Patent Publication Number: US-7906114-B2

Title: Compositions for promoting wound healing and treating psoriasis

Description:
PRIORITY CLAIM 
     This application is a divisional application of U.S. patent application Ser. No. 12/033,204, filed Feb. 19, 2008, now U.S. Pat. No. 7,674,832, which is a continuation-in-part of U.S. patent application Ser. No. 10/952,003, filed Sep. 28, 2004, now U.S. Pat. No. 7,335,638, which is a continuation-in-part of International Patent Application No. PCT/BR03/0049, filed Mar. 28, 2003 and published in English on Oct. 9, 2003 as WO 03/082324 (priority to both Ser. No. 10/952,003 and PCT/BR03/0049 being claimed herein), and further claims priority to U.S. Provisional Application Ser. No. 60/890,980, filed Feb. 21, 2007, all of which are incorporated by reference in their entireties herein. 
    
    
     1. INTRODUCTION 
     The present invention relates to compositions and methods for promoting healing of cutaneous, mucosal and/or mucocutaneous lesions by topical application of a protein capable of removing sialic acid residues and a plant extract comprising nucleic acid containing particles. 
     2. BACKGROUND 
     Mycoplasmas represent some of the smallest self-replicating microorganisms, and have unique properties among the prokaryotes. These properties include the need for cholesterol to maintain their membrane envelope, and the absence of an external wall. Mycoplasmas are known to cause pulmonary infection in humans, and it is widely known that mycoplasmas can cause disease in most animals including humans as well as animals of commercial importance such as cattle, swine, and fowl. (Razin et al., 1998, Microbiol. and Molecular Biology Review, 62(4):1094-1156; Maniloff et al. Eds., 1992, Mycoplasmas, Molecular Biology and Pathogenesis, American Society for Microbiology, Washington). 
     Frequent co-occurrence of mycoplasma with other microorganisms, such as chlamydia, has been observed in diseases involving cell proliferation (International Patent Application No. PCT/BR01/00083, filed Jul. 3, 2001 and BR PI 0002989-0, filed Jul. 3, 2001). This association appears to increase the virulence of both pathogens. Mycoplasmal lipoproteins are potent macrophage activators and have a comparable activity and distribution in mollicutes as the LPS of Gram-negative bacteria. (Razin et al. Eds., 2002, Molecular biology and pathogenicity of mycoplasmas, Kluwer Academic/Plenum Publishers, New York). Recently described toll-like receptors (TLR) in macrophages that are activated by products from pathogens such as mycoplasmal lipoproteins and LPS from bacteria have been demonstrated to be important for activation of the immune system and it appears that the efficacy of the immune response depends on which concomitant TLR s are activated. (Akira et al., 2001, Nature Immunol. 2:675-680). 
     Archaea are the most ancient microorganisms existing in nature, but have been characterized only recently. See, Woese et al., Proc Natl. Acad. Sci. U.S.A. 74: 5088-5090 (1977). They inhabit extreme environments and are constituted by lipid monolayer membranes. Rich alkaline atmosphere with sodium ions and metals prevents proliferation of other bacteria, but is favorable to archaea&#39;s growth. Archaea have been isolated from alkaline waters from the Dead Sea, the Great Salt Lake and Yellowstone National Park. They have a small size, can—just barely—be viewed with an optical microscope, and observation of structural details requires electron microscopy. See, Howland et al., The surprising archaea. Discovering another domain of life, Oxford University Press (New York, 2000). Some are considered hyperthermophilic as they survive in very high temperatures. 
     Another unusual characteristic of some archaea is that they appear to use metal as an energy source. See, Amend et al., F.E.M.S. Microbiol. Rev. 25: 175-243 (2001). It is considered that archaea usually need an anaerobic or nearly anaerobic environments to carry out oxidation-reduction reactions with participation of different chemical compounds, including metals. 
     Recently, a new kind of extremely small archaea, which is dependent on bigger archaea, was described and named nanoarchaea. See, Huber J et al., Nature 417: 63-67 (2002). With the exception of archaea that reside in the mammalian intestine and produce methane gases, there is no report of archaea existing within plants or animals. See, Florin T H J et al., Am. J. Gastroenterol. 95: 2872-2879 (2000). 
     Cutaneous lesions, for example, psoriasis and radiodermatitis resulting from radiotherapy, present obstacles to successful wound healing, as the lesions result from continued exposure to causative factors such as radiation therapy for cancer, or immune system dysfunction in psoriasis. In particular, the persistence of radiodermatitis lesions often requires the suspension of radiation therapy during cancer treatment in order to permit healing of the lesions. Such interruptions can be detrimental to the successful treatment of cancer. 
     3. SUMMARY OF THE INVENTION 
     The present invention relates to compositions and methods for promoting healing of cutaneous, mucosal and/or mucocutaneous lesions by topical application of a protein capable of removing sialic acid residues and a plant extract comprising nucleic acid containing particles. In various embodiments of the invention, the composition is a topical gel, cream, or ointment comprising a protein capable of removing sialic acid residues, such as a neuraminidase enzyme and/or a trans-sialidase enzyme, and one or more purified plant extracts. In additional embodiments, the composition further comprises a metal chelator. Administration of the topical formulation to the lesion promotes healing. 
    
    
     
       4. BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1A-F  shows a decrease in edema and redness of radiodermatitis skin lesions following treatment with a trans-sialidase gel and an orchid extract gel.  FIG. 1A-C  show three different patients presenting radiodermatitis degree 4, represented by inflammation and cutaneous ulcers (arrow) in a woman suffering from breast cancer (A), a woman with malignant melanoma (B) and a man with larynx cancer (C).  FIG. 1D-F  show the skin lesions of the same patients described in (A-C) following one week of treatment with trans-sialidase and orchid extract gels (arrow). The trans-sialidase gel was administered topically once per day to the lesions in an amount of 1.0 ml/50 cm 2  of cutaneous tissue each morning of treatment. The orchid extract gel was administered topically once per day to the lesions in an amount of 1.0 ml/50 cm 2  of cutaneous tissue each evening of treatment. The  FIG. 2A-D  shows lesions in a 59 year old woman suffering from psoriasis of the scalp before (A-B) and following (C-D) topical treatment with trans-sialidase and orchid extract gels. (A-B) show psoriatic lesions (arrows) characterized by silvery scales on bright red, well-demarcated plaques on the scalp. The plaques were usually associated with hair loss. (C-D) show the same lesions as in A-B following two months of treatment with trans-sialidase and orchid extract gels. The trans-sialidase gel was administered topically once per day to the lesions in an amount of 1.0 ml/50 cm 2  of cutaneous tissue each morning of treatment. The orchid extract gel was administered topically once per day to the lesions in an amount of 1.0 ml/50 cm 2  of cutaneous tissue each evening of treatment. 
         FIG. 3  shows the nucleotide sequence of a plasmid encoding the catalytic trans-sialidase unit of trans-sialidase from  Trypanosoma cruzi  (SEQ ID NO:3). The letters in capital represent the pET14b plasmid and the underlined letters correspond to the position of the oligonucleotides used to amplify the  Trypanosoma cruzi  clone. 
         FIG. 4  shows the amino acid sequence of the protein encoded by the nucleic acid sequence depicted in  FIG. 3  (SEQ ID NO:4). In bold are the amino acids not found in the original clone. 
         FIG. 5  shows small dark electron-dense nanoarchae of between 0.03-0.15 μm in diameter. 
         FIG. 6  shows dark medium sized electron-dense archaea of between 0.5-1.1 μm in diameter, and large clear, empty archaea of between 1.0-2.4 μm in diameter. 
         FIG. 7  shows clear, empty archaea of between 0.15-2.0 μm in diameter. 
     
    
    
     SEQUENCE LISTING 
     The specification further incorporates by reference the Sequence Listing submitted herewith via EFS on Mar. 3, 2010. Pursuant to 37 C.F.R. §1.52(e)(5), the Sequence Listing text file, identified as 0685280113seqlist.txt, is 8,981 bytes and was created on Mar. 3, 2010. The Sequence Listing, electronically filed herewith, does not extend beyond the scope of the specification and thus does not contain new matter. 
     5. DETAILED DESCRIPTION OF THE INVENTION 
     For purposes of clarity, and not by way of limitation, the detailed description of the invention is divided into the following subsections: 
     (i) compositions for treating lesions; and 
     (ii) methods of treatment. 
     5.1 Compositions for Treating Cutaneous Lesions 
     The present invention provides for compositions and methods that promote the healing of wounds, in particular, cutaneous, mucosal, and/or mucocutaneous lesions. Specifically, it has been found that the treatment of lesions with compositions of the invention promotes the healing of the lesions believed to be due to, without being bound to any particular theory, a decrease in the association of a mycoplasma and/or one or more non-mycoplasma microorganisms with the lesion being treated. 
     In non-limiting embodiments of the invention, the composition comprises a carrier, such as a gel, cream, ointment, or lotion which further comprises a protein capable of removing sialic acid residues, such as a neuraminidase enzyme, a trans-sialidase enzyme, or a combination thereof, and/or one or more purified plant extracts, and optionally, a metal chelator. According to the invention, application of the composition to a lesion inhibits or prevents the attachment of a mycoplasma and/or a non-mycoplasma microorganisms to the host cells. 
     The term “lesion” as described herein means any structural disturbance, defect or abnormality to the cuticle, skin, mucosa, or mucocutaneous tissues of a subject. Lesions include, for example, but not limited to, structural defects, for example, lacerations and surgical wounds, radiodermatitis or injury resulting from exposure to radiation, psoriasis, rashes, moles, cysts, pimples, warts, burns, irritations, abrasions, baldness, seborreic keratosis, keloid scars, chronic dermatitis, fibrosis, sclerosis, cutaneous thickening, scars, cutaneous discontinuities, and ulcers. 
     The term “composition,” as used herein means one or more agents or combinations thereof effective to promote the healing of a lesion and/or decrease the presence of a mycoplasma and non-mycoplasma microorganism with the lesion. In one embodiment, the composition inhibits the ability of the mycoplasma and non-mycoplasma to associate with a substrate, for example, but not limited to, a lesion. In another non-limiting embodiment, the composition inhibits the association of a mycoplasma and a non-mycoplasma microorganism. 
     The term “carrier,” as used herein means any agent the composition is mixed with to facilitate topical administration of the composition to a subject. Examples of carriers include, but is not limited to, gels, creams, ointments, liquids, or powders. In a preferred embodiment, the carrier is a gel. 
     The term “mycoplasma” as used herein means a microorganism of the genus  Mycoplasma  such as, but not limited to,  Mycoplasma  ( M .)  buccale, M. faucium, M. fermentans, M. genitalium, M. hominis, M. lipophilum, M. oral, M. penetrans, M. pneumoniae, M. salivarium , or  M. spermatophilum . The one or more additional non-mycoplasma microorganism may be a bacteria, archaea or virus, for example, but not limited to, spirochete or chlamydia such as  Chlamydia pneumoniae . According to the invention, the mycoplasma and non-mycoplasma may be attached to a substrate, for example, but not limited to, a lesion. In a further non-limiting embodiment, the mycoplasma and non-mycoplasma are attached to the substrate by sialic acid. 
     In a preferred embodiment of the invention, the protein capable of removing sialic acid residues is a trans-sialidase or neuraminidase enzyme. 
     In certain non-limiting embodiments, the composition comprises a neuraminidase enzyme of, for example but not limited to,  Bacteroides fragilis, Streptococcus pneumoniae, Streptococcus oralis, Arthrobacter ureafaciens, Clostridium perfringens, Mycoplasma alligatoris, Arcanobacterium pyogenes, Clostridium sordellii, Pseudomonas aeruginosa, Micromonospora viridifaciens, Vibrio cholerae. Streptomyces avermitilis, Influenza virus, Streptomyces coelicolor, Flavobacteriales bacterium , and  Solibacter usitatus.    
     In another non limiting embodiment, the protein comprises a trans-sialidase, for example, the trans-sialidase enzyme of  Trypanosoma brucei.    
     In a preferred embodiment of the invention, the composition comprises the trans-sialidase enzyme of  Trypanosoma cruzi , or a portion or variant of the native enzyme which has trans-sialidase activity. 
     Alternatively, the trans-sialidase enzyme may be a recombinant trans-sialidase enzyme. 
     According to the invention, the recombinant trans-sialidase is as described in International Patent Publication WO/2002/002050 by Higuchi et al., published Jan. 10, 2002; and U.S. Pat. No. 7,108,851 by Higuchi et al., issued Sep. 19, 2006. For example, the trans-sialidase gene may be obtained from a genomic clone, isolated from a commercially available lambda Zap®II library (Stratagene, //www.stratagene.com) of  T. cruzi  Y strain (Silva and Nussenzweig, 1953, Folia Clin Biol 20: 191-203), as described in Uemura et al. (Uemura et al., 1992, EMBO J 11: 3837-3844). From the original lambda clone, which expresses enzymatic activity, an SK plasmid containing the trans-sialidase gene may be generated (SK-154-0). The preferred plasmid used is pTSII, which corresponds to a fragment of the original gene (clone 154-0) amplified through PCR, and inserted into the sites Ndel and BamHl of the vector pET14b (Novagen—//www.novagen.com). The PCR product may be amplified using SK-154-0 as a template with the following primers: 
     a) TSPET14: 5′-GGAATTCCATATGGCACCCGGATCGAGC (SEQ ID NO:1) 
     b) RT154: 5′-CGGATCCGGGCGTACTTCTTTCACTGGTGCCGGT (SEQ ID NO:2) 
     The resulting PCR product should have a nucleic acid sequence as set forth in  FIG. 3  (SEQ ID NO:3), and a corresponding amino acid sequence as depicted in  FIG. 4  (SEQ ID NO:4). The resulting plasmid may be transformed into the  Escherichia coli  BLB21 DE3. The construct can be made in two steps due to an internal BamHl site in the trans-sialidase gene. The PCR product may be treated with BamHl and Ndel enzymes, and the resulting fragments fractionated by electrophoresis on an agarose gel. The separated fractions may then be purified from the gel with the Sephaglass purification kit (Amersham-Pharmacia). The 5′ Ndel-BamHl digestion fragment may be ligated into the pET14b vector which has been pre-digested with BamHl and Ndel. The ligation products may be used to transform K12 DH5a  E. coli  cells. The plasmid containing  E. coli  cells may be selected and the plasmid purified by methods known in the art. The purified construct may be treated with BamH1, shrimp alkaline phosphatase, and ligated with the BamHI-BamHI-3′ fragment purified from the fractionation gel. The ligation products may then be used to transform K12 DH5a  E. coli  cells, from which clones expression of trans-sialidase may be selected and purified. The final plasmid may be confirmed by restriction analysis and used to transform the BLB21 DE3 pLys strain of  E. coli , from which recombinant trans-sialidase enzyme can be purified, as described in International Patent Publication WO/2002/002050 by Higuchi et al., published Jan. 10, 2002; and U.S. Pat. No. 7,108,851 by Higuchi et al., issued Sep. 19, 2006. 
     Alternatively, the trans-sialidase enzyme may be purified from a culture of  Trypanosoma cruzi , such as, for example, a culture according to Kloetzel et al. (Kloetzel et al., 1984, Rev. Inst. Med. Trop. Sao Paulo., 26:179-85). Supernatant from the culture may be filtered through a 1 μm pore filter in a vacuum chamber. The enzyme may be further purified by filtering the supernatant through a 0.22 μm filter and then precipitating the filtrate with a 50% (NH 4 ) 2 SO 4  solution. The precipitates may then be dialyzed against phosphate-buffered saline, and passed through a tresyl-agarose column comprising an immobilized anti-trans-sialidase monoclonal or polyclonal antibody. The column may be washed with phosphate-buffered saline, followed by an additional wash with 10 mM sodium phosphate, pH 6.5. The trans-sialidase may then be eluted with a 3.5 mM MgCl 2 , 10 mM sodium phosphate, pH 6.0 solution. The fractions eluted from the column may be filtered through a Sephadex G-25 column equilibrated with 20 mM Tris-HCl, pH 8.0, to remove the MgCl 2 . The trans-sialidase may be further purified by passage through a Mono Q column equilibrated in 20 mM Tris-HCl, pH 8.0, and eluted with a linear gradient from 0 to 1 mM NaCl in the same buffer. 
     The purified enzyme derived from the culture should comprise 400 kDa multimeric aggregates. The enzymatic activity of the purified trans-sialidase may be measured according to methods described in International Patent Publication WO/2002/002050 by Higuchi et al., published Jan. 10, 2002; and U.S. Pat. No. 7,108,851 by Higuchi et al., issued Sep. 19, 2006. 
     In non-limiting embodiments, the purified trans-sialidase has an enzymatic activity of between 0.1 and 10 U/ml, more preferably between 1.0 and 5.0 U/ml, and most preferably 1.3 U/ml. 
     In a further non-limiting embodiments, the plant extract may be derived from, for example but not limited to,  Allium sativum  (garlic),  Ginkgo biloba , tomato, orchid, guava, ginseng, for example  Pfaffia paniculata  (Brazilian ginseng); tobacco; or  Zingiber officinale  (ginger), wherein the orchid is preferably of the genus  Cymbidium , for example, yellow or green orchids from the genus  Cymbidium  ( Cymbidium  ssp.). Alternatively, the orchid is of the genus  Dendrobium , for example,  Dendrobium nobile  or  Dendrobium moschatum.    
     The extract from plants may be obtained by adding a solvent, such as, for example, alcohol, to the plant tissue, for example, but not limited to, roots, cloves, flower petals, or leaves which may be chopped, or macerated prior to mixture with the solvent. The solvent may be mixed with the plant tissue in a proportion of between 1:99 and 60:40, more preferably between 15:85 and 50:50 and most preferably between 30-40:70-60 of plant mass:alcohol. The solvent can be an alcohol, for example, ethanol, methanol, or grain alcohol, and can have a concentration of between 60% and 100%, more preferably between 70% and 95%, and most preferably about 92% alcohol. The plant/alcohol mixture may be aged in a dark, anaerobic environment for a period of time between 15 days and 24 months, more preferably between 1 and 12 months, and most preferably 10 months. 
     According to the invention, the extract derived from plant comprises particles containing nucleic acid (DNA and/or RNA), wherein the particle is an archaea (preferably non-pathogenic) and/or a nanoarchaea, and further wherein the particle is present in an amount effective to prevent or inhibit the growth of a mycoplasma and one or more non-mycoplasma microorganisms. Aging of the plant/alcohol mixture increases the concentration of particles in the mixture. 
     The plant/alcohol mixture may be purified, and the concentration of nanoparticles may be increased through one or more filtrations. The mixture can be filtered through pores of between 0.5 μm and 50 μm, more preferably between 5 μm and 20 μm, and most preferably 11 μm, for example, but not limited to Whatman qualitative filter paper grade 1, diameter 24 cm, pore size 11 μm. Vacuum chambers can also be used separately, or in addition to other filtration methods. Additionally, glass microfiber filters can be used, for example, but not limited to, a 47 mm diameter glass microfiber filter with a pore size of 1.1 μm. Man filtration methods are known in the art can be used to filter the aged plant/alcohol mixture. 
     In a non-limiting embodiment, the plant/alcohol mixture can be subjected to additional aging during the filtration process. For example, olive oil may be added to the filtrate to create a 1% olive oil filtrate mixture, followed by an additional month of storage in a dark anaerobic environment. 
     Furthermore, the composition may comprise particles and/or nanoparticles containing DNA or RNA, wherein the particles are a non-pathogenic archaea and/or a nanoarchaea, and further wherein the particle is present in amounts effective to prevent or inhibit the growth of a mycoplasma and one or more non-mycoplasma microorganisms. The nanoparticles may be between 5-500 nm, more preferably between 15-250 nm, and most preferably between 30-150 nm in diameter. Alternatively, the composition may comprise medium particles of between 500 nm and 1.1 μm in diameter. Additionally, the compositions may comprise one or a combination of both small and medium particles. The size of a particle can enlarge or decrease depending on the concentration of water and ions in a solution comprising the particles, such as, for example, Na+ or Ca+. 
     According to the invention, the purity of the plant extract may be determined by microscopic examination of the filtered, aged, plant extract, as described in U.S. Patent Application Publication No. 20050142116. For example, the filtered, aged plant extract can be stained with any DNA or RNA dye known in the art, such as acridine orange, bisbenzimide H 33342 (Hoechst), or 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI); and viewed with an immunofluorescence optical microscope, an electron microscope, or any other microscope known in the art. Two forms of archaea, having different morphological characteristics may be identified. One type comprising an electron-dense content may be between about 0.03-0.15 μm (nanoparticle) and about 0.5-1.1 μm in diameter (medium particle) ( FIGS. 5 and 6 , respectively). A second type may comprises a clear, empty content, and may be about 0.15-2.4 μm in diameter ( FIGS. 6 and 7 ). The clear, empty archaea are similar in morphology to the pathogenic archaea associated with lesions, while the electron dense archaea comprise the non-pathogenic archaea and nanoarchaea comprising DNA or RNA. Brilliant red particles, which may comprise metallic ions, may also adhere to the surface of the archaea. Optimum purity may be achieved when predominantly, preferably essentially, only fast moving electron-dense nanoparticles are visible. The presence of clear, empty archaea or large brilliant red particles of about 0.15-0.24 μm and at a concentration of, for example, ≧1.0 large brilliant red particle/visual field, indicates suboptimal purity. In cases of suboptimal purity, the filtered aged plant extract is subjected to additional filtration, for example, tangential flow filtration in the Minitan Ultrafiltration System (Millipore, Bedford, Mass., USA), using the microporous membrane packet (30,000 NMWL). In preferred embodiments, the compositions of the invention comprise a greater number of electron dense archaea (nanoparticles and medium particles) than empty, clear archaea; and a greater number of archaea not associated with large brilliant red particles than those associated with large brilliant red particles. 
     According to the invention, the purified plant extract may comprise an enriched population of particles. The concentration of particles may be between 1×10 5  and 1×10 10  particles/ml, more preferably between 1×10 6  and 1×10 9  particles/ml, and most preferably about 1×10 7  particles/ml. 
     In certain non-limiting embodiments, the composition comprises a metal chelator, for example, but not limited to, Nitrilotriacetate (NTA), diphenylthiocarbazone(dithizone), histidine, the lipophilic metal chelator DP-109, ethylene glycol tetraacetic acid (EGTA), ethylenediaminetetraacetic acid (EDTA), DMPS (2,3-dimercapto-1-propanesulfonate), Lysinoalanine, Synthetic lysinoalanine (N-ε-DL-(2-amino-2-carboxyethyl)-L-lysine), tetracycline, alpha lipoic acid (ALA), Dimercaptosuccinic acid, (DMSA), 2,3-Dimercapto-1-propanesulfonic acid (DMPS), Calcium disodium versante (CaNa 2 -EDTA), D-penicillamine, Deferoxamine, Defarasirox, Dimercaprol (SAL), the calcium salt of diethylene triamine pentaacetic acid (DTPA), or any other metal chelator known in the art. In a preferred embodiment, the metal chelator is pyrrolidine dithiocarbamate (PDTC). In a preferred embodiment, the metal chelator is pyrrolidine dithiocarbamate (PDTC). The composition of the invention may comprise the metal chelator in a concentration of between about 0.01 and 10 mg/ml, more preferably between about 0.5 and 5 mg/ml, more preferably between about 1 and 2 mg/ml, and most preferably about 1.5 mg/ml. 
     In a non-limiting embodiment, the compositions of the invention comprise combinations of trans-sialidase, a metal chelator, and one or more purified plant extracts as shown in Table I. 
     
       
         
           
               
             
               
                 TABLE I 
               
               
                   
               
               
                 Combinations of trans-sialidase, a metal chelator, and one or more  
               
               
                 purified plant extracts encompassed by the invention. 
               
               
                 Combinations of trans-sialidase (TS), pyrrolidine 
               
               
                 dithiocarbamate (PDTC), and purified plant extracts 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                   
                 TS 
               
               
                   
                   
                 TS + PDTC 
               
               
                   
                   
                 TS + PDTC +  Allium sativum  (AS) 
               
               
                   
                   
                 TS + PDTC +  Ginkgo biloba  (GB) 
               
               
                   
                   
                 TS + PDTC +  Zingiber officinale  (ZO) 
               
               
                   
                   
                 TS + PDTC + orchid extract (OE) 
               
               
                   
                   
                 TS + PDTC + AS + GB 
               
               
                   
                   
                 TS + PDTC + AS + ZO 
               
               
                   
                   
                 TS + PDTC + AS + OE 
               
               
                   
                   
                 TS + PDTC + AS + GB + ZO 
               
               
                   
                   
                 TS + PDTC + AS + GB + OE 
               
               
                   
                   
                 TS + PDTC + AS + GB + ZO + OE 
               
               
                   
                   
                 TS + PDTC + AS + ZO + OE 
               
               
                   
                   
                 TS + PDTC + GB + ZO 
               
               
                   
                   
                 TS + PDTC + GB + OE 
               
               
                   
                   
                 TS + PDTC + GB + ZO + OE 
               
               
                   
                   
                 TS + PDTC + ZO + OE 
               
               
                   
                   
                 TS + AS 
               
               
                   
                   
                 TS + GB 
               
               
                   
                   
                 TS + ZO 
               
               
                   
                   
                 TS + OE 
               
               
                   
                   
                 TS + AS + GB 
               
               
                   
                   
                 TS + AS + ZO 
               
               
                   
                   
                 TS + AS + OE 
               
               
                   
                   
                 TS + AS + GB + ZO 
               
               
                   
                   
                 TS + AS + GB + OE 
               
               
                   
                   
                 TS + AS + GB + ZO + OE 
               
               
                   
                   
                 TS + AS + ZO + OE 
               
               
                   
                   
                 TS + GB + ZO 
               
               
                   
                   
                 TS + GB + OE 
               
               
                   
                   
                 TS + GB + ZO + OE 
               
               
                   
                   
                 TS + ZO + OE 
               
               
                   
               
            
           
         
       
     
     5.2 Methods of Treatment 
     The present invention provides for compositions and methods that promote the healing of wounds, in particular, cutaneous, mucosal, and/or mucocutaneous lesions. Specifically, it has been found that the treatment of lesions with compositions of the invention promotes the healing of the lesions believed to be due to, without being bound to any particular theory, a decrease in the association of a mycoplasma and/or one or more non-mycoplasma microorganisms with the lesion being treated. In a non-limiting embodiment of the invention, the composition may be administered topically to a lesion as a lotion, cream, liquid, paste, ointment, powder, or any other carrier known in the art. In a preferred embodiment of the invention, the composition is applied as a topical gel. 
     In one non-limiting embodiment, the composition of the invention is applied in an amount effective to promote healing of wounds, in particular, cutaneous, mucosal, and/or mucocutaneous lesions, for example, but not limited to, structural defects, for example, lacerations and surgical wounds, radiodermatitis or injury resulting from exposure to radiation, psoriasis, rashes, moles, cysts, pimples, warts, burns, irritations, abrasions, baldness, seborreic keratosis, keloid scars, chronic dermatitis, fibrosis, sclerosis, cutaneous thickening, scars, cutaneous discontinuities, and ulcers. 
     The term “treatment” as defined herein means a reduction in lesion size, inflammation, irritation, scaling, scar formation, fibrosis, sclerosis, and cutaneous thickening. In a non-limiting embodiment, the treatment may reduce the presence of moles, cysts, pimples, warts, burns, irritations, abrasions, baldness, seborreic keratosis, keloid scars, chronic dermatitis, cutaneous discontinuities, structural defects, lacerations and surgical wounds. 
     In a non-limiting embodiment, the methods and compositions of the invention are effective for treating a lesion, wherein topical administration of the composition may be effective to reduce the size of the lesion by at least about 1%, 5%, 10%, 25%, 50%, 75%, 90%, or 100%. 
     The present invention provides for formulations, to be applied topically to a cutaneous, mucous, or mucocutneous lesion. The composition may comprise between about 1×10 −8  and 1×10 −4  U/ml, or between about 1×10 −7  and 1×10 −5  U/ml, or between about 1×10 −6  and 5×10 −6  U/ml, and most preferably about 2×10 −6  U/ml of neuramidase and/or trans-sialidase activity. The composition may also comprise between about 100 and 1×10 7  plant extract derived particles/ml, or between about 1×10 3  and 1×10 6  particles/ml, or between about 1×10 4  and 5×10 5  particles/ml and most preferably about 3×10 5  particles/ml. Optionally, the composition may comprise a metal chelator, for example, but not limited to, PDTC, NTA, diphenylthiocarbazone(dithizone), histidine, DP-109, EGTA, EDTA, DMPS, Lysinoalanine, Synthetic lysinoalanine, tetracycline, ALA, Dimercaptosuccinic acid, DMSA, Calcium disodium versante, D-penicillamine, Deferoxamine, Defarasirox, Dimercaprol, or DTPA, in a concentration of between about 0.01 and 10 mg/ml, or between about 0.5 and 5 mg/ml, or between about 1 and 2 mg/ml, and most preferably about 1.5 mg/ml. 
     In specific non-limiting embodiments, the composition comprises 2.6×10 −6  U/ml neuramidase and/or trans-sialidase activity, and/or 3×10 5  plant derived particles/ml. 
     Alternatively, the composition may comprise between about 0.01 and 10 U/ml, or between about 0.2 and 5 U/ml, or between about 0.5 and 2 U/ml and most preferably about 1.0 U/ml of neuramidase and/or trans-sialidase activity. 
     In one embodiment of the invention, the gel is a trans-sialidase gel, wherein the trans-sialidase gel is created by mixing trans-sialidase enzyme with a gel, for example, but not limited to, a non-ionic, anionic, or cationic gel. In one preferred embodiment, the gel is a hydroxyethylcellulose gel, for example, Natrosol® gel The trans-sialidase enzyme may be diluted with a suitable dilutant, for example, but not limited to, MilliQ purified water, distilled water, or thermal water, prior to mixing with the carrier. In a preferred embodiment, the dilutant is MilliQ purified water. When diluted, the trans-sialidase is diluted at a trans-sialidase:dilutant ratio of between 1:1 and 1:2,000,000, more preferable between 1:100 and 1:1,000,000, more preferably between 1:1000 and 1:500,000, and most preferably 1:250,000. The resulting trans-sialidase dilution, or alternatively, undiluted enzyme, may then be mixed with a gel at a trans-sialidase:gel ratio of between 1:1000 and 1000:1, more preferably between 1:100 and 100:1, more preferably between 1:10 and 10:1 and most preferably 1:1. 
     In one, non-limiting example, the trans-sialidase gel is made by diluting a purified trans-sialidase enzyme 1:250,000 in purified water, for example, MilliQ purified water. The diluted trans-sialidase is then mixed into Natrosol® gel (Dermavita, Brusque, Santa Catarina, Brazil), at a concentration of 1:1 and stored at 4° C. The gel may be applied topically to a subject in an amount of 1.0 ml/50 cm 2  of cutaneous surface. 
     In one non-limiting embodiment of the invention, the trans-sialidase enzymatic activity of the trans-sialidase gel is between 100 and 100,000 CPM, more preferably between 1000 and 20,000 CPM, more preferably between 1,500 and 10,000 CPM, and most preferably between 2,000-5,000 CPM, wherein 30,000 CPM corresponds to 0.36 nmoles of transferred sialic acid in a 30 min assay, which is further defined as one (1) unit (U). 
     In a non-limiting embodiment of the invention, the trans-sialidase gel may be administered topically to a subject in need of treatment, wherein the gel is administered one or more times per day. For example, the trans-sialidase gel may be administered once, twice, three, four, five, or 6 or more times per day. In a non-limiting example, the trans-sialidase gel may be applied topically once per day in the morning or evening during treatment. 
     In another non-limiting embodiment, the gel is a purified plant extract gel, preferably an orchid extract gel. The purified plant extract may be diluted with a suitable dilutant, for example, but not limited to, MilliQ purified water, distilled water, or thermal water, prior to mixing with the carrier. When diluted, the purified plant extract is diluted at a plant extract:dilutant ratio of between 1:100 and 100:1, more preferably between 1:50 and 50:1, more preferably between 1:10 and 10:1, and most preferably 1:5. The diluted or undiluted purified plant extract may be mixed with a gel, for example, but not limited to, an anionic gel. In a preferred embodiment, the gel is a hydroxyethylcellulose gel, for example, Natrosol® gel. 
     In a further non-limiting embodiment, the gel and the diluted or undiluted purified plant extract are mixed to achieve a gel with a final plant extract concentration of between 0.1% and 99%, more preferably between 5% and 80%, more preferably between 10% and 50%, and most preferably 15%. 
     In a non-limiting example, the purified plant extract is diluted 1:5 in thermal water (e.g. from Irai-RS, Brazil, or another thermal water source), which was previously boiled and filtered. The diluted plant extract is then mixed with an anionic gel (Dermavita, Brusque, Santa Catarina, Brazil; or another thermal water source) until the mixture achieved a final concentration of 15% plant extract. The gel may be applied typically to a subject in an amount of 1.0 ml/50 cm 2  of cutaneous surface. 
     In a non-limiting embodiment of the invention, the purified plant extract gel may be administered topically to a subject in need of treatment, wherein the gel is administered one or more times per day. For example, the purified plant extract gel may be administered once, twice, three, four, five, or 6 or more times per day. In a non-limiting example, the purified plant extract gel may be applied topically once per day in the morning or evening during treatment. 
     In another non-limiting embodiment of the invention, both the purified plant extract gel and the trans-sialidase gel may be administered topically to a subject in need of treatment, wherein the gels are administered one or more times per day, for example, the purified plant extract gel and the trans-sialidase gel may be administered once, twice, three, four, five, or 6 or more times per day. In a further non-limiting embodiment, the purified plant extract gel and the trans-sialidase gel may be administered simultaneously or in series. In a non-limiting example, the trans-sialidase gel may be applied topically once per day in the morning and the purified plant extract gel may be applied topically once per day in the evening during treatment. Alternatively, the gels may be applied topically at the same time. 
     In an additional non-limiting embodiment of the invention, the composition comprises both trans-sialidase and one or more purified plant extracts, for example, a gel comprising both a trans-sialidase gel and a purified plant extract gel. The two gels may be combined to produce a gel with a final trans-sialidase gel:purified plant extract gel ratio of between about 99:1 and 1:99, more preferably between 90:10 and 10:90, more preferably between 85:15 and 15:85, and most preferably 80:20. 
     The gel mixture comprising both the trans-sialidase gel and the purified plant extract gel may comprise a trans-sialidase enzymatic activity of between about 1×10 −8  and 1×10 −4  U/ml, or between about 1×10 −7  and 1×10 −5  U/ml, or between about 1×10 −6  and 5×10 −6  U/ml, and preferably about 2×10 −6  U/ml. The gel mixture may further comprises between about 100 and 1×10 7  plant extract derived particles/ml, or between about 1×10 3  and 1×10 6  particles/ml, or between about 1×10 4  and 1×10 5  particles/ml and preferably about 6×10 4  particles/ml. 
     In a specific, non-limiting embodiment, the gel comprises both a trans-sialidase enzymatic activity of 2.08 U/ml and 6×10 4  plant derived particles/ml. 
     In a non-limiting embodiment, the gel comprising both trans-sialidase and one or more purified plant extracts may be administered topically to a subject in need of treatment, wherein the gel is administered one or more times per day. For example, the gel may be administered once, twice, three, four, five, or 6 or more times per day. In a non-limiting example, the gel may be applied topically once per day in the morning or evening during treatment. 
     In a non-limiting embodiment of the invention, the composition is applied to a lesion in an amount between about 0.1 ml/50 cm 2  and 100 ml/50 cm 2 , or between about 0.5 ml/50 cm 2  and 50 ml/50 cm 2 , and most preferably about 1.0 ml/50 cm 2  of cutaneous surface. 
     In an alternative embodiment, the composition is applied to the lesion in an amount between about 0.1 ml/5 cm 2  and 100 ml/5 cm 2 , or between about 0.5 ml/5 cm 2  and 50 ml/5 cm 2 , and most preferably about 1.0 ml/5 cm 2  of cutaneous surface. 
     In a non-limiting example, the methods and compositions of the invention may be effective for treating radiodermatitis lesions. Daily topical administration of the trans-sialidase gel once per day in the morning, and the purified plant extract gel once per day in the evening during a treatment period which may be between one week, two weeks, one month, two months, or three months and one year may be effective to reduce the lesions. The gels may be administered in an amount of between about 1.0 ml/5 cm 2  and 1.0 ml/50 cm 2 , or between about 1.0 ml/5 cm 2  and 50 ml/50 cm 2 , or between about 1.0 ml/5 cm 2  and 100 ml/50 cm 2 , wherein the trans-sialidase gel comprises an enzymatic activity of between about 1×10 −8  and 1×10 −4  U/ml, or between about 1×10 −7  and 1×10 −5  U/ml, or between about 1×10 −6  and 5×10 −6  U/ml, and preferably about 2.6×10 −6  U/ml. The purified plant extract gel may comprise between about 100 and 1×10 7  plant extract derived particles/ml, or between about 1×10 3  and 1×10 6  particles/ml, or between about 1×10 4  and 5×10 5  particles/ml and preferably about 3×10 5  particles/ml. Alternatively, the gel may be a mixture of both the trans-sialidase gel and the purified plant extract gel wherein the gel mixture comprises a trans-sialidase enzymatic activity of between about 1×10 −8  and 1×10 −4  U/ml, or between about 1×10 −7  and 1×10 −5  U/ml, or between about 1×10 −6  and 5×10 −6  U/ml, and preferably about 2.08×10 −6  U/ml, and further wherein the gel mixture comprises between about 100 and 1×10 7  plant extract derived particles/ml, or between about 1×10 3  and 1×10 6  particles/ml, or between about 1×10 4  and 1×10 5  particles/ml and preferably about 6×10 4  particles/ml. 
     The treatment may be administered between intervals of radiation treatment for cancer. Such treatment may be effective to reduce the size of radiodermatitis lesions by about 1%, 5%, 10%, 25%, 50%, 75%, 90%, or 100%. 
     In another non-limiting example, the methods and compositions of the invention may be effective for treating psoriasis. Daily topical administration of the trans-sialidase gel once per day in the morning, and the purified plant extract gel once per day in the evening during a treatment period which may be between one week, two weeks, one month, two months, or three months and one year may be effective to reduce the psoriatic lesions. The gels may be administered in an amount of between about 1.0 ml/5 cm 2  and 1.0 ml/50 cm 2 , or between about 1.0 ml/5 cm 2  and 50 ml/50 cm 2 , or between about 1.0 ml/5 cm 2  and 100 ml/50 cm 2 , wherein the trans-sialidase gel comprises an enzymatic activity of between about 1×10 −8  and 1×10 −4  U/ml, or between about 1×10 −7  and 1×10 −5  U/ml, or between about 1×10 −6  and 5×10 −6  U/ml, and preferably about 2.6×10 −6  U/ml. The purified plant extract gel may comprise between about 100 and 1×10 −7  plant extract derived particles/ml, or between about 1×10 3  and 1×10 6  particles/ml, or between about 1×10 4  and 5×10 5  particles/ml and preferably about 3×10 5  particles/ml. Alternatively, the gel may be a mixture of both the trans-sialidase gel and the purified plant extract gel wherein the gel mixture comprises a trans-sialidase enzymatic activity of between about 1×10 −8  and 1×10 −4  U/ml, or between about 1×10 −7  and 1×10 −5  U/ml, or between about 1×10 −6  and 5×10 −6  U/ml, and preferably about 2.08×10 −6  U/ml, and further wherein the gel mixture comprises between about 100 and 1×10 7  plant extract derived particles/ml, or between about 1×10 3  and 1×10 6  particles/ml, or between about 1×10 4  and 1×10 5  particles/ml and preferably about 6×10 4  particles/ml. 
     The treatment may be administered between intervals of radiation treatment for cancer. Such treatment may be effective to reduce the size of radiodermatitis lesions by about 1%, 5%, 10%, 25%, 50%, 75%, 90%, or 100%. 
     6 EXAMPLES 
     Example 1 
     Treatment of Radiodermatitis and Inflamed Cutaneous Lesions 
     In the following study, thirty-one patients presenting severe skin lesions caused by radiodermatitis after radiotherapy were treated with daily topical application of a trans-sialidase gel and an orchid gel to the lesions. Application of the gels decreased the edema and redness of the radiodermatitis lesions. 
     Materials and Methods 
     Production of Purified Plant Extract 
     Orchid flowers,  Cymbidium  ssp.,  Dendrobium nobile  and  Dendrobium moschatum  were immersed whole in 92% ethanol, in a 30:70 proportion plant weight:ethanol. The mixture was stored in a dark, anaerobic environment (in a sealed bottle), for at least 10 months. Following storage, the mixture was passed through Whatman qualitative filter paper grade 1, diameter 24 cm, pore size 11 um. Olive oil was then added at a volume of 10 ml per 1000 ml of the filtrate and stored for at least one additional month to increase the concentration of nanoparticles in the mixture. The mixture was then filtered a second time in Whatman qualitative filter paper grade 1. The filtrate was next filtered in a vacuum chamber, with a 47 mm diameter glass microfiber filter, pore size of 1.1 μm. 
     To determine the purity of the extract, 10 μl of the extract was mixed with 5 μl of acridine orange and placed on a glass microscope slide. The mixture was examined on an immunofluorescence optical microscope at 400× magnification. The extract quality was considered optimum when only fast moving small nanoparticles (30-150 nm) were visible. If large brilliant red particles (0.15-0.24 um) were observed in the mixture at a concentration of ≧1.0 large particle/visual field, the extract was submitted to tangential flow filtration in the Minitan Ultrafiltration System (Millipore, Bedford, Mass., USA), using the microporous membrane packet (30,000 NMWL) that concentrates large particles. The filtrate was then used in the experiments. 
     Production of Orchid Gel 
     The purified orchid extract was diluted 1:5 in thermal water (from Irai-RS, Brazil), which was previously boiled and filtered. The diluted orchid extract was then mixed in an anionic gel (Dermavita, Brusque, Santa Catarina, Brazil) until the mixture achieved a final concentration of 15% orchid extract. 
     Production of Trans-Sialidase 
     Purified trans-sialidase was produced from the  Escherichia coli  BLB21 DE3 inserted with the pTSII plasmidium, as described previously (International Patent application no. PCT/BR01/00083, filed Jul. 3, 2001). 
     Production of Trans-Sialidase Gel 
     Pure recombinant trans-sialidase was diluted 1:250,000 in MilliQ purified water. The diluted trans-sialidase was then mixed into Natrosol® gel (Dermavita, Brusque, Santa Catarina, Brazil), at a concentration of 1:1 and stored at 4° C. The trans-sialidase enzymatic activity of the gel mixture was between 2,000-5,000 CPM. 
     Production of a Trans-Sialidase-Orchid Gel Mixture. 
     A combined mixture of the orchid extract gel and trans-sialidase gel was created by mixing the two together. The two gels were combined to produce a final gel mixture that was 20% orchid extract gel and 80% trans-sialidase gel, or was in an orchid extract gel:trans-sialidase gel ration of 1:1, 1:2.5, 1:6 or 1:20. 
     Treatment with Orchid Extract Gel and Trans-Sialidase Gel 
     Thirty one patients presenting severe skin lesions including edema and redness, in the most severe cases associated with ulcers, caused by radiodermatitis after radiotherapy (median 5040 cGy; radiodermatitis degree 2-4) were treated with orchid extract gel and trans-sialidase gel. Treatment with the gels was administered between intervals of radiotherapy. Patients received treatment with the gels for a one year period. 26 patients were receiving radiotherapy for malignant neoplasia, and 5 patients were receiving radiotherapy for different inflammatory cutaneous discontinuities (Table II). Patients were treated with daily topical application (1.0 ml/50 cm2 of cutaneous surface) of trans-sialidase gel (once in the morning) and orchid gel (once in the evening). An additional 7 patients presenting radiodermatitis grade 3-4 lesions received treatment with a combined orchid extract gel and trans-sialidase gel during the treatment period. 
     Results 
     The 7 patients treated with the combined mixture of orchid extract gel and trans-sialidase gel, and 30 of the 31 patients treated with the once daily applications of the trans-sialidase gel and the orchid extract gel separately exhibited a decrease of edema and redness within two to eight days of treatment, along with healing of ulcers ( FIG. 1 ). Untreated lesions require between at least 20-30 days for healing to occur. One patient who was receiving chemotherapy in combination with radiotherapy during the study did not exhibit a decrease in edema or redness. 
     
       
         
           
               
             
               
                 TABLE II 
               
             
            
               
                   
               
               
                 Clinical data of the 31 patients treated with  
               
               
                 trans-sialidase gel and orchid extract gel. 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Radiodermatitis 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Case 
                 Sex/age 
                 Diagnosis 
                 degree 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 1. 
                 F/50y 
                 Breast cancer 
                 2 
               
               
                   
                 2. 
                 F/42 
                 Breast cancer 
                 2 
               
               
                   
                 3. 
                 F/54 
                 Breast cancer 
                 2 
               
               
                   
                 4. 
                 F/63 
                 Breast cancer 
                 3 
               
               
                   
                 5. 
                 F/69 
                 Breast cancer 
                 3 
               
               
                   
                 6. 
                 F/62 
                 Breast cancer 
                 2 
               
               
                   
                 7. 
                 M/32 
                 Melanoma 
                 2 
               
               
                   
                 8. 
                 F/58 
                 Breast cancer 
                 2 
               
               
                   
                 9. 
                 M/54 
                 Larynx cancer 
                 3 
               
               
                   
                 10. 
                 F/38 
                 Melanoma 
                 4 
               
               
                   
                 11. 
                 F/62 
                 Breast cancer 
                 3 
               
               
                   
                 12. 
                 F/45 
                 Breast cancer 
                 3 
               
               
                   
                 13. 
                 M/67 
                 Rectum cancer 
                 4 
               
               
                   
                 14. 
                 F/64 
                 Breast cancer 
                 2 
               
               
                   
                 15. 
                 F/38 
                 Breast cancer 
                 3 
               
               
                   
                 16. 
                 F/49 
                 Breast cancer 
                 2 
               
               
                   
                 17. 
                 F/28 
                 skin dehiscence 
                 — 
               
               
                   
                 18. 
                 F/28 
                 Breast cancer 
                 3 
               
               
                   
                 19. 
                 F/46 
                 Breast cancer 
                 4 
               
               
                   
                 20. 
                 F/63 
                 Breast cancer 
                 2 
               
               
                   
                 21. 
                 F/50 
                 Breast cancer 
                 3 
               
               
                   
                 22. 
                 F/70 
                 Breast cancer 
                 2 
               
               
                   
                 23. 
                 F/84 
                 Breast cancer 
                 2 
               
               
                   
                 24. 
                 F/58 
                 Breast cancer 
                 2 
               
               
                   
                 25. 
                 F/57 
                 Thymoma 
                 3 
               
               
                   
                 26. 
                 F/50 
                 Breast cancer 
                 2 
               
               
                   
                 27. 
                 F/62 
                 foreign body reaction 
                 — 
               
               
                   
                 28. 
                 M/62 
                 fistulae 
                 — 
               
               
                   
                 29. 
                 M/61 
                 skin dehiscence 
                 — 
               
               
                   
                 30. 
                 F/51 
                 Legs chronic dermatitis 
                 — 
               
               
                   
                 31. 
                 M/71 
                 Chemotherapy ulcers 
                 — 
               
               
                   
               
            
           
         
       
     
     Example 2 
     Treatment of Psoriasis and Psoriatic Arthritis with Trans-Sialidase Gel and Orchid Extract Gel 
     A 59 years old woman with severe psoriasis of the scalp, as well as many other parts of the body, was treated with the trans-sialidase and orchid extract gels. The two gels were prepared as described previously. The patient was treated by topical application of the gels once per day for two months. The trans-sialidase gel was applied in the morning, and the orchid extract gel was applied at night during each day of treatment. Following treatment, the psoriasis of the scalp regressed, exhibiting a decrease in the presence of the skin lesions ( FIG. 2 ). 
     A 47 year old woman with severe psoriatic arthritis received treatment with the trans-sialidase and orchid extract gels, prepared as previously described. The psoriatic arthritis presented as inflammation of the joints associated with psoriasis of the scalp, arms, hands, and legs. The psoriatic arthritis was characterized by retraction and immobilization of the fingers, and difficulty in raising the arms. Immunohistochemical analysis performed on cutaneous biopsies as previously described in Higuchi et al., 2006, APMIS 114:338-344, revealed a high presence of  Mycoplasma pneumoniae  and  Chlamydia pneumoniae  antigens on the affected tissue. The patient was treated for one year with both trans-sialidase and orchid extract gels. Each gel was applied once per day with the trans-sialidase gel applied in the morning, and the orchid extract gel applied at night. Following one year of treatment, the patient experienced a decrease in the presence of skin lesions, and an increase in mobility of the fingers and arms. 
     Example 3 
     Treatment of Joint and Column Pain with Trans-Sialidase Gel and Orchid Extract Gel 
     Ten patients suffering from joint and column pain, which was associated with articulation inhibition, were treated with the orchid extract gel or a mixture of both the orchid extract gel and the trans-sialidase gel for more than one year. 
     Three patients that were at least 65 years of age topically applied the orchid extract gel each time joint or column pain was experienced. Treated patients reported pain relief to be immediate, or within one hour after treatment with the orchid gel, and the relief lasted at least one day. 
     Seven patients aged 35 to 65 years with articular and muscle pain due to arthritis of different etiologies, bursitis and idiopathic myalgia, were treated with a combined orchid extract gel and trans-sialidase gel mixed at a ration of 1:1. The combined gel was applied twice a day (once every twelve hours) during a 15-30 day treatment period. Pain relief occurred in less than one hour following treatment and persisted during the entire treatment period. 
     Various publications are cited herein, the contents of which are hereby incorporated by reference in their entireties.