Smad7 mice had an intact oral epithelial layer while wild-type (wt) mice had oral ulcers. pH2AX, a marker for DNA damage (green), was obvious in wt epithelium (K14, red) of oral mucositis, but was significantly reduced in Smad7 transgenic lesion.
- CD45 stained for total leukocytes (brown), showed severe inflammation in wild-type lesion but a significant reduction in Smad7 transgenic lesion. CD45 immunostaining has non-specific staining in the stratum corneum. For immunostaining, wild-type epithelial images were taken adjacent to the ulcer and Smad7 images were taken from an area with the most leukocytes close to the basement membrane.
- FIG. 2 Smad7 transgene expression reversed psoriasis of K5.TGFpi wt mice.
- Upper panel gross appearance of a 3 month-old K5.TGFpi wt /K5.Smad7 mouse (smooth skin) and its KS.TGFpiTM 1 littermate (inflamed skin, eyes and ears).
- Lower panel while KS.TGFpi ⁇ skin exhibited epidermal hyperplasia and skin inflammation, K5.Smad7 skin and K5.Smad7/TGFpi wt skin are similar to normal wild-type (WT) skin.
- FIG. 3 Tat-Smad7 protein transduction to keratinocytes. Left and middle panels: Tat-Smad7 staining (green) in transduced cells, counterstained by a keratin K14 antibody (red). Right: pSmad2 (green) stains vehicle-treated (control) keratinocytes but not in Tat-Smad7 transduced cells. Tat-Smad7 staining using a V5 antibody showed nuclear and/or cytoplasmic staining of transduced cells but not in control cells.
- FIG. 4 Local Tat-Smad7 protein delivery to oral mucosa. Left: PBS buffer treated buccal mucosa. Right: buccal mucosa 24h after oral Tat-Smad7 treatment. Tat-Smad7 was stained using a V5 antibody (counterstained by a K14 antibody).
- FIG. 5 Abrogation of skin inflammation by Tat-Smad7 protein.
- FIG. 6 Accelerated body weight recovery by orally treating Tat-Smad7 protein after 20Gy irradiation. Daily Tat-Smad7 treatment began on day 5 after radiation. Irradiation with 16Gy gives similar results (not shown). *: p ⁇ 0.05.
- FIG. 7 Oral Tat-Smad7 treatment accelerated healing of oral mucositis.
- Smad7 protein can be easily produced recombinantly and used directly or encapsulated into delivery vehicles such as patches, solutions and gels that transduce target cells and exert bioactivities.
- delivery vehicles such as patches, solutions and gels that transduce target cells and exert bioactivities.
- this type of Smad7 delivery and thus should show therapeutic efficacies with little side effects.
- viral vector delivery, as well as in vivo induction and activation of Smad7 also are contemplated.
- Smad7 Mothers against decapentaplegic homolog 7 (Smad7) was previously identified as an antagonist of TGF- ⁇ signaling by several mechanisms including: (a) blockade of TGF- ⁇ receptor-mediated phosphorylation and nuclear translocation of signaling Smads; (b) increased degradation of TGF- ⁇ receptors and signaling Smads through specific ubiquitin-proteasome pathways and (c) inhibition of signaling Smads for their binding to Smad binding elements (SBEs). Smad7 also antagonizes other signaling pathways, like the NF- ⁇ pathway.
- Smad7 protein is encoded by the SMAD7 gene (SEQ ID NOS: 1 and 2). Like many other TGF- ⁇ family members, Smad7 is involved in cell signalling. It is a TGF- ⁇ type 1 receptor antagonist. It blocks TGF- ⁇ and activin associating with the receptor, blocking access to Smad2. It is an inhibitory Smad (I-SMAD) and is enhanced by SMURF2. Smad7 also enhances muscle differentiation.
- the present invention relates to Smad7 protein compositions.
- the present invention also relates to fragments of the polypeptide that retain the anti-inflammatory activity. Fragments may be generated by genetic engineering of translation stop sites within the coding region (discussed below). Alternatively, treatment of the Smad7 molecule with proteolytic enzymes, known as proteases, can produces a variety of N-terminal, C-terminal and internal fragments.
- fragments may be purified according to known methods, such as precipitation (e.g., ammonium sulfate), HPLC, ion exchange chromatography, affinity chromatography (including immunoaffinity chromatography) or various size separations (sedimentation, gel electrophoresis, gel filtration).
- Variants of Smad7 are also contemplated - these can be substitutional, insertional or deletion variants.
- Deletion variants lack one or more residues of the native protein which are not essential for activity, including the truncation mutants described above.
- Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide, such as stability against proteolytic cleavage, without the loss of other functions or properties.
- Substitutions of this kind preferably are conservative, that is, one amino acid is replaced with one of similar shape and charge.
- Conservative substitutions are well known in the art and include, for example, each amino acid can be changed or substituted with a different amino acid. .
- the hydropathic index, hydrophilicity, charge and size are normally considered.
- a specialized kind of variant is the fusion protein.
- This molecule generally has all or a substantial portion of the native molecule, linked at the N- or C-terminus, to all or a portion of a second polypeptide.
- fusions may employ leader sequences from other species to permit the recombinant expression of a protein in a heterologous host.
- Another useful fusion includes the addition of a functionally active domain, such as an antibody epitope, to facilitate purification of the fusion protein.
- Another type of fusion includes attaching a domain that can act as the target for an activating or inactivating ligand, thereby permitting control of the fusion protein's function once delivered to a subject.
- Such domains include, for example, steroid ligand binding (e.g., ER, PR, GR), which can be activated by small molecules, e.g., 4-hydroxyl tomaxifen or RU486 that are either uniquely able to activate those steroid ligand binding domains and/or do not exist in nature and will therefore enable full control of the Smad7 function by the presence of these small molecules.
- steroid ligand binding e.g., ER, PR, GR
- small molecules e.g., 4-hydroxyl tomaxifen or RU486 that are either uniquely able to activate those steroid ligand binding domains and/or do not exist in nature and will therefore enable full control of the Smad7 function by the presence of these small molecules.
- a fusion protein finding particular utility in the present invention is a fusion including a protein transduction domain (PTD), also called a cell delivery domain or cell transduction domain.
- PTD protein transduction domain
- Such domains have been described in the art and are generally characterized as short amphipathic or cationic peptides and peptide derivatives, often containing multiple lysine and arginine resides (Fischer, 2007).
- PTD protein transduction domain
- TAT protein from HIV
- HSV VP 16 cell delivery domain
- the present invention also provides, in another embodiment, genes encoding Smad7.
- genes encoding Smad7 In addition to the identified SMAD7 gene (SEQ ID NO: 2), it should be clear that the present invention is not limited to the specific nucleic acids disclosed herein. As discussed below, a "Smad7 gene" may contain a variety of different bases and yet still produce a corresponding polypeptide that is functionally indistinguishable from, and in some cases structurally identical to, the human gene disclosed herein. 1. Nucleic Acids Encoding Smad7
- Nucleic acids according to the present invention may represent an entire
- the nucleic acid may be derived from genomic DNA, i.e., cloned directly from the genome of a particular organism. In preferred embodiments, however, the nucleic acid would comprise complementary DNA (cDNA). Also contemplated is a cDNA plus a natural intron or an intron derived from another gene; such engineered molecules are sometime referred to as "mini-genes.” At a minimum, these and other nucleic acids of the present invention may be used as molecular weight standards in, for example, gel electrophoresis.
- cDNA is intended to refer to DNA prepared using messenger RNA
- mRNA as template.
- mRNA mRNA
- a nucleic acid encoding a Smad7 refers to a nucleic acid molecule that has been isolated free of total cellular nucleic acid.
- the invention concerns a nucleic acid sequence essentially as set forth in SEQ ID NO:2.
- the term "as set forth in SEQ ID NO:2” means that the nucleic acid sequence substantially corresponds to a portion of SEQ ID NO:2.
- the term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine, and also refers to codons that encode biologically equivalent amino acids, as discussed in the following pages.
- Sequences that are essentially the same as those set forth in SEQ ID NO:2 also may be functionally defined as sequences that are capable of hybridizing to a nucleic acid segment containing the complement of SEQ ID NO:2 under standard conditions.
- the DNA segments of the present invention include those encoding biologically functional equivalent Smad7 proteins and peptides, as described above.
- Such sequences may arise as a consequence of codon redundancy and amino acid functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded.
- functionally equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by man may be introduced through the application of site- directed mutagenesis techniques or may be introduced randomly and screened later for the desired function, as described below.
- expression vectors are employed to express the
- the expression vectors are used in gene therapy. Expression requires that appropriate signals be provided in the vectors, and which include various regulatory elements, such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in host cells. Elements designed to optimize messenger RNA stability and translatability in host cells also are defined. The conditions for the use of a number of dominant drug selection markers for establishing permanent, stable cell clones expressing the products are also provided, as is an element that links expression of the drug selection markers to expression of the polypeptide.
- expression construct is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed.
- the transcript may be translated into a protein, but it need not be.
- expression includes both transcription of a gene and translation of mRNA into a gene product. In other embodiments, expression only includes transcription of the nucleic acid encoding a gene of interest.
- vector is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated.
- a nucleic acid sequence can be "exogenous,” which means that it is foreign to the cell into which the vector is being introduced or that the sequence is homologous to a sequence in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found.
- Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs).
- plasmids include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs).
- YACs artificial chromosomes
- expression vector refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. In some cases, R A molecules are then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes.
- Expression vectors can contain a variety of "control sequences,” which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism, including promoters and enhancers. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions, such as transcription termination signals and poly-adenylation sites.
- viral vectors have led to the development and application of a number of different viral vector systems (Robbins et al., 1998).
- Viral systems are currently being developed for use as vectors for ex vivo and in vivo gene transfer.
- adenovirus, herpes-simplex virus, lentiviruses, retrovirus and adeno-associated virus vectors are being evaluated currently for treatment of diseases such as cancer, cystic fibrosis, Gaucher disease, renal disease and arthritis (Robbins and Ghivizzani, 1998; Imai et al., 1998; U.S. Patent 5,670,488).
- the various viral vectors present specific advantages and disadvantages, depending on the particular gene-therapeutic application.
- Suitable non-viral methods for nucleic acid delivery for transformation of an organelle, a cell, a tissue or an organism for use with the current invention are believed to include virtually any method by which a nucleic acid (e.g., DNA) can be introduced into an organelle, a cell, a tissue or an organism, as described herein or as would be known to one of ordinary skill in the art.
- a nucleic acid e.g., DNA
- Such methods include, but are not limited to, direct delivery of DNA such as by injection (U.S.
- Patent 5,384,253, incorporated herein by reference by calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et a/., 1990); by using DEAE-dextran followed by polyethylene glycol (Gopal, 1985); by direct sonic loading (Fechheimer et al., 1987); by liposome mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991); by microprojectile bombardment (PCT Application Nos. WO 94/09699 and 95/06128; U.S.
- compositions discussed above Numerous expression systems exist that comprise at least a part or all of the compositions discussed above.
- Prokaryote- and/or eukaryote-based systems can be employed for use with the present invention to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Many such systems are commercially and widely available.
- the insect cell/baculovirus system can produce a high level of protein expression of a heterologous nucleic acid segment, such as described in U.S. Patents 5,871,986 and 4,879,236, both herein incorporated by reference, and which can be bought, for example, under the name MAXBAC ® 2.0 from INVITROGEN ® and BACPAC TM BACULOVIRUS EXPRESSION SYSTEM FROM CLONTECH®.
- a heterologous nucleic acid segment such as described in U.S. Patents 5,871,986 and 4,879,236, both herein incorporated by reference, and which can be bought, for example, under the name MAXBAC ® 2.0 from INVITROGEN ® and BACPAC TM BACULOVIRUS EXPRESSION SYSTEM FROM CLONTECH®.
- expression systems include STRATAGENE®'S COMPLETE CONTROLTM Inducible Mammalian Expression System, which involves a synthetic ecdysone- inducible receptor, or its pET Expression System, an E. coli expression system.
- INVITROGEN ® Another example of an inducible expression system is available from INVITROGEN ® , which carries the T-PvEXTM (tetracycline -regulated expression) System, an inducible mammalian expression system that uses the full-length CMV promoter.
- INVITROGEN ® also provides a yeast expression system called the Pichia methanolica Expression System, which is designed for high-level production of recombinant proteins in the methylotrophic yeast Pichia methanolica.
- a vector such as an expression construct, to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide.
- Primary mammalian cell cultures may be prepared in various ways. In order for the cells to be kept viable while in vitro and in contact with the expression construct, it is necessary to ensure that the cells maintain contact with the correct ratio of oxygen and carbon dioxide and nutrients but are protected from microbial contamination. Cell culture techniques are well documented.
- One embodiment of the foregoing involves the use of gene transfer to immortalize cells for the production of proteins.
- the gene for the protein of interest may be transferred as described above into appropriate host cells followed by culture of cells under the appropriate conditions.
- the gene for virtually any polypeptide may be employed in this manner.
- the generation of recombinant expression vectors, and the elements included therein, are discussed above.
- the protein to be produced may be an endogenous protein normally synthesized by the cell in question.
- Examples of useful mammalian host cell lines are Vero and HeLa cells and cell lines of Chinese hamster ovary, W138, BHK, COS-7, 293, HepG2, NIH3T3, RIN and MDCK cells.
- a host cell strain may be chosen that modulates the expression of the inserted sequences, or modifies and process the gene product in the manner desired. Such modifications ⁇ e.g., glycosylation) and processing ⁇ e.g., cleavage) of protein products may be important for the function of the protein.
- Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to insure the correct modification and processing of the foreign protein expressed.
- a number of selection systems may be used including, but not limited to, HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase genes, in tk-, hgprt- or aprt- cells, respectively.
- antimetabolite resistance can be used as the basis of selection for dhfr, that confers resistance to; gpt, that confers resistance to mycophenolic acid; neo, that confers resistance to the aminoglycoside G418; and hygro, that confers resistance to hygromycin.
- the terms “cell,” “cell line,” and “cell culture” may be used interchangeably. All of these terms also include their progeny, which is any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations.
- "host cell” refers to a prokaryotic or eukaryotic cell, and it includes any transformable organisms that is capable of replicating a vector and/or expressing a heterologous gene encoded by a vector.
- a host cell can, and has been, used as a recipient for vectors.
- a host cell may be "transfected” or “transformed,” which refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
- a transformed cell includes the primary subject cell and its progeny.
- Host cells may be derived from prokaryotes or eukaryotes, depending upon whether the desired result is replication of the vector or expression of part or all of the vector- encoded nucleic acid sequences.
- Numerous cell lines and cultures are available for use as a host cell, and they can be obtained through the American Type Culture Collection (ATCC), which is an organization that serves as an archive for living cultures and genetic materials (atcc.org).
- ATCC American Type Culture Collection
- An appropriate host can be determined by one of skill in the art based on the vector backbone and the desired result.
- a plasmid or cosmid for example, can be introduced into a prokaryote host cell for replication of many vectors.
- Bacterial cells used as host cells for vector replication and/or expression include DH5a, JM109, and KC8, as well as a number of commercially available bacterial hosts such as SURE ® Competent Cells and SOLOPAC TM Gold Cells (STRATAGENE ® , La Jolla).
- bacterial cells such as E. coli LE392 could be used as host cells for phage viruses.
- Examples of eukaryotic host cells for replication and/or expression of a vector include HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC 12. Many host cells from various cell types and organisms are available and would be known to one of skill in the art. Similarly, a viral vector may be used in conjunction with either a eukaryotic or prokaryotic host cell, particularly one that is permissive for replication or expression of the vector.
- Some vectors may employ control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells.
- One of skill in the art would further understand the conditions under which to incubate all of the above described host cells to maintain them and to permit replication of a vector.
- Also understood and known are techniques and conditions that would allow large-scale production of vectors, as well as production of the nucleic acids encoded by vectors and their cognate polypeptides, proteins, or peptides.
- a chronic wound is a wound that does not heal in an orderly set of stages and in a predictable amount of time the way most wounds do; wounds that do not heal within three months are often considered chronic.
- Chronic wounds seem to be detained in one or more of the phases of wound healing. For example, chronic wounds often remain in the inflammatory stage for too long. In acute wounds, there is a precise balance between production and degradation of molecules such as collagen; in chronic wounds this balance is lost and degradation plays too large a role.
- Chronic wounds may never heal or may take years to do so. These wounds cause patients severe emotional and physical stress as well as creating a significant financial burden on patients and the whole healthcare system. Acute and chronic wounds are at opposite ends of a spectrum of wound healing types that progress toward being healed at different rates. The vast majority of chronic wounds can be classified into three categories: venous ulcers, diabetic, and pressure ulcers. A small number of wounds that do not fall into these categories may be due to causes such as radiation poisoning or ischemia.
- Venous and arterial ulcers are characterized by Venous and arterial ulcers. Venous ulcers, which usually occur in the legs, account for about 70% to 90% of chronic wounds and mostly affect the elderly. They are thought to be due to venous hypertension caused by improper function of valves that exist in the veins to prevent blood from flowing backward. Ischemia results from the dysfunction and, combined with reperfusion injury, causes the tissue damage that leads to the wounds.
- Diabetic ulcers Another major cause of chronic wounds, diabetes, is increasing in prevalence. Diabetics have a 15% higher risk for amputation than the general population due to chronic ulcers. Diabetes causes neuropathy, which inhibits nociception and the perception of pain. Thus patients may not initially notice small wounds to legs and feet, and may therefore fail to prevent infection or repeated injury. Further, diabetes causes immune compromise and damage to small blood vessels, preventing adequate oxygenation of tissue, which can cause chronic wounds. Pressure also plays a role in the formation of diabetic ulcers.
- Pressure ulcers Another leading type of chronic wounds is pressure ulcers, which usually occur in people with conditions such as paralysis that inhibit movement of body parts that are commonly subjected to pressure such as the heels, shoulder blades, and sacrum. Pressure ulcers are caused by ischemia that occurs when pressure on the tissue is greater than the pressure in capillaries, and thus restricts blood flow into the area. Muscle tissue, which needs more oxygen and nutrients than skin does, shows the worst effects from prolonged pressure. As in other chronic ulcers, reperfusion injury damages tissue.
- Chronic wounds may affect only the epidermis and dermis, or they may affect tissues all the way to the fascia. They may be formed originally by the same things that cause acute ones, such as surgery or accidental trauma, or they may form as the result of systemic infection, vascular, immune, or nerve insufficiency, or comorbidities such as neoplasias or metabolic disorders.
- the reason a wound becomes chronic is that the body's ability to deal with the damage is overwhelmed by factors such as repeated trauma, continued pressure, ischemia, or illness.
- Ischemia is an important factor in the formation and persistence of wounds, especially when it occurs repetitively (as it usually does) or when combined with a patient's old age. Ischemia causes tissue to become inflamed and cells to release factors that attract neutrophils such as interleukins, chemokines, leukotrienes, and complement factors.
- neutrophils While they fight pathogens, neutrophils also release inflammatory cytokines and enzymes that damage cells.
- ROS Reactive Oxygen Species
- the enzymes and ROS produced by neutrophils and other leukocytes damage cells and prevent cell proliferation and wound closure by damaging DNA, lipids, proteins, the ECM, and cytokines that speed healing.
- Neutrophils remain in chronic wounds for longer than they do in acute wounds, and contribute to the fact that chronic wounds have higher levels of inflammatory cytokines and ROS. Since wound fluid from chronic wounds has an excess of proteases and ROS, the fluid itself can inhibit healing by inhibiting cell growth and breaking down growth factors and proteins in the ECM.
- Chronic wounds also differ in makeup from acute wounds in that their levels of proteolytic enzymes such as elastase. and matrix metalloproteinases (MMPs) are higher, while their concentrations of growth factors such as Platelet-derived growth factor and Keratinocyte Growth Factor are lower.
- proteolytic enzymes such as elastase. and matrix metalloproteinases (MMPs) are higher, while their concentrations of growth factors such as Platelet-derived growth factor and Keratinocyte Growth Factor are lower.
- MMPs matrix metalloproteinases
- GFs growth factors
- Chronic wounds such as diabetic and venous ulcers are also caused by a failure of fibroblasts to produce adequate ECM proteins and by keratinocytes to epithelialize the wound. Fibroblast gene expression is different in chronic wounds than in acute wounds.
- Excess matrix metalloproteinases which are released by leukocytes, may also cause wounds to become chronic. MMPs break down ECM molecules, growth factors, and protease inhibitors, and thus increase degradation while reducing construction, throwing the delicate compromise between production and degradation out of balance. 2. Acute Wounds/Trauma
- Trauma can also be described as both unplanned, such as an accident, or planned, in the case of surgery. Both can be characterized by mild to severe tissue damage, blood loss and/or shock, and both may lead to subsequent infection, including sepsis.
- the present invention provides to treatment of trauma, including both pre-treatment (in the case of a medical procedure) and treatment after trauma injury as occurred.
- Surgery uses operative manual and instrumental techniques on a patient to investigate and/or treat a pathological condition such as disease or injury, to help improve bodily function or appearance, or sometimes for some other reason.
- a pathological condition such as disease or injury
- the present invention can address trauma resulting from surgeries, as defined further below.
- a procedure is considered surgical when it involves cutting of a patient's tissues or closure of a previously sustained wound.
- Other procedures that do not necessarily fall under this rubric such as angioplasty or endoscopy, may be considered surgery if they involve common surgical procedure or settings, such as use of a sterile environment, anesthesia, antiseptic conditions, typical surgical instruments, and suturing or stapling. All forms of surgery are considered invasive procedures; so-called noninvasive surgery usually refers to an excision that does not penetrate the structure being addressed (e.g., laser ablation of the cornea) or to a radiosurgical procedure (e.g., irradiation of a tumor). Surgery can last from minutes to hours.
- Surgical procedures are commonly categorized by urgency, type of procedure, body system involved, degree of invasiveness, and special instrumentation.
- Elective surgery is done to correct a non- life-threatening condition, and is carried out at the patient's request, subject to the surgeon's and the surgical facility's availability.
- Emergency surgery is surgery which must be done quickly to save life, limb, or functional capacity. Exploratory surgery is performed to aid or confirm a diagnosis.
- Therapeutic surgery treats a previously diagnosed condition.
- Amputation involves cutting off a body part, usually a limb or digit.
- Replantation involves reattaching a severed body part.
- Reconstructive surgery involves reconstruction of an injured, mutilated, or deformed part of the body.
- Cosmetic surgery is done to improve the appearance of an otherwise normal structure.
- Excision is the cutting out of an organ, tissue, or other body part from the patient.
- Transplant surgery is the replacement of an organ or body part by insertion of another from different human (or animal) into the patient. Removing an organ or body part from a live human or animal for use in transplant is also a type of surgery.
- Minimally invasive surgery involves smaller outer incision(s) to insert miniaturized instruments within a body cavity or structure, as in laparoscopic surgery or angioplasty.
- an open surgical procedure requires a large incision to access the area of interest.
- Laser surgery involves use of a laser for cutting tissue instead of a scalpel or similar surgical instruments.
- Microsurgery involves the use of an operating microscope for the surgeon to see small structures.
- Robotic surgery makes use of a surgical robot, such as Da Vinci or Zeus surgical systems, to control the instrumentation under the direction of the surgeon.
- the present invention contemplates the treatment of a variety of autoimmune and/or inflammatory disease states such as spondyloarthropathy, ankylosing spondylitis, psoriatic arthritis, reactive arthritis, enteropathic arthritis, ulcerative colitis, Crohn's disease, irritable bowel disease, inflammatory bowel disease, rheumatoid arthritis, juvenile rheumatoid arthritis, familial Mediterranean fever, amyotrophic lateral sclerosis, Sjogren's syndrome, early arthritis, viral arthritis, multiple sclerosis, or psoriasis.
- spondyloarthropathy ankylosing spondylitis
- psoriatic arthritis reactive arthritis
- enteropathic arthritis ulcerative colitis
- Crohn's disease irritable bowel disease
- inflammatory bowel disease rheumatoid arthritis
- juvenile rheumatoid arthritis juvenile rheumatoid arthritis
- familial Mediterranean fever amyotrophic lateral sclerosis
- Various forms of cancer therapy including chemotherapy, radiation, and cytokines, are associated with toxicity, sometimes severe, in the cancer patient.
- the present invention seeks to reduce this toxicity using the pharmaceutical compositions of the present invention, thereby reducing or alleviating discomfort on the part of the patient, as well as permitting higher doses of the therapy.
- Oral cancer the 6 th most common cancer worldwide, is a subtype of head and neck cancer, is any cancerous tissue growth located in the oral cavity. It may arise as a primary lesion originating in any of the oral tissues, by metastasis from a distant site of origin, or by extension from a neighboring anatomic structure, such as the nasal cavity or the oral cancers may originate in any of the tissues of the mouth, and may be of varied histologic types: teratoma, adenocarcinoma derived from a major or minor salivary gland, lymphoma from tonsillar or other lymphoid tissue, or melanoma from the pigment-producing cells of the oral mucosa.
- oral cancers There are several types of oral cancers, but around 90% are squamous cell carcinomas, originating in the tissues that line the mouth and lips. Oral or mouth cancer most commonly involves the tongue. It may also occur on the floor of the mouth, cheek lining, gingiva (gums), lips, or palate (roof of the mouth). Most oral cancers look very similar under the microscope and are called squamous cell carcinoma. These are malignant and tend to spread rapidly.
- Oral mucositis is a chronic oral ulceration. This disease frequently occurs in radiation-treated patients of all cancer types, patients radiation-treated for organ transplants (to eliminate rejection of the transplants), and patients undergoing routine chemotherapy. Severe oral mucositis is extremely painful and impairs food/liquid intake, hence is often the most severe complication of cancer therapy. Oral mucositis is a major factor in determining the maximum dose possible of radiation and chemotherapy to the head and neck region; it can significantly complicate cancer treatment, extend hospitalization, decrease quality of life and increase costs. Currently, there is no established therapy to effectively treat severe oral mucositis.
- Kepivance® a recombinant protein of human keratinocyte growth factor (KGF)
- KGF human keratinocyte growth factor
- Other potential therapies include topical rinses, such as viscous 2% lidocaine rinses, or baking soda and saline solutions, or a cocktail solution, for instance BAX (lidocaine, diphenhyramine, sorbitol and Mylanta).
- Other investigative or mucoprotective adjuvant therapies include but are not limited to, beta carotene, tocopherol, laser irradiation, prophylactic brushing the oral mucosa with silver-nitrate, misoprostol, leucovorin, systemic KGF, pentoxifylline, allopurinol mouthwash, systemic sucralfate, chlorhexidine gluconate, and cryotherapy.
- Chemotherapy and radiation induced gut mucositits is an inflammatory condition that arises as a result of the acute death of rapidly dividing intestinal epithelial cells.
- Most chemotherapeutic drugs used for treatment of solid tumors, alone, in a combination of drugs, or with radiation, will result in the death of a large number of intestinal epithelial cells.
- the clinical manifestations of the ensuing mucositis include digestive symptoms such as nausea and vomiting, serious diarrhea, acute weight loss and wasting. This is fast becoming one of the limiting factors for administering chemotherapy for many cancer patients.
- the ability of Tat-Smad7 to protect intestinal epithelial cells from either chemotherapeutic agents, radiation, or a combinations of those, will significantly decrease the undesirable side effects of cancer therapies, and enable more aggressive ways to treat the disease with existing tools.
- Bone marrow failure syndromes are a set of conditions that develop when the hematopoietic stem cell compartment is compromised and fails to give rise to normal cell types. Bone marrow failure occurs as a result of inherited genetic abnormalities, exposure to a noxious substance, such as toxins, chemicals or viruses. Although the nature and identity of environmental factors that can lead to the development of acquired bone marrow failure is still not completely understood, a few factors have been linked to the development of acquired bone marrow failure among military personnel including exposure to mustard gas, ionizing radiation, and infectious agents such as visceral leishmaniasis or African trypanosomiasis.
- This process may involve contacting the cells/subjects with the both agents/therapies at the same time, e.g., using a single composition or pharmacological formulation that includes both agents, or by contacting the cell/subject with two distinct compositions or formulations, at the same time, wherein one composition includes the Smad7 agent and the other includes the other agent.
- the Smad7 agent may precede or follow the other treatment by intervals ranging from minutes to weeks.
- agents suitable for use in a combined therapy against an inflammatory disorder include steroids, glucocorticoids, non- steriodal anti-inflammatory drugs (NSAIDS; including COX-1 and COX-2 inhibitors), aspirin, ibuprofen, and naproxen.
- NSAIDS non- steriodal anti-inflammatory drugs
- Analgesics are commonly associated with antiinflammatory drugs but which have no anti-inflammatory effects.
- An example is paracetamol, called acetaminophen in the U.S. and sold under the brand name of Tylenol.
- a particular agent for combination use is an anti-TGF- ⁇ antibody.
- the invention may be applied as a combination to with cancer therapies. While cancer therapies address the cancer, they unfortunately cause serious side effects.
- cancer therapies address the cancer, they unfortunately cause serious side effects.
- the Smad7 agents of the present invention can be used advantageously in combination with such cancer therapies. This process may involve contacting the cells, organ or patient with the agents/therapies at the same time, including by contacting the cells, organ or patient with a single composition or pharmacological formulation that includes both agents, or with two distinct compositions or formulations at the same time, wherein one composition includes the Smad7 agent and the other includes the other agent.
- the compositions can be delivered at different times, including repeated doses of one or both agents.
- Agents or factors suitable for use in a combined therapy include any chemical compound or treatment method that induces DNA damage when applied to a cell. Such agents and factors include radiation and waves that induce DNA damage such as, irradiation, microwaves, electronic emissions, and the like.
- a variety of chemical compounds, also described as "chemotherapeutic” or “genotoxic agents,” are intended to be of use in the combined treatment methods disclosed herein.
- chemotherapeutic or “genotoxic agents”
- agents are intended to be of use in the combined treatment methods disclosed herein.
- treating cancer according to the invention one would contact the tumor cells with an agent in addition to the expression construct. This may be achieved by irradiating the localized tumor site; alternatively, the tumor cells may be contacted with the agent by administering to the subject a therapeutically effective amount of a pharmaceutical composition.
- SERMs selective estrogen receptor antagonists
- Tamoxifen 4-hydroxy Tamoxifen (Afimoxfene)
- Falsodex Raloxifene
- Bazedoxifene Raloxifene
- Clomifene Femarelle
- Lasofoxifene Ormeloxifene
- Toremifene Toremifene
- Chemotherapeutic agents contemplated to be of use include, e.g., camptothecin, actinomycin-D, mitomycin C.
- the invention also encompasses the use of a combination of one or more DNA damaging agents, whether radiation-based or actual compounds, such as the use of X-rays with cisplatin or the use of cisplatin with etoposide.
- the agent may be prepared and used as a combined therapeutic composition, or kit, by combining it with a MUC1 peptide, as described above.
- Heat shock protein 90 is a regulatory protein found in many eukaryotic cells.
- HSP90 inhibitors have been shown to be useful in the treatment of cancer.
- Such inhibitors include Geldanamycin, 17-(Allylamino)-17-demethoxygeldanamycin, PU-H71 and Rifabutin.
- Agents that directly cross-link DNA or form adducts are also envisaged. Agents such as cisplatin, and other DNA alkylating agents may be used. Cisplatin has been widely used to treat cancer, with efficacious doses used in clinical applications of 20 mg/m for 5 days every three weeks for a total of three courses. Cisplatin is not absorbed orally and must therefore be delivered via injection intravenously, subcutaneously, intratumorally or intraperitoneally.
- Agents that damage DNA also include compounds that interfere with DNA replication, mitosis and chromosomal segregation.
- chemotherapeutic compounds include adriamycin, also known as doxorubicin, etoposide, verapamil, podophyllotoxin, and the like. Widely used in a clinical setting for the treatment of neoplasms, these compounds are administered through bolus injections intravenously at doses ranging from 25-75 mg/m at 21 day intervals for doxorubicin, to 35-50 mg/m for etoposide intravenously or double the intravenous dose orally.
- Microtubule inhibitors such as taxanes, also are contemplated.
- Epidermal growth factor receptor inhibitors such as Iressa, mTOR, the mammalian target of rapamycin, also known as FK506-binding protein 12-rapamycin associated protein 1 (FRAPl) is a serine/threonine protein kinase that regulates cell growth, cell proliferation, cell motility, cell survival, protein synthesis, and transcription. Rapamycin and analogs thereof (“rapalogs”) are therefore contemplated for use in combination cancer therapy in accordance with the present invention.
- TNF-a tumor necrosis factor-alpha
- TNF-a tumor necrosis factor-alpha
- TNF-alpha a cytokine involved in systemic inflammation and a member of a group of cytokines that stimulate the acute phase reaction.
- the primary role of TNF is in the regulation of immune cells. TNF is also able to induce apoptotic cell death, to induce inflammation, and to inhibit tumorigenesis and viral replication.
- nucleic acid precursors and subunits also lead to DNA damage.
- nucleic acid precursors have been developed.
- agents that have undergone extensive testing and are readily available are particularly useful.
- agents such as 5-fluorouracil (5-FU) are preferentially used by neoplastic tissue, making this agent particularly useful for targeting to neoplastic cells.
- 5-FU is applicable in a wide range of carriers, including topical, however intravenous administration with doses ranging from 3 to 15 mg/kg/day being commonly used.
- Models and resources developed herein can provide unique tools for analytical studies to identify biomarkers and therapeutic targets related to Smad7 overexpression and control, for example, downstream molecules turned on or bound by Smad7 can be identified as additional therapeutic targets for example, to treat oral mucositis, psoriasis and other conditions aggravated by TGF- ⁇ activities and NFKB activities.
- compositions - proteins, expression vectors, virus stocks, proteins and drugs - in a form appropriate for the intended application.
- this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.
- compositions of the present invention comprise an effective amount of the vector to cells, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions also are referred to as inocula.
- pharmaceutically or pharmacologically acceptable refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human.
- pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
- the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the vectors or cells of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions.
- compositions of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention will be via any common route so long as the target tissue is available via that route. This includes oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions, described supra. Of particular interest is direct intratumoral administration, perfusion of a tumor, or admininstration local or regional to a tumor, for example, in the local or regional vasculature or lymphatic system, or in a resected tumor bed.
- Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
- the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
- the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
- the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
- the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
- a coating such as lecithin
- surfactants for example, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sorbic acid, thimerosal, and the like.
- isotonic agents for example, sugars or sodium chloride.
- Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
- Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
- dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
- the preferred methods of preparation are vacuum-drying and freeze- drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
- pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
- the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
- compositions of the present invention may be formulated in a neutral or salt form.
- Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. [000113] The formulations are easily administered in a variety of dosage forms.
- the polypeptides of the present invention may be incorporated with excipients and used in the form of non-ingestible mouthwashes and dentifrices.
- a mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution).
- the active ingredient may be incorporated into an antiseptic wash containing sodium borate, glycerin and potassium bicarbonate.
- the active ingredient may also be dispersed in dentifrices, including: gels, pastes, powders and slurries.
- the active ingredient may be added in a therapeutically effective amount to a paste dentifrice that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.
- oral delivery materials can also include creams, salves, ointments, patches, liposomes, nanoparticles, microparticles, timed- release formulations and other materials known in the art for delivery to the oral cavity and/or to the skin of a subject for treatment and/or prevention of a condition disclosed herein.
- Certain embodiments concern using a biodegradable oral patch delivery system or gelatinous material.
- These compositions can be a liquid formulation or a pharmaceutically acceptable delivery system treated with a formulation of these compositions, and may also include activator/ inducers .
- a patch contemplated herein may be a slowly dissolving or a time -released patch.
- a slowly dissolving patch can be an alginate patch.
- a patch may contain a detectible indicator dye or agent such as a fluorescent agent.
- a tag e.g. detectible tag such as a biotin or fluorescently tagged agent
- a treatment molecule in order to detect the molecule after delivery to the subject.
- one or more oral delivery patches or other treatment contemplated herein may be administered to a subject 3 times daily, 2 times daily, once a day, every other day, weekly, and the like, depending on need of the subject assessed by a health professional.
- Patches contemplated herein may be oral-biodegradable patches or patches for exterior use that may or may not degrade.
- Patches contemplated herein may be 1 mm, 2 mm, 3 mm, 4 mm to 5 mm in size or more depending on need.
- skin patches are contemplated herein for use in a subject suffering from psoriasis.
- Smad7 can be delivered topically using vehicles such as glycerol, carboxymethycellulose. It can also use transdermal system (e.g., commercially available from 3M) for delivery. Subcutaneous injection into the lesion (in normal saline or PBS) can also be used
- kits contemplated herein may include compositions discussed above for treating a subject having a condition contemplated herein, such as oral mucositis or psoriasis.
- the kits can include one or more containers containing the therapeutic compositions. Any of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container, into which compositions may be preferably and/or suitably aliquoted. Kits herein may also include a kit for assessing biological targets that contribute to a condition contemplated herein.
- Smad7 transgenic mice have been described with multiple functions of Smad7. This mouse model is crucial for understanding that Smad7 has a potent effect in oral mucositis. Smad7 promotes healing of oral wounds. Based on these findings, if Smad7 can provide protection from oral mucositis can be assessed. Smad7 transgenic mice and their littermates were exposed to radiation. In non-transgenic littermates, all died of oral mucositis in 10 days due to loss of oral intake-associated dehydration and starvation. In contrast, none of the Smad7 transgenic mice formed oral mucositis (FIG. 1). Based on this data, local pharmacologic delivery of Smad7 can be used to treat oral mucositis. In other embodiments, a Smad7 expression plasmid with either replication-free viral vector or non-viral vector, can be used to treat oral mucositis.
- Smad7 promotes healing mainly through promoting epithelial cell migration into the wounds and protecting cells against DNA damage (FIG. 1), the latter is tumor suppressive.
- Smad7 has been shown to inhibit cancer metastasis and unpublished data show Smad7 overexpression in mice does not increase susceptibility to cancer. Given that unresolved inflammation is one of the major causes of failed healing in oral mucositis, the strong effect of Smad7 can significantly promote healing of oral mucositis.
- transgenic mice were generated with severe skin psoriasis, and these mice die within 6 months due to severe itch-associated wasting syndrome.
- Smad7 transgenic mice the same showing resistance to radiation-induced oral mucositis in FIG. 1
- skin inflammation was absent and the mice lived a normal lifespan (FIG. 2).
- Smad7 transgene expression level is only 2-fold of endogenous Smad7, suggesting that a low dose of Smad7 is sufficient for therapeutic effect.
- Smad7 oral patch/gel can be administered by patients with minimal systemic effects.
- the inventors generated a recombinant human Smad7 (98% homology to mouse Smad7) with an N-terminal Tat-tag (Tat-Smad7) that allows proteins to permeate the cell membrane and reach the nucleus in a matter of seconds (Cardarelli et al., 2008; Kalvala et al., 2010; Brooks et al., 2005).
- the human Smad7 cDNA nucleotide sequence was altered to optimize codons for bacterial protein production and a 5' Tat-tag (9 aa) was added.
- the Tat.Smad7 was cloned into the pETlOl protein expression vector (Invitrogen) for protein production in E. coli.
- the vector contains a V5 epitope for protein identification using a V5 antibody and 6XHis tag for protein purification at the 3' of the Tat-Smad7 protein.
- the inventors tested purified Tat-Smad7 protein transduction at a concentration of 7.5 g/ml. In less than 5 minutes after exposure to Tat-Smad7, nearly 100% of cells showed nuclear Tat- Smad7 (FIG. 3), which was retained at least the first 2 h after transduction. Later, Tat-Smad7 was also detected in the cytoplasm (FIG. 3), consistent with its ability to move between the nucleus and the cytoplasm (Zhang et al, 2007).
- Tat-Smad7 transduced keratinocytes abrogated Smad2 phosphorylation (FIG. 3), suggesting it is functionally intact. Consistent with its long half-life, Tat-Smad7 was still detectable in cells 36 hr after withdrawal of the 5-min Tat- Smad7 treatment (not shown).
- Tat-Smad7 To determine if this is rapid enough for Tat-Smad7 to penetrate oral mucosa in vivo, the inventors treated 5 mice orally with 10 g of Tat-Smad7 in 10 ⁇ phosphate buffered saline (PBS) buffer once, with an hour food/water restriction after treatment, and excised oral mucosa for Tat-Smad7 detection 24 h after treatment. Tat-Smad7 was detected uniformly in epithelial and stromal cells of oral mucosa (FIG. 4). These data suggest that local Tat-Smad7 delivery quickly achieves cellular uptake and should not require more than daily application.
- PBS phosphate buffered saline
- Tat-Smad7 Although the inventors have an endotoxin removal step in the purification process, they further assessed whether Tat-Smad7 is functional in vivo, and if potential endotoxin contamination from production in bacteria poses a toxicity risk.
- the inventors s.c- injected Tat-Smad7 (10 ⁇ g/mouse, 3 times/wk) into K5.TGF i mice which exhibit severe skin inflammation (Li et al., 2004). In all three, K5.TGF i mice in this pilot experiment, Tat- Smad7 treatment significantly alleviated skin inflammation (FIG. 5) and no obvious side effects were observed.
- Tat-Smad7 protein accumulated in the dermis and throughout the epidermis (FIG. 5).
- Tat-Smad7 -transduced K5.TGF i skin showed reduced pSmad2 and NFKB p50 in both the epidermis and the stroma (FIG. 5).
- Tat-Smad7 can treat radiation-induced oral mucositis through oral delivery.
- the inventors irradiated mouse cranial facial area and treated mice with Tat-Smad7 afterwards.
- Seven-to-nine week old C3H female mice were anesthetized and exposed to 16Gy, 20Gy or 25Gy head irradiation with an RS2000 X-ray irradiator.
- mice were treated daily with either 10 ⁇ g Tat.Smad7 dissolved in 50% glycerol/PBS or 50% glycerol/PBS (control), each group contained 4 mice, beginning on day 5 after irradiation when tissue damage in oral mucosa occurs.
- Tat-Smad7 treated mice recovered body weight more rapidly than control mice with 16Gy or 20Gy irradiation (FIG. 6); control mice with 25Gy irradiation were all euthanized for humane reasons hence body weight recovery could not be monitored. Histology shows that by day 9 after 20Gy or 25Gy radiation, control mice developed oral mucositis (open ulcers, FIG. 7). In contrast, Tat- Smad7 treated mice had damaged oral mucosa with thinning epithelial layers but no ulcer formation (FIG. 7). Continual treatment of 20Gy irradiated mice with Tat-Smad7 till day 13 accelerated repair.
- Tat-Smad7 protein was delivered primarily to oral epithelial cells (FIG. 8).
- Cells positive for pH2AX, a DNA damage marker were significantly reduced in Tat-Smad7- treated oral mucosa (FIG. 8).
- apoptotic cells as determined by TUNEL assay, were also significantly reduced in Tat-Smad7 treated oral mucosa (FIG. 8).
- Infiltrated leukocytes, identified by CD45 staining were prominent in irradiated oral mucosa treated with vehicle control but were significantly reduced in Tat- Smad7 treated mucosa (FIG. 8).
- CD31 staining to highlight vessels shows enlarged vessels in irradiated oral mucosa treated with vehicle control but normal vessel sizes in Tat-Smad7 treated oral mucosa (FIG. 8).
- Tat-Smad7 local delivery can treat severe oral mucositis through preventing tissue damage, improving epithelial healing and reducing inflammation and vessel damage in the stroma.
Abstract
Description
Claims
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EP19167590.9A EP3536337A1 (en) | 2010-09-22 | 2011-09-21 | Therapeutic applications of smad7 |
CA2849382A CA2849382C (en) | 2010-09-22 | 2011-09-21 | Therapeutic applications of smad7 for the treatment of oral mucositis |
US13/822,173 US9084746B2 (en) | 2010-09-22 | 2011-09-21 | Therapeutic applications of SMAD7 |
CN201510882397.6A CN105457016B (en) | 2010-09-22 | 2011-09-21 | Therapeutic uses of SMAD7 |
EP11827421.6A EP2618829B1 (en) | 2010-09-22 | 2011-09-21 | Smad7 for use in the treatment of oral mucositis or psoriasis |
CN201180051033.3A CN103501803B (en) | 2010-09-22 | 2011-09-21 | The treatment use of SMAD7 |
JP2013530249A JP6124791B2 (en) | 2010-09-22 | 2011-09-21 | Therapeutic application of Smad7 |
IL225406A IL225406B (en) | 2010-09-22 | 2013-03-21 | Therapeutic applications of smad7 |
US14/750,557 US9474784B2 (en) | 2010-09-22 | 2015-06-25 | Therapeutic applications of SMAD7 |
US15/282,022 US10350265B2 (en) | 2010-09-22 | 2016-09-30 | Therapeutic applications of Smad7 |
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