Antisense oligomers for treating Androgenetic Alopecia

[Dave001]

A couple of weeks ago, I found an interesting new study published on the use of antisense oligomers for treating local androgen dependent disorders when searching Medline. Abstract is below.

Even more interesting, is that yesterday, while searching through some old posts in Google's archive, I came across a message from Dr. Proctor, in which he mentioned a patent application by a San Diego based company for treating male pattern baldness using nucleoside oligomers. I searched the U.S. patent database and found the patent to which he referred: "Compositions and methods of treatment of androgen-associated baldness using antisense oligomers." Harper, et al. Patent 5,877,160. March 2, 1999.
Assignee: Genta Incorporated (San Diego, CA)
Appl. No.: 483464
Filed: June 7, 1995

Edit: I forgot to include a link to the patent. Here is the URL:
http://patft.uspto.gov/netacgi/nph-Pars ... er=5877160

Granted, it is unlikely that there is any relationship between the U.S patent holder and the _German _ study authors below, but it may be evidence that this particular method of treatment is being actively investigated and pursued. Remember what Dr. P has said many times: the rule of thumb is for companies to patent, rather than publish anything they may be working on.

For those of you whom are unfamiliar with the concept of using antisense oligomers, it is basically what you would call "gene therapy," in the present context. In this case, the objective is to inhibit or alter expression of the androgen receptor or 5-alpha-reductase by manipulating upstream gene coding.

Abstract

Exp Dermatol. 2005 Feb;14(2):156.

Control of androgen receptor expression in human keratinocytes and in a reconstituted human epidermis model with selective antisense oligonucleotides.

Fimmel S, Bonte F, Kurfurst R, Zouboulis C.

Department of Dermatology, Campus Benjamin Franklin, Charite-Universitaetsmedizin Berlin, Berlin, Germany.

Association of locally increased androgen activity and skin disorders is obvious in acne and androgenetic alopecia in males. In addition, testosterone was unexpectedly found to perturb the epidermal barrier. Blockade of androgen action via androgen receptor (AR) antagonism accelerates wound healing in aged individuals. Androgen activity on skin can classically be inhibited by systemic administration of compounds, which have strong affinity for AR and antagonize androgen binding to AR molecules. In this study we applied a new technology to realize the same purpose: We tested the activity of antisense oligonucleotides against the AR in primary human foreskin keratinocytes, human non-foreskin keratinocytes from young (30 y) and older (60 y) female donors, and reconstituted human epidermis (SkinEthic model). Reconstituted human epidermis is similar to in vivo human epidermis and features a functional permeability barrier. To transfer the antisense oligonucleotides into human keratinocytes an optimum liposome-mediated transfection system with Poly-L-ornithine (12 mug/ml) over 4 h was used. The transfection efficiency was assessed using FITC-labeled (ACTG)(5) random oligonucleotides, which were localized in cytoplasmic structures of the keratinocytes. AR expression on the protein level was investigated by Western blotting. Transient transfection of foreskin keratinocytes with phosphorothioate antisense oligonucleotides (PTO) revealed a reduction of AR expression ( approximately 25%) compared to native keratinocytes after 14 h recovery time. The AR knock down in epidermal keratinocytes of the compared women was stronger in the older, more differentiated keratinocytes. After 24 h, AR expression level have returned back to the level of non-transfected cells. The effect could be reestablished by repetition of transfection. PTO and 2?O-methylribosyl (MRO) antisense oligonucleotides decreased AR expression at levels varying between 46% and 70% in the air-lifted reconstituted human epidermis after 18 h recovery time. The successful inhibition of AR expression in human keratinocytes and reconstituted human epidermis is the first step to develop topically efficient compounds with oligonucleotides.

PMID: 15679604

 

[thin=depressed]

Here's a nice complementary patent fairly unknown and interesting to compare the gene effect trying to be established.

Cell growth accelerator and cell growth method using the same

United States Patent: 6,333,193

Inventors: Shiba; Toshikazu (Sapporo, JP)
Assignee: Nipro Corporation (Osaka, JP)

Appl. No.: 384384
Filed: August 27, 1999

Foreign Application Priority Data: Aug 28, 1998[JP] (10-242416); Sep 24, 1998[JP] (10-288869)




Abstract

Provided are a cell growth accelerator containing a polyphosphoric acid and a cell growth method in which cells are grown by adding the cell growth accelerator to a medium for cells of animals and plants or by using a glass or plastic cell cultivator containing or coated with the cell growth accelerator. When substances are produced in vitro using culture cells, the cell culture and the cell growth acceleration are conducted at low cost safely and efficiently by adding the cell growth accelerator containing the polyphosphoric acid to the medium for cells of animals and plants or by using the cell cultivator containing or coated with the cell growth accelerator.

