Title:  Methods of increasing the efficacy of peroxides

United States Patent:  6,737,070

Issued:  May 18, 2004

Inventors:  Burkhart; Craig N. (4556 Crossfields Rd., Toledo, OH 43623)

Appl. No.:  091607

Filed:  March 6, 2002

Abstract

A method is provided for long term culture of proliferating hepatocytes that retain hepatic function to produce a hepatic cell culture. Hepatocytes and nonparenchymal cells are co-cultured ex vivo on a matrix coated with a molecule that promotes cell adhesion, proliferation or survival, in the presence of growth factors, resulting in a long-term culture of proliferating hepatocytes that retain hepatic function. The co-culturing method results in the formation of matrix/hepatic cell clusters that may be mixed with a second structured or scaffold matrix that provides a three-dimensional structural support to form structures analogous to liver tissue counterparts. The method can be used to form bio-articial livers through which a subjects blood is perfused. In an embodiment, the hepatocytes and nonparenchymal cells are derived from disaggregated liver tissue and are co-cultured in the presence of epidermal growth factor or heptocyte growth factor and beads coated with extracellular matrix protein, Alternatively, the hepatic cell culture may be implanted into the body of a recipient host having a hepatic disorder. Such hepatic disorders, include, for example, cirrhosis of the liver, induced hepatitis, chronic hepatitis, primary sclerosing cholangitis and alpha₁ antitrypsin deficiency.

SUMMARY OF THE INVENTION

The present invention relates to a novel tissue culture system that provides for long term culture of proliferating hepatocytes that retain their capacity to express hepatic function. The invention generally relates to compositions and methods for generating long term cultures of hepatocytes that can be used to produce three-dimensional hepatic cell culture systems. Such hepatic cell culture systems can be used to form bio-artificial livers that function as perfusion devices. Alternatively, the three-dimensional hepatic cell cultures may be implanted into a subject having a liver disorder.

The method of the present invention comprises the co-culturing of hepatocytes and nonparenchymal cells in the presence of growth factors and a matrix material coated with at least one biologically active molecule that promotes cell adhesion, proliferation or survival. The co-culturing method results in the formation of matrix/hepatic cell clusters containing a mixture of replicating hepatocytes and nonparenchymal cells. The method of the present invention may further comprise the mixing of the matrix/hepatic cell clusters in combination with a second structured, or scaffold matrix, that provides a three-dimensional structural support to form structures analogