HK1174565B - Method of treating scars and ss-catenin-mediated disorders using nefopam compounds - Google Patents

Description

Methods of treating scar and beta-catenin mediated diseases using nefopam compounds

Technical Field

The present invention relates generally to the treatment of scar and beta-catenin mediated diseases.

Background

The fibroproliferative process is a group of diseases characterized by an excessive proliferation of mesenchymal fibroblast-like spindle cells. They range from hypertrophic wounds to tumor progression such as invasive fibromatosis (AF).

During wound healing, several cell types and signaling pathways are activated to reconstitute the epithelial and dermal layers of the skin. After skin injury, three consecutive distinct but overlapping courses begin: inflammation, proliferation and remodeling. During the proliferative phase, mesenchymal fibroblast-like cells accumulate in the epidermal components of the skin and the epithelial barrier layer is engineered (Singer1999, Martin1997, McClain 1996). Beta-catenin has been shown to regulate epithelial and mesenchymal cell activity, so it can promote proliferation and differentiation of epidermal mesenchymal cells and reduce metastasis of epithelial keratin cells (Cheon 2002). Mouse models have shown that β -catenin is able to regulate the size of the final wound, while β -catenin levels induced by lithium treatment lead to wound healing of larger size (Cheon 2006). Also, transgenic mice expressing stable β -catenin in mesenchymal cells under the control of a tetracycline-regulated promoter have been produced. Wounded mice that had healed with proliferative skin wounds were compared to wild-type control mice (Cheon 2002). This suggests the importance of β -catenin in mesenchymal cells and its crucial role in wound healing.

Another β -catenin mediated fibroproliferative disease is invasive fibromatosis (AF), also known as desmoid. AF is a locally invasive soft tissue tumor comprising mesenchymal fibroblast-like spindle cells. AF occurs as a sporadic lesion or as a familial syndrome such as familial multiple adenocarcinoma (FAP). Beta-catenin stabilization is prevalent in AF, which is manifested by increased levels of beta-catenin and increased beta-catenin-mediated transcriptional activity. In addition, β -catenin stabilization was sufficient to cause AF, as shown in the mouse model using genes that were overexpressed in β -catenin stabilization (Cheon 2002). This suggests a key role for β -catenin in fibroproliferative diseases and its importance in mesenchymal cells.

In addition to the role of β -catenin in fibroproliferative diseases, numerous studies have shown that dysregulation of β -catenin expression is an important event in the genesis of a large number of malignant tumors such as colon, melanoma, hepatocellular carcinoma, ovarian, endometrial, medulloblastoma, pilomoblastoma, and prostate cancer. Beta-catenin mutations represent a critical step in the progression of this group of cancers, suggesting an important role in the control of cell proliferation or cell death (as described by Polakisp. Themanywaysof Wntincer. CurrorOpinGenetDev. 2007.2 months; 17 (1): 45-51).

In view of the above, it would be desirable to develop new methods for the effective treatment of conditions and diseases that may be associated with β -catenin.

Summary of The Invention

Nefopam and analogues thereof have now been found to be beneficial in the treatment of β -catenin mediated disorders such as fibroproliferative disorders and in the treatment of scar tissue.

Accordingly, in one aspect of the present invention there is provided a method of treating a β -catenin-mediated disease or condition in a mammal, comprising administering nefopam, or a functionally equivalent analogue, prodrug, salt or solvate thereof, to the mammal.

In another aspect of the invention, a method of treating scar tissue or reducing scar tissue formation comprises administering to the tissue a therapeutically effective amount of nefopam, or a pharmaceutically acceptable analogue, salt, solvate or prodrug thereof.

In an alternative aspect, there is provided a product comprising packaging and a composition comprising nefopam or a functionally equivalent analogue, salt, solvate or prodrug thereof.

In another aspect, there is provided a novel use of nefopam or a functionally equivalent analogue, salt, solvate or prodrug thereof, for the manufacture of a medicament for the treatment of a β -catenin-mediated disease or condition or scar tissue.

Brief Description of Drawings

The invention will now be further described with reference to the following figures:

figure 1A is a bar graph showing the activity of cultured standard fibroblasts and cultured cells from two proliferative wounds measured using SRB assay after treatment with DMSO (control) or nefopam. Percent cell survival is given as mean and 95% confidence interval. There was a significant reduction in the percent of cell survival in cultures treated with nefopam compared to DMSO control proliferative wound cell cultures, however, cell survival in normal fibroblast cultures remained relatively unchanged (asterisks indicate significance compared to normal fibroblast cultures).

FIG. 1B is a Western blot analysis of β -catenin levels in proliferating wound cell cultures. Nefopam treated groups showed a substantial reduction in β -catenin levels compared to DMSO treated controls.

Fig. 2 is a graph comparing the number of invasive fibromatosis (AF) neoplasia in nefopam untreated (left) or treated or DMSO control male Apc +/Apc1638N mice, illustrating the number of epithelial-derived polyps in the upper gastrointestinal tract under the same treatment conditions. 1) Untreated (n ═ 11), 2) 0.1% DMSO (n ═ 10), and 3) nefopam (n ═ 10) at 40mg/kg body weight.

Figure 3A is a western blot (92kDa) of β -catenin levels in extracts (n ═ 5) from primary cell cultures of human invasive fibromatosis (AF) tumors after 5 days of treatment with either 0.1% DMSO (control) or nefopam. β -catenin levels were also determined in primary fibroblast cultures cultured with Wnt3a with or without nefopam. The experiments were performed in triplicate. Actin expression was expressed as a control in lysates.

Figure 3B is a graph of densitometric analysis of protein level data showing near five-fold reduction in total levels of β -catenin in cell cultures from human AF tumors treated with 0.1% DMSO (control) or nefopam. Shown as mean and 95% confidence interval. Statistically significant differences (p < 0.05) compared to the control group are indicated by asterisks.

Figure 4A is a graph showing the mean and 95% confidence intervals for cell activity from human AF tumor primary cells treated with DMSO (n-5) or nefopam (n-5) for 5 days. Cell viability was measured by staining cells with trypan blue dye and counting the number of all live (clear) and dead (blue) cells. Nefopam significantly reduced the number of live cells while the number of dead cells was unchanged. Statistically significant differences (p < 0.05) compared to the control group are indicated by asterisks.

Figure 4B is a graph showing the percentage of BrdU-positive/DAPI-positive cells compared to total DAPI-positive cells as a measure of proliferation in primary cell cultures derived from human invasive fibromatosis tumors (n-2) after treatment with DMSO or nefopam in triplicate for 5 days. Nefopam significantly reduced entry of BrdU into the cells. Shown as mean and 95% confidence interval. Statistically significant differences (p < 0.05) compared to the control group are indicated by asterisks.

FIG. 5A shows Western blot analysis extracted from immortalized human fibroblast lysates. A significant reduction in β -catenin levels was observed in nefopam treated cells compared to DMSO treated cells. GAPDH expression is expressed as a lysate internal control.

FIG. 5B is a graph of densitometry data consistent with the Western blot data of FIG. 5A.

FIG. 6A is a Western blot analysis of protein levels of β -catenin in cell cultures of wounds 14 days after wounding of Tcf mice. GAPDH expression is expressed as a lysate internal control.

Figure 6B is a graph of normal scar size in mice with full-thickness ring wounds treated with nefopam (nefopam) formulated with vehicle or vehicle only (control), treated by systemic administration of 40mg/kg daily for two weeks. The graph represents the mean and 95% confidence intervals for the surface area diameter of skin wounds produced using a 4mm biopsy punch. The diameter of the wound after nefopam treatment was significantly smaller relative to the control treatment (asterisks indicate significant differences).

Figure 7 is a line graph showing the relative protein levels of β -catenin compared to uninjured tissue after a period of time during normal wound healing (normal) and in proliferative wounds (hyperplasia). The normal pattern of elevation and decline of β -catenin protein levels during normal wound healing is dysregulated in proliferative wounds, demonstrating a significant prolongation of the time for which β -catenin levels are elevated.

Figure 8 is a graph of the mean and 95% confidence interval of the diameter of the surface area of the hypertrophic scar of the skin after four weeks of injury. A biopsy punch was used to create a full thickness annular wound of 4mm diameter. The wound diameter is given in mm. Asterisks indicate statistically significant differences in the size of the scars recorded (p < 0.01) when compared to TGF- β treatment, where injection of TGF- β when wounded is known to cause an increase in hypertrophic scar size.

Figure 9 is a graph of nefopam topical formulations at different concentrations in three different vehicles: carboxymethylcellulose (CMC), petrolatum and carboxypropylmethylcellulose. These three vectors were tested in vivo in a mouse model to determine the most effective formulation for delivery of nefopam across the skin. Formulations of the petrolatum matrix carrier were determined to exhibit enhanced nefopam release properties as measured by nefopam levels in skin and serum.

Figure 10 is a graph of relative scar surface area measured at arbitrary units (scar size after injury is considered 100 arbitrary units). Full-thickness puncture wounds of 4mm diameter were treated topically with either vehicle control cream or 1% nefopam cream formulated in a petrolatum vehicle, twice a day for 14 days. The data represent the mean and standard deviation of 10 wounds per treatment.

Detailed description of the preferred embodiments

There is provided a method of treating a β -catenin-mediated disease in a mammal comprising administering nefopam or a functionally equivalent analogue thereof to the mammal.

As used herein, the term "β -catenin-mediated disease or condition" refers to a disease or condition characterized by the accumulation of fibrous tissue ("fibrosis") including, but not limited to, fibroproliferative diseases such as epidermal scars, including hypertrophic scars, hypertrophic scars and keloids, which are at the stage of formation or have formed, and invasive fibromatosis, e.g., sporadic lesions or familial syndromes such as familial multiple adenocarcinoma (FAP), liver fibrosis, lung fibrosis (e.g., silicosis, asbestosis), kidney fibrosis (including diabetic nephropathy), glomerulosclerosis, tendinopathy (leddhoese disease) and Dupuytren's Contracture (DC), and malignant tumors such as colon cancer, colorectal cancer, melanoma, hepatocellular carcinoma, ovarian cancer, endometrial cancer, medulloblastoma, pilomoblastoma, and prostate cancer.

The term "nefopam" refers to 5-methyl-1-phenyl-1, 3,4, 6-tetrahydro-2, 5-tolyloxaxine and pharmaceutically acceptable functionally equivalent analogs, prodrugs, salts and solvates thereof. The term "functionally equivalent", as it is used with respect to analogs, prodrugs, salts and solvates of nefopam, refers to the ability of the selected compound to modulate β -catenin. The degree to which a selected compound can modulate β -catenin may vary from compound to compound.

The term "analog" as used herein refers to a compound having the following general formula (1),

wherein R is₁Is H, C₁-C₆Alkyl radicals, substituted by F or C₃-C₆Cycloalkyl or C₂-C₄Alkenyl is optionally substituted; a is O, CH₂Or S (O)_nWherein n is 0-2; w, X, Y and ZIs N, CH or CR₃And the others are CH; r₂Is C₅-C₆Heteroaryl group, C₅-C₁₀Cycloalkyl or cycloalkenyl, optionally containing one or more substituents selected from O, N and S (O)_nWherein n is 0-2 and is substituted by R₃Optionally substituted; or phenyl in one or more positions is selected from halogen, CN, CF₃、C₁-C₆Alkyl and OR₁Optionally substituted or the phenyl group is fused to a five or six membered ring which may be carbocyclic, heterocyclic (containing 1-2 heteroatoms selected from O, N and S), aromatic or heteroaromatic (containing 1-2 heteroatoms selected from O and N); r₃Selected from halogens; CF (compact flash)₃；CN；OR₅；SO₂N(R₅)₂；COR₅；CO₂R₅；CON(R₅)₂；NR₁COR₄；NR₁SO₂R₄；NR₁CO₂R₄；NR₁CON(R₅)₂(ii) a With R₃Substituted OC₁-C₆An alkyl group; unsubstituted R₃Optionally substituted C₁-C₆An alkyl group; unsubstituted R₃Optionally substituted C₃-C₆A cycloalkyl group; unsubstituted R₃Optionally substituted C₂-C₆An alkenyl group; unsubstituted R₃Optionally substituted C₂-C₆An alkynyl group; unsubstituted R₃Optionally substituted aryl; and a five or six membered aromatic heterocyclic ring containing 1 to 4 heteroatoms selected from N and O; r₄Is C₁-C₆Alkyl radical, C₂-C₆Alkenyl radical, C₂-C₆Alkynyl, C₃-C₆Cycloalkyl, aryl or heteroaryl; and R₅Is H, C₁-C₆Alkyl radical, C₂-C₆Alkenyl radical, C₂-C₆Alkynyl, C₃-C₆Cycloalkyl, aryl or heteroaryl with another R₅The same or different; or a pharmaceutically acceptable salt thereof; wherein R is₁Is H, C₁-C₆Alkyl radicals, substituted by F or C₃-C₆Cycloalkyl or C₂-C₆Alkenyl is optionally substituted; r₂And R₃Identical or different and are H, halogen, CN, CF₃、C₁-C₆Alkyl OR OR₁Or R is₂And R₃Forming a five or six membered ring, which may be carbocyclic, heterocyclic (containing 1-2 heteroatoms selected from O, N and S), aromatic or heteroaromatic (containing 1-2 heteroatoms selected from O and N); w, X, Y and one of Z is N or CR₄And the others are each CH; r₄Is a halogen atom, CF₃、CN、OR₇、SO₂N(R₆)₂、COR₆、CO₂R₆、CON(R₆)₂、NR₁COR₅、NR₁SO₂R₅、NR₁CO₂R₅、NR₁CON(R₆)₂Quilt R₄Optionally substituted OC₁-C₆Alkyl radical, by R₄Optionally substituted C₁-C₆Alkyl radical, by R₄Optionally substituted C₃-C₆Cycloalkyl radicals, by R₄Optionally substituted C₂-C₆Alkenyl, by R₄Optionally substituted C₂-C₆Alkynyl, by R₄An optionally substituted aryl or a five or six membered aromatic heterocyclic ring containing 1 to 4 heteroatoms selected from N and O, linked through carbon or nitrogen; r₅Is C₁-C₆Alkyl radical, C₂-C₆Alkenyl radical, C₂-C₆Alkynyl, C₃-C₆Cycloalkyl, aryl or heteroaryl; each R₆(they may be the same or different) is H, C₁-C₆Alkyl radical, C₂-C₆Alkenyl radical, C₂-C₆Alkynyl, C₃-C₆Cycloalkyl, aryl or heteroaryl; and R is₇Is aryl or heteroaryl; or a pharmaceutically acceptable salt thereof; wherein R is₁Is H, C₁-C₆Alkyl, by F or C₃-C₆Cycloalkyl or C₂-C₄Alkenyl is optionally substituted; a is O, CH₂Or S (O)_nWherein n is 0-2; w, X, Y and Z is N, CH or CR₃And the others are CH; r₂Is C₅-C₆Heteroaryl group, C₅-C₁₀Cycloalkyl or cycloalkenyl, optionally containing one or more substituents selected from O, N and S (O)_nWherein n is 0-2 and is substituted by R₃Optionally substituted; or phenyl in one or more positions is selected from halogen, CN, CF₃、C₁-C₆Alkyl and OR₁Optionally substituted or the phenyl group is fused to a five or six membered ring which may be carbocyclic, heterocyclic (containing 1-2 heteroatoms selected from O, N and S), aromatic or heteroaromatic (containing 1-2 heteroatoms selected from O and N); r₃Selected from halogens; CF (compact flash)₃；CN；OR₅；SO₂N(R₅)₂；COR₅；CO₂R₅；CON(R₅)₂；NR₁COR₄；NR₁SO₂R₄；NR₁CO₂R₄；NR₁CON(R₅)₂(ii) a With R₃Substituted OC₁-C₆An alkyl group; unsubstituted R₃Optionally substituted C₁-C₆An alkyl group; unsubstituted R₃Optionally substituted C₃-C₆A cycloalkyl group; unsubstituted R₃Optionally substituted C₂-C₆An alkenyl group; unsubstituted R₃Optionally substituted C₂-C₆An alkynyl group; unsubstituted R₃Optionally substituted aryl; and a five or six membered aromatic heterocyclic ring containing 1 to 4 heteroatoms selected from N and O; r₄Is C₁-C₆Alkyl radical, C₂-C₆Alkenyl radical, C₂-C₆Alkynyl, C₃-C₆Cycloalkyl, aryl or heteroaryl; and R₅Is H, C₁-C₆Alkyl radical, C₂-C₆Alkenyl radical, C₂-C₆Alkynyl, C₃-C₆Cycloalkyl, aryl or heteroaryl with another R₅The same or different; or pharmaceutically acceptable thereofSalt; wherein R is₁Is H, C₁-C₆Alkyl, by F or C₃-C₆Cycloalkyl or C₂-C₄Alkenyl is optionally substituted; a is O, CH₂Or S (O)_nWherein n is 0-2; w, X, Y and Z is N, CH or CR₃And the others are CH; r₂Is C₅-C₆Heteroaryl group, C₅-C₁₀Cycloalkyl or cycloalkenyl, optionally containing one or more substituents selected from O, N and S (O)_nWherein n is 0-2 and is substituted by R₃Optionally substituted; or phenyl in one or more positions is selected from halogen, CN, CF₃、C₁-C₆Alkyl and OR₁Optionally substituted or the phenyl group is fused to a five or six membered ring which may be carbocyclic, heterocyclic (containing 1-2 heteroatoms selected from O, N and S), aromatic or heteroaromatic (containing 1-2 heteroatoms selected from O and N); r₃Selected from halogens; CF (compact flash)₃；CN；OR₅；SO₂N(R₅)₂；COR₅；CO₂R₅；CON(R₅)₂；NR₁COR₄；NR₁SO₂；NR₁CO₂R₄；NR₁CON(R₅)₂(ii) a With R₃Substituted OC₁-C₆An alkyl group; unsubstituted R₃Optionally substituted C₁-C₆An alkyl group; unsubstituted R₃Optionally substituted C₃-C₆A cycloalkyl group; unsubstituted R₃Optionally substituted C₂-C₆An alkenyl group; unsubstituted R₃Optionally substituted C₂-C₆An alkynyl group; unsubstituted R₃Optionally substituted aryl; and a five or six membered aromatic heterocyclic ring containing 1 to 4 heteroatoms selected from N and O; r₄Is C₁-C₆Alkyl radical, C₂-C₆Alkenyl radical, C₂-C₆Alkynyl, C₃-C₆Cycloalkyl, aryl or heteroaryl; and R₅Is H, C₁-C₆Alkyl radical, C₂-C₆Alkenyl radical, C₂-C₆Alkynyl, C₃-C₆Cycloalkyl, aryl or heteroaryl with another R₅The same or different; or a pharmaceutically acceptable salt thereof; or wherein: r₁Is H, C₁-C₆Alkyl, by F or C₃-C₆Cycloalkyl or C₂-C₄Alkenyl is optionally substituted; r₂And R₃Are identical or different and are each H, halogen, CN, CF₃、C₁-C₆Alkyl OR OR₁Or R is₂And R₃Forming a five or six membered ring, which may be carbocyclic, heterocyclic (containing 1-2 heteroatoms selected from O, N and S), aromatic or heteroaromatic (containing 1-2 heteroatoms selected from O and N); and W, X, Y and one of Z is N, CH or CR₄And the others are CH; r₄Is halogen; CF (compact flash)₃；CN；OR₇；SO₂N(R₆)₂(wherein each R is₆Same or different), COR₆、CO₂R₆、CON(R₆)₂(wherein R is₆Same or different), NR₁COR₅、NR₁SO₂R₅、NR₁CO₂R₅、NR₁CON(R₆)₂(wherein each R is₆Same or different), unsubstituted R₄Optionally substituted OC₁-C₆Alkyl, unsubstituted R₄Optionally substituted C₁-C₆Alkyl, unsubstituted R₄Optionally substituted C₃-C₆Cycloalkyl, unsubstituted R₄Optionally substituted C₂-C₆Alkenyl, unsubstituted R₄Optionally substituted C₂-C₆Alkynyl, unsubstituted R₄Optionally substituted aryl, or R₄Is a five or six membered aromatic heterocyclic ring containing 1 to 4 heteroatoms selected from N and O; r₅Is C₁-C₆Alkyl radical, C₂-C₆Alkenyl radical, C₂-C₆Alkynyl, C₃-C₆Cycloalkyl, aryl or heteroaryl; r₆Can be H, C₁-C₆Alkyl radical、C₂-C₆Alkenyl radical, C₂-C₆Alkynyl, C₃-C₆Cycloalkyl, aryl or heteroaryl; and R is₇Is aryl or heteroaryl; or a pharmaceutically acceptable salt thereof. Other analogues of nefopam are described in WO2004/056788, WO2005/103019 and US2006/0019940, the contents of which are incorporated herein by reference. Nefopam and analogues thereof can be prepared using chemical synthesis methods well known in the art. Furthermore, nefopam is commercially available.

The term "prodrug" refers to a compound (e.g., prodrug) that is converted in vivo to produce a compound having the structure nefopam or a pharmaceutically acceptable analog, salt, hydrate, or solvate thereof. This conversion may occur by various mechanisms (e.g., by metabolic or chemical processes), such as, for example, by hydrolysis in the blood. The term "salt", as used herein, means acid salts formed with inorganic and/or organic acids, and base salts formed with inorganic and/or organic bases. Pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salts are preferred, although other salts may also be used. A "solvate" is formed by mixing nefopam or an analogue thereof in a solvent, preferably a pharmaceutically acceptable solvent.

The method includes the treatment of a beta-catenin-mediated disease in a mammal. The terms "treat", "treating" and "treatment" are used broadly herein to refer to methods of favorably altering a targeted disease, including moderating or reversing the disease process, reducing the severity of the disease, preventing or curing the disease. The term "mammal" is used herein to include both human and non-human mammals.

Also provided is a method of treating epidermal scars, including scars resulting from cuts, abrasions, infections, acne, burns, surgery, and the like, hypertrophic scars, keloids, scars including stroma and cells derived from stroma, any of which may or may not be beta-catenin-mediated. The method comprises administering a therapeutically effective amount of nefopam compound to a target site. Methods of treating a scar that is developing or has developed include reducing the size of the scar (e.g., at least about 5-10%, preferably at least about 20% and more preferably at least about 25% or more) or the prevalence of the scar (e.g., height of the scar, redness, etc.) and thereby improving its appearance. In this regard, one skilled in the art will appreciate that scar assessment scales, such as the Manchester scale, may be used to assess the improvement of a particular scar. The Manchester scale evaluates the color, matte or shiny appearance, appearance (flushing of surrounding skin to scar/pimple), texture (normal to hard), margin (clear or unclear), size and number (single or multiple) compared to surrounding skin (Disability & Rehabilitation, 2009, volume 31, phase 25: 2055-.

Thus, nefopam compounds may be used in cosmetic treatment to reduce scar tissue and improve the appearance of the scar and surrounding area, and may provide additional cosmetic features, such as an anti-wrinkle effect.

In another embodiment, a method of treating a tumor is provided. Tumor therapy includes inhibition of tumorigenesis and tumor cell proliferation. The method is effective for tumors from dysregulated β -catenin expression, such as invasive fibromatosis, and tumors from various cancers, such as colon, melanoma, hepatocellular carcinoma, ovarian, endometrial, and prostate. The method comprises administering to a mammal in need of treatment, i.e., a mammal having a tumor, an effective amount of nefopam, an analog thereof, or a pharmaceutically acceptable salt, solvate or prodrug thereof.

While not wishing to be bound by any particular theory, treatment according to the present invention may be achieved by modulating or modulating β -catenin expression at the nucleic acid level, or β -catenin activity at the protein level.

Administering to a mammal a therapeutically effective dose of nefopam according to the present invention. The term "therapeutically effective" as it is used herein for dosages refers to dosages effective to treat beta-catenin-mediated diseases without causing undesirable adverse side effects. The term "administering" refers to any suitable method of providing nefopam to a mammal, and will depend on the dosage form to be used as described. For example, the dose may be administered orally, by injection, mucosally, and topically, as will be described in more detail.

Thus, a therapeutically effective dose according to the method is in the range of about 0.0001 to about 1500mg, for example, in the range of about 0.0001-100 mg. However, as will be appreciated by those skilled in the art, the therapeutically effective amount of nefopam or an analogue thereof will vary depending on a number of factors including, but not limited to, the type of disease to be treated, the nature and severity of the disease, the mammal to be treated, the symptoms of the mammal being treated, the compound used for treatment, and the route of administration.

Nefopam can be administered in a composition according to the methods of the invention, alone or in combination with a pharmaceutically acceptable adjuvant or carrier. The term "pharmaceutically acceptable" means acceptable for use in the pharmaceutical arts, i.e., without unacceptable toxicity, or otherwise unsuitable for administration to a mammal. Examples of pharmaceutically acceptable excipients include, but are not limited to, diluents, excipients, and the like. Reference may be made to "Remington's: the science and practice of pharmacy, 21 edition, lippincott williams & Wilkins, 2005, which is commonly used as a guide for pharmaceutical preparations. The choice of excipients depends on the desired mode of administration of the composition. In one embodiment of the invention, the compounds are formulated for administration by infusion, or subcutaneous or intravenous injection, and are accordingly used as aqueous solutions in sterile and pyrogen-free form and optionally buffered or made isotonic. Thus, the compound may be administered in distilled water or, more desirably, in saline, phosphate buffered saline or a 5% dextrose solution. Compositions for oral administration in the form of tablets, capsules, lozenges, solutions or suspensions in aqueous or non-aqueous liquids, oil-in-water or water-in-oil liquid emulsions, elixirs or syrups are prepared using excipients which include sugars, such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives, including sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate; powdered gum tragacanth; malt; gelatin; talc powder; stearic acid; magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil and corn oil; polyols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; agar; alginic acid; water; isotonic saline and phosphate buffer. Wetting agents, lubricants such as sodium lauryl sulfate, stabilizers, tableting agents, disintegrants, antioxidants, preservatives, colorants and flavoring agents may also be present. In another embodiment, the composition for topical administration may be formulated as a cream, lotion, or ointment. For such topical administration, the composition may include a suitable base such as a triglyceride base. The cream, lotion or ointment may also contain surfactants and other cosmetic additives such as skin softeners and the like as well as fragrances. Aerosols, e.g. for nasal administration, may also be prepared, wherein a suitable propellant vehicle is used. The compositions of the present invention may also be administered as a bolus, electuary or paste. Compositions for mucosal administration, including oral, nasal, rectal or vaginal administration, may also be included to treat infections affecting these areas. The compositions generally include one or more suitable non-irritating excipients or carriers including, for example, cocoa butter, polyethylene glycol, suppository wax, salicylic acid, or other suitable carriers. Other adjuvants may also be added to the composition without regard to how it is to be administered, for example, may help to extend its shelf life.

According to the methods of the present invention, nefopam compounds may be administered in a convenient manner by a number of routes including, but not limited to, oral, subcutaneous, intravenous, intraperitoneal, intranasal, enteral, topical, sublingual, intramuscular, intraarterial, intramedullary, intrathecal, inhalation, ophthalmic, transdermal, vaginal or rectal means. Nefopam compounds can also be administered to cells in an in vitro treatment regimen. Depending on the route of administration, the nefopam compound may be coated or encased in a protective material to avoid degradation, for example by enzymes, acids or other conditions that may affect its therapeutic activity.

In one embodiment, nefopam, or an analogue thereof, may be applied topically to the target site, for example, a scar being formed or already formed, which is attached to a biocompatible device, polymer or other substrate, such as a bandage, dressing (dressing), polymeric mesh, implant, device or other cosmetically-related product. The bioengineered epidermal fibroblasts/keratinocytes expressing nefopam compounds may also be applied to the target site. A suitable matrix or polymeric mesh, such as an artificial or non-artificial skin graft, may be selectively impregnated in the nefopam compound for the target site to achieve a slow release of the compound over a period of time for sustained treatment of the target site.

The nefopam compounds of the present invention may be administered in controlled release formulations using established methods, including, for example, monolithic devices (monolithichevice) controlled by dissolution or diffusion, microbead embedded systems, osmotic pressure control systems, and modified membrane coating systems incorporating suitable polymeric and non-polymeric hydrophilic and hydrophobic materials. Suitable controlled release formulations may include hydrophilic materials including, but not limited to, acrylic or methacrylic acid polymers or copolymers, alkyl vinyl polymers, cellulose, hydroxyalkyl cellulose, carboxyalkyl cellulose, polysaccharides, alginates, pectins, starches and derivatives, natural and artificial gums, polycarbophil, chitosan. Suitable hydrophobic materials include, but are not limited to, hydrophobic polymers, waxes, fats, long chain fatty acids, their corresponding esters, their corresponding ethers, and mixtures thereof.

In another embodiment, the nefopam compound may be administered in combination with one or more additional therapeutic agents, including, for example, an anti-scarring agent; wound healing agents such as growth factors, e.g., epidermal growth factor, bFCF, PDGF; platelets, epidermal fibroblasts, and keratinocytes; chemotherapeutic agents such as, but not limited to, rapamycin, troglitazone, rosiglitazone, celecoxib, retinoids, and iressa. In this regard, nefopam may be administered in a separate formulation, or in a combined formulation with an additional therapeutic agent.

In addition, the methods of the present invention can be used in combination with other treatments, for example, in combination with radiation therapy in the treatment of malignant tumors, or in combination with laser therapy to treat scar tissue such as normal scars, hypertrophic scar tissue, and the like.

In another aspect of the invention, there is provided an article of manufacture comprising packaging and a composition comprising nefopam as described herein. The package may be labeled to indicate that the composition is suitable for use in treating a beta-catenin-mediated disease, or may be labeled to indicate that the composition is suitable for use in treating a developing or developed scar.

The invention is described by reference to the drawings and specific embodiments, which are not to be construed as limiting.

Examples

The following materials and methods were used in the examples discussed below.

Apc⁺/Apc^1638NMouse models of AF and methods of treatment. The generation and phenotype of Apc/Apc1638N mice are well characterized. These mice harbor the targeted variation in codon 1638 of the Apc gene as a result of neomycin insertion in the antisense primer localization of exon 15. Male mice developed an average of 45 AF lesions and 6 gastrointestinal polyps by age 6 months, while female mice developed significantly lower numbers of AF lesions. Male Apc/Apc1638N mice were divided into three study groups: untreated (n-11), 0.1% DMSO (n-10), and nefopam (n-10) at a dose of 40mg/kg body weight. Weaning of Apc/Apc1638N mice 2 months later by daily passage was initiatedOral gavage was performed for 3 months. At the time of dissection, AF tumors and intestinal polyps were scored visually. AF tumor and normal tissues were collected for protein extraction and solidified for histological examination.

Tcf reporter mice and wound assays. A Tcf-reporter gene containing the downstream region of the lacZ gene of the c-Fos minimal promoter and three consensus Tcf-binding motifs was constructed. After the β -catenin/Tcf complex binds to the Tcf motif, lacZ expression is activated. Tcf mice were wounded as described previously: two full-thickness skin wounds of 4mm diameter were created using an epidermal biopsy punch (miltex instruments company, York, PA, USA). Wounded Tcf mice were divided into two study groups: a control group, which received daily intraperitoneal injections of saline; and nefopam group, which received intraperitoneal injections of 40mg/kg body weight daily. After 14 days of injury, the wound size was examined and wound tissue was collected for RNA and protein extraction and solidified for histological examination.

Human AF tumor and normal fascia tissue samples. Samples of human invasive fibromatosis tumors were obtained at the time of surgery by hospitale for rick children, Toronto. Tumor tissue and surrounding normal fascia tissue from the same patient were collected and processed immediately after surgical resection. The tissues were cryopreserved and stored under liquid nitrogen.

Cell culture studies. Primary cell cultures from human AF tumor and normal fascia tissue samples were established. Monolayer cell cultures were cultured in DMEM supplemented with 10% fetal bovine serum and incubated at 37 ℃ in 5% CO₂And (4) maintaining. Cells were split when fused and experiments were performed between the first and fifth passages. Prior to experimental studies, cells were seeded overnight and treatment was started on the following day (day 0), where cells were treated with media control 0.1% DMSO with or without 250 μm nefopam prepared in DMEM medium.

Cell activity, proliferation and apoptosis assays were performed. Cell activity was measured using the trypan blue dye exclusion method. Cells were stained with trypan blue dye at a 1: 1 ratio and all live (clear) and dead (blue) cells were counted. Proliferation was measured using the 5-bromo-2-deoxy-uridine (BrdU) incorporation method. BrdU-incorporated cells were identified using rabbit monoclonal anti-BrdU antibodies and horse anti-mouse antibodies conjugated to alkaline phosphatase after 12 hours incubation of BrdU. The presence of BrdU was detected using alkaline phosphatase substrate. The percentage of positively stained nuclei in total nuclei was analyzed in a 10-fold high field.

Protein extraction and western blot analysis. Tissue samples were washed twice with PBS and lysed with reporter gene assay lysis buffer (Roche). Lysates were centrifuged at 16,000Xg for 5 minutes to remove cell debris and quantified using 2, 2' -bicinchoninic acid (BCA) protein assay (Pierce). Equal amounts of total protein were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane (Amersham), and immunoblotted overnight at 4 ℃ with primary antibodies directed against phosphoGSK3 β (Ser9, rabbit polyclonal, new englandbiolabs), β -catenin (mouse monoclonal, UpstateBiotechnology), total GSK3 β (mouse monoclonal, transduction laboratories), and GAPDH (mouse monoclonal, UpstateBiotechnology). Horseradish peroxidase (HRP) -labeled secondary antibodies and enhanced chemiluminescence (Amersham) were used to detect hybridization. Optical density measurements were performed using alphaeasef fc software (AlphaInnotech). Three western blots were performed to ensure reproducibility.

And (5) carrying out statistical analysis. Data in this work are presented as mean ± 95% confidence intervals. All studies were performed at least three times to ensure reproducibility.

Example 1: nefopam treatment reduced proliferative wound cell activity.

Compounds were screened to identify that they met two criteria: 1) inhibiting cellular activity of fibroblasts obtained from a proliferative wound, which exhibit β -catenin activation; and 2) exhibit little to no effect on normal epidermal fibroblast culture. The biological relevance of the screen is important because the cells used for screening are from patients with hyperplastic wounds and healthy tissue. The experiment was repeated three times in 96-well plates, each containing 4000 cells, treated with between 0.11.0 or 10 μ M compound or DMSO as control. Sulforhodamine b (SRB) assay was used to measure cellular activity. Compounds detected in the initial screen were further tested using samples of a larger pool from which nefopam was determined (see figure 1A).

Western blot analysis was used to analyze β -catenin levels from cell cultures of nefopam or control treated hypertrophic scars. Nefopam was observed to substantially reduce the protein levels of β -catenin in cell cultures from proliferative wounds (see figure 1B). GAPDH expression was included as an internal control.

Example 2: nefopam reduced the number of AF tumors formed in Apc/Apc1638N mice.

It was investigated whether nefopam treatment could modulate the phenotype of AF damage in vivo. At 6 months of age, the number of AF tumors formed was significantly reduced in nefopam treated male Apc/Apc1638N mice compared to untreated mice or mice treated with 0.1% DMSO (8.18 ± 1.77vs13.2 ± 2.30 or 12.09 ± 1.31, p < 0.03, see figure 2). There was no significant difference in the number of polyps from the epithelium in the upper segment of the gastrointestinal tract (see fig. 2). This indicates that nefopam inhibits tumorigenesis and is also specific for mesenchymal cells.

Example 3: nefopam reduces the β -catenin level of human AF tumor cells.

AF tumors are characterized by elevated levels of beta-catenin. To examine whether nefopam has the ability to modulate β -catenin levels, primary cell cultures from several human AF tumors were investigated. Western blot analysis using antibodies against total β -catenin demonstrated a significant reduction in the amount of 92kDa protein, consistent with total β -catenin, as a result of 5 days of nefopam treatment, see figure 3A. Densitometric analysis showed that total β -catenin levels were reduced by nearly 5-fold in nefopam-treated human AF tumor cell cultures compared to those treated with 0.1% DMSO (see figure 3B). Actin expression was measured as a lysate internal control.

Example 4: nefopam decreases cell activity and cell proliferation in human AF tumor cells.

To determine how nefopam can alter AF cell behaviour, primary cell cultures from several human AF tumors were investigated. First, the effect of nefopam on cell activity in human AF tumors was investigated. A significant reduction in the number of viable cells was observed for the human AF tumor cell cultures after nefopam treatment compared to cultures treated with 0.1% DMSO (p < 0.05). There was no significant difference in the number of dead cells counted as a result of nefopam treatment on the tumors (p < 0.05) (see figure 4A).

The effect of nefopam on primary cell culture proliferation was studied after demonstrating that β -catenin levels are involved in the modulation of proliferation rates in mesenchymal cells. The percentage of BrdU +/DAPI + cells compared to total DAPI + cells was measured using the BrdU incorporation method. It was observed that nefopam treated human AF tumors contained significantly fewer proliferating cells as determined by BrdU incorporation (p < 0.05, see figure 4B).

Taken together, these results indicate that nefopam preferentially inhibits the number of active AF cells by reducing the proliferation rate.

Example 5: nefopam reduces β -catenin levels in human primary fibroblast cultures.

Proliferative wounds are characterized by elevated levels of β -catenin during the proliferative phase. The data described herein indicate that nefopam is able to modulate β -catenin levels, particularly in cells from the stroma. To demonstrate that nefopam is able to modulate β -catenin levels in mesenchymal cells, immortalized human fibroblasts were treated with nefopam (see figure 5A). Nefopam treatment resulted in an approximately 4-fold reduction in total β -catenin levels in human primary fibroblast cultures as determined by densitometry analysis compared to 0.1% DMSO-treated cultures (p < 0.05, see fig. 5B). Additional controls included in the experiment were Wnt3 a-treated cells (known to increase β -catenin expression) and nefopam effect on cells treated with Wnt3 a.

Example 6: systemic administration of nefopam reduced β -catenin levels and wound size in Tcf mice.

The wound tissue from Tcf mice was then studied by examining the effect of nefopam on β -catenin levels during wound healing. Creating a skin wound using a biopsy punch procedure resulted in a 4mm diameter full thickness annular wound. Measurements were made in mm. Western blot with antibodies against total β -catenin (see figure 6A) showed a reduction in β -catenin levels in cells cultured from Tcf mouse wounds treated with nefopam 14 days after wounding compared to the control group.

In addition, wound examination after dissection showed nefopam treated mice had scars significantly smaller in diameter (asterisks indicate significant difference, p < 0.001) compared to vehicle (saline) treated controls after 14 days of injury (see figure 6B).

Example 7: systemic administration of nefopam reduces TGF- β induced hypertrophic scar size.

β -catenin levels are known to increase early in wound healing and subsequently decrease throughout late stages relative to uninjured tissue. The normal increase and decrease in β -catenin levels during proliferative wound healing is dysregulated, with a significant prolonged increase in β -catenin levels being observed (see figure 7).

Medicine in medicineFollowing the screening study, mice were used for in vivo experiments of nefopam effect. The oral and intraperitoneal routes of administration were evaluated (40mg/kg body weight per day; 0.1% DMSO as a control). Nefopam was identified in serum by detection using HPLC in all routes of administration (data not disclosed). A 4mm full-thickness puncture wound was made on the skin and nefopam or control was administered daily after injury. To determine whether nefopam was effective in treating hypertrophic scars, a mouse hypertrophic scar model was used in which TGF-once was injected prior to injury creating hypertrophic scarsImportantly, the same nefopam treatment method as described above, compared to without TGF-The treated control scar resulted in smaller scars (see figure 8). Nefopam is therefore able to reduce scar size in all hyperplastic and normal wound repairs.

Example 8: various carriers can be used for local delivery of nefopam.

For skin wounds, the ideal product is a topical dosage form of nefopam. Topical formulations of nefopam were prepared and evaluated using the following vehicle: carboxymethylcellulose (CMC), petrolatum and carboxypropylmethylcellulose. The three vehicles were tested in vivo to determine that the formulation was effective in delivering nefopam through the skin. The results are illustrated in fig. 9.

Example 9: topical administration of nefopam reduced the size of normal scars in mice.

Skin wounds were created in Tcf mice using the biopsy punch procedure described above. A 6mm diameter full-thickness puncture wound was topically treated twice daily with either a control cream or a 1% nefopam cream for up to 21 days. Nefopam treatment was observed to reduce scar size by about one-third compared to the control group. Table 1 shows the average size (measured in mm) of normal wounds in a mouse model 21 days after topical application of 1% nefopam cream formulated in a petrolatum vehicle or a control cream of vehicle alone on day 0 and daily. The average size of 4 wounds per group is provided.

Table 1.

		Day 0	Day 21
				Control	Average size of wound (mm)	6	3.116666667
1% nefopam	Average size of wound (mm)	6	2.02

A 4mm puncture wound was created in Tcf mice, which were then topically treated twice daily with either 1% nefopam cream or control cream for up to 14 days. The surface area of the scar formed after 14 days of treatment was measured using arbitrary units (arbitraryunits), where 100 arbitrary units represent treatment of the control cream. Ten wounds were measured for each treatment and data are presented as mean and standard deviation. It was observed that the scar on mice receiving nefopam treatment was significantly smaller than those receiving control treatment (p < 0.05) where the control treatment was 0% nefopam (see figure 10).

While the invention has been described with reference to illustrative embodiments and examples, this description is not intended to be construed in a limiting sense. Accordingly, various modifications of the illustrative embodiments, as well as other embodiments of the invention, will be apparent to persons skilled in the art upon reference to this description. It is therefore contemplated that the appended claims will cover any such modifications or embodiments. Further, all claims are hereby incorporated into the description of the preferred embodiments by reference.

Any publications, patents and patent applications mentioned herein are incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

Claims (14)

1. Use of nefopam, or a salt thereof, that is effective to modulate β -catenin in the manufacture of a medicament for the treatment of scar tissue or to reduce scar tissue formation.

2. Use according to claim 1, wherein the scar tissue is associated with fibromatosis or hypertrophic diseases.

3. Use according to claim 1, wherein the amount of scar tissue is reduced and/or the appearance of scar tissue is improved.

4. Use according to claim 1, wherein the scar is selected from the group consisting of hypertrophic scars, surgical scars, scars resulting from cuts or abrasions, scars resulting from infections, scars resulting from acne or burn scars.

5. The use of claim 1, wherein the medicament is for topical administration.

6. The use of claim 5, wherein the medicament comprises a nefopam compound applied to a biocompatible matrix.

7. Use according to claim 6, wherein the biocompatible matrix is selected from the group consisting of a dressing, a bandage, an implant, a device, a bioengineered epidermal fibroblast or keratinocyte and a polymer matrix.

8. Use according to claim 7, wherein the biocompatible matrix is a modified membrane coating system mixed with a hydrophilic material selected from acrylic or methacrylic polymers or copolymers, alkyl vinyl polymers, polysaccharides, or polycarbophil, or a hydrophobic material selected from hydrophobic polymers, waxes, fats, long chain fatty acids, esters of long chain fatty acids, ethers of long chain fatty acids and mixtures of acrylic or methacrylic polymers or copolymers, alkyl vinyl polymers, polysaccharides, polycarbophil, hydrophobic polymers, waxes, fats, long chain fatty acids, esters of long chain fatty acids and ethers of long chain fatty acids.

9. Use according to claim 8, wherein the polysaccharide is selected from the group consisting of cellulose, alginate, pectin, starch, natural and artificial gums or chitosan.

10. Use according to claim 9, wherein the cellulose is selected from hydroxyalkyl celluloses, carboxyalkyl celluloses.

11. The use of claim 1, wherein the medicament comprises one or more additional therapeutic agents.

12. The use of claim 11, wherein the additional therapeutic agent is selected from the group consisting of an anti-scarring agent, a wound-healing agent, and a chemotherapeutic agent.

13. The use of claim 1, wherein the medicament comprises a pharmaceutically acceptable adjuvant or carrier.

14. The use of claim 1, wherein the medicament is prepared in a dosage form in a range of 0.0001 to 1500 mg.