DETAILED DESCRIPTION OF THE INVENTION

The polyphosphoric acid useful in the present invention includes a linear condensed polyphosphoric acid obtained through dehydrocondensation of an orthophosphoric acid, a side chain polyphosphoric acid in which an organic group is introduced into a side chain, and a cyclic polyphosphoric acid. Especially preferable is a linear condensed polyphosphoric acid represented by formula H(n+2) (Pn O3n+1), wherein n is an integer of at least 2, and it is preferably between 5 and 5,000, more preferably between 15 and 2,000, having a structure in that two or more PO4 tetrahedrons are linearly bound with an oxygen atom held in common. A polyphosphate is a compound having a molecular structure wherein a hydrogen of a hydroxyl group of a polyphosphoric acid is replaced with a metal, such as sodium or potassium. In a phosphoric acid in which n is 1 in the above formula, no effect of the invention is provided. The cell growth accelerator of the invention may contain additives in addition to the polyphosphoric acid.

The polyphosphoric acid stabilizes the cell growth factor which all plant and animal cells secrete in a small amount to accelerate cell growth or to enhance the linkage between cells and the cell growth factor. The polyphosphoric acid promotes activation of hair matrix cells and bring forth hair growth promotion. The amount of the polyphosphoric acid in the hair growth promotor is between 1x10-7 and 50% by weight, preferably between 0.001 and 10% by weight. When the content of the phosphoric acid is less than 1x10-7 % by weight, no effect of hair growth promotion is observed. When it exceeds 50% by weight, it gives rise to problems with preparation, and the product cannot be used as a hair growth promotor.

Further, in this invention, the polyphosphoric acid is mixed with various substances of which the effect of hair growth promotion has been recognized. That is, the polyphosphoric acid is mixed with at least one agent selected from the group consisting of a cell activator, a blood flow stimulant, a skin stimulant, a humectant and an anti-inflammatory agent.

Examples of a cell activator include panthotenic acid and its derivatives, photosensitive element No. 301, carrot extract, biotin, mononitroguaiacol, allantoin and glyceride pentadecanoate. The amount of the cell activator in the hair growth promotor is preferably between 0.001 and 10% by weight.

Examples of a blood flow stimulant to be mixed with the polyphosphoric acid include acetylcholine, carpronium chloride, extract of Swertia japonica, Guinea pepper tincture, hinokitiol, cepharanthine, benzyl nicotinate, garlic extract, ligusticum extract, gentian extract, .gamma.-oryzanol, licorice, minoxidil, cnidii rhizoma, Panax japonicus C. A. Mey, Panax ginseng C. A. Mey, ginger, Rhmannia root, aloe, spironolactone, Vitamin B6 hydrochloride, D-camphor, DL-camphor, DL-.alpha.-tocopherol, iodized garlic extract, DL-.alpha.-tocopherol linoleate, inositol hexanicotinate, Vitamin E derivatives, sodium dextran sulfate, nicotinic acid, DL-.alpha.-tocopherol nicotinate, butoxyethyl nicotinate, methyl nicotinate, vanillylamide nonanoate, DL-.alpha.-tocopherol succinate, DL-.alpha.-tocopherol acetate, cantharis tincture and ginger tincture. The amount of blood flow stimulant is preferably between 0.001 and 10% by weight.

Further, examples of a skin stimulant to be mixed with the phosphoric acid include 1(L)-menthol, peppermint oil, benzyl nicotinate, vanillylamide nonylate and camphor. The amount of a skin stimulant in the hair growth promotor is preferably between 0.001 and 5% by weight.

Still further, examples of a humectant to be mixed with the polyphosphoric acid include glycerin, propylene glycol, hyaluronic acid and its salt, sodium pyrrolidonecarboxylate, chondroitin sulfate, Mini-Sasanishiki extract, vegetative wasp extract and saffron extract. The amount of humectant in the hair growth promotor is preferably between 0.001 and 5% by weight.

Furthermore, examples of an anti-inflammatory agent to be mixed with the polyphosphoric acid include glycyrrhizic acid derivatives, licorice extract, disodium carbenoxolone, guaiazulene, diphenhydramine hydrochloride, lithospermus root extract, rose fruit extract, hydrocortisone acetate and predonisolone. The amount of anti-inflammatory agent in the hair growth promotor is preferably between 0.001 and 3% by weight.

The hair growth promotor of the invention can contain, in addition to these components, an antibacterial agent, a keratolytic agent, an estrogen, an antiseborrheic agent and a nutrient. Examples of an antibacterial agent include benzalkonium chloride, photosensitive element No. 201, a chlorhexidine gluconate solution, chloroxylenol, trichlorocarbanilide, halocarvan and mononitroguaiacol. Examples of a keratolytic acid include salicylic acid, resorcin and lactic acid. Examples of an estrogen include estron, estradiol and ethinyl estradiol. Examples of an antiseborrheic agent include pyridoxine and its derivatives, sulfur, thioxolone, and lecithin. Examples of a nutrient include amino acids, cystine, cysteine, methionine, serine and vitamins.

Moreover, the hair growth promotor of the invention can contain an oil, a surfactant, a polyhydric alcohol, an antioxidant, a metal ion chelating agent, a pigment and a flavor as required. Examples of an oil include isopropylene myristate, lecithin and squalane. Examples of a surfactant include polyoxyethylenesorbitan fatty acid ester, sorbitan fatty acid ester, polyoxyethylene fatty acid ester and glycerin fatty acid ester. Examples of a polyhydric alcohol include propylene if glycol, glycerin and macrogol. Examples of an antioxidant include dibutylhydroxytoluene and isopropyl gallate. Examples of a metal ion chelating agent include ethylenediamine tetraacetate and its salt.

The hair growth promotor of the invention is used by coating a suitable amount thereof one or more times a day on a scalp or a section in which hair growth is expected. The form of the hair growth promotor in the invention is not particularly limited. For example, it can be used as a hair tonic, a hair lotion, a hair cream, an aerosol, an ointment, a shampoo or a hair treatment reagent including a rinse.

As stated above, the polyphosphoric acid incorporated in the hair growth promotor of the invention has an excellent effect on hair growth promotion, particularly in comparison with the conventional product.

Further, the polyphosphoric acid promotes the formation of new bone tissue. Bone morphogenetic protein, a filler for cosmetic surgery, and/or the natural substance containing bone morphogenetic proteins is mixed with a polyphosphoric acid, and the mixture can locally be administered as an implant or a device. In this case, the product to be administered is occluded or injected in a physiologically acceptable viscous form free from a pyrogenic substance and suitable for feeding into a fractured bone site. Consequently, a hard or soft bone structure is formed in the fractured bone site, providing a matrix which can be re-absorbed into the body in an optimum state. The bone regeneration material of the invention is used, as an osterogenic preparation containing the polyphosphoric acid, in preventive applications such as improvements in the reduction of an occlusive fracture or a complicated fracture and the placement of artificial joints.

Further, the osterogenic preparation induces the bone formation and is used in the restoration of an innate or traumatic defective portion or a defective portion caused by a tumor incision.

In vitro, culture cells secrete a trace amount of a cell growth factor in the culture solution. However, under the ordinary incubation conditions, a mixture of a cell growth factor in the form of serum has to be externally supplemented. No efficient growth of cells is observed in a serum-free environment. When polyphosphoric acid is added to a medium for as incubation of cells of animals and plants, cells which ordinarily cannot be grown in a serum-free environment can be grown in the serum-free medium. The cultured cells can be grown in the serum-free free medium or the medium having a low serum concentration by making use of the property of polyphosphoric acid. The concentration of the polyphosphoric acid used in the medium is between 1 nM and 100 mM, preferably between 10 nM and 10 mM.

A serum-free medium obtained by adding polyphosphoric acid to a medium for cells of animals and plants or a medium obtained by adding phosphoric acid and a small amount of serum to a medium for cells of animals and plants is used. The amount of serum added to the medium in the invention is between 0 and 10% by weight, preferably between 0 and 5% by weight. The medium used in the invention can be applied to various culture cells. It can be applied to primary culture cells or strain cells derived from organs such as the liver, the pancreas, the kidney, the lung, the stomach and the spleen, primary culture cells or strain cells derived from leucocytes such as lymphocytes and tissues such as nerves, muscles, skins and bones, and various tumor cells. In this case, the cells referred to include cells of all kinds of organisms including arthropods (insects) and plants as well as embryonic and fetal cells.

Various myeloma cells and hybridoma cells that are commonly used for monoclonal-antibody production, are also applicable. Examples include epithelial keratinocytes, melanocytes, vascular endothelial cells, vascular smooth muscle cells, hair matrix cells, osteoblasts, chondrocytes, amnion cells, fetal kidney cells, fetal lung cells, and strain cells such as Hela cells, FL cells, KB cells, HEp-2 cells, WI-38 cells, MA104 cells, BSC-1 cells, Vero cells, CV-1 cells, BHK-21 cells, RK-13 cells, Raji cells, R388D1 cells, Ralb/3T3 cells, CHO-K1, EB-3, EI-38, HEL cells, hair loss-60 cells, K562 cells, MPC-11 cells, MRC-5 cells, Namalva cells and L cells. Especially, fibroblast growth factor (FGF) demand cell strains, normal human epithelial fibroblasts, normal human gingival fibroblast cells, normal human epithelial cells and mouse cell strains are preferably used. Polyphosphoric acid can also be applied to cell culture systems preparing a artificial organ, such as an artificial liver, pancreas, skin, etc.

The cell growth factor, which is stabilized with the polyphosphoric acid or of which the linkage with cells is enhanced with the polyphosphoric acid, includes various growth factors. Of these, .beta.-FGF (basic fibroblast growth factor), .alpha.-FGF (acidic fibroblast growth factor), FGF-7 (keratinocyte growth factor), PDGF (platelet-derived growth factor), EGF (epidermal growth factor), a vascular endothelial growth factor and pleiotrophine are preferable. These cell growth factors are mixed with the polyphosphoric acid either individually or in combination, and the mixture is added to a medium for animal cell culture to exhibit a high growth acceleration ability compared with that in adding the cell growth factor alone. The concentration of the growth factor used is between 0.1 and 1000 ng/ml depending on the type.

Examples of substances other than the growth factor which accelerate with polyphosphoric acid include all substances that adjust the physiological activity of cells by extracellularly acting on cells (hereinafter referred to as "physiologically active factors"), such as cytokines, a chemotactic factor, hormones, a differentiation inducing factor, a morphogenetic factor, an angiogenetic factor, an angiogenesis inhibitor, a hemopoietic factor, a TGF-.beta. superfamily, TNF, INF and a plant growth factor. These physiologically active factors are mixed with the polyphosphoric acid either individually or in combination, and the mixture is added to a medium for incubation of cells of animals and plants, whereby the growth of culture cells and the control of the physiological activity of cells can be controlled. Substances containing the cell growth factor may be commercial products. The amount of the physiologically active factor is preferably between 1 pg/ml and 1 mg/ml depending on the type.

The medium used in the invention is a medium containing saccharides, amino acids, vitamins and salts, which is ordinarily used to incubate cells of animals and plants. Examples of the medium include Eagle's MEM medium, modified Eagle's MEM medium to which amino acids, vitamins and inorganic salts are added, increased or decreased to adapt to the cells to be incubated, Dulbecco's modified Eagle's medium, Iskov medium, RPMI 1640 medium, Ham F10 medium, Ham F12 medium, MCDB131 medium, MCBD151 medium, MCBD152 medium, MCBD153 medium, MCBD201 medium, MCBD302 medium, GIT medium and MEDIUM199. Further, the medium of the invention may contain additives ordinarily used for cell cultures, such as antibiotics, fungicides, buffers, pigments and agars. Examples of the medium for incubation of plant cells include Murashige and Skoog medium, B5 medium, Nagata and Takebe medium, Kao and Michayluk (8p) medium, Nagy and Maliga (K) medium, Shepard (CL) medium and Chupeau (To).

Examples of the cell incubator used in the invention include a petri dish, a bottle, a flask,.a test tube, a beaker and hollow fibers made of plastics such as polystyrene, polyethylene, polyvinyl chloride, polyester, polycarbonate, acetyl cellulose and polyacrylate and a glass. Either the polyphosphoric acid is added to the incubator materials and the mixture is formed into an incubator, or the surface of the incubator is coated with a film containing the polyphosphoric acid.

The stabilization of the physiologically active factor or the stabilization of the linkage between the cells and the physiologically active factor with the polyphosphoric acid can be applied not only to the culture cells but also to the tissue cells of animals including humans. The polyphosphoric acid stabilizes the functioning of a very small amount of the physiologically active factor which the cells in the tissue secrete, thereby efficiently increasing the growth or the physiological activity of the cells in the tissue and repairing or regenerating the tissue.

The polyphosphoric acid-containing medium and incubator in the invention has properties of accelerating the growth of culture cells in a serum-free state or in a state containing a small amount of a serum or a physiologically active factor, and these can be applied to production of various substances from culture cells. Further, the cell growth accelerator and the pharmaceutical composition containing the polyphosphoric acid accelerate tissue repair and can accelerate the healing of injuries and burns, treatment of periodontal diseases and recovery after surgical operations.

Claim 1 of 9 Claims

What is claimed is:

1. A cell growth accelerator composition comprising a linear condensed polyphosphoric acid represented by the formula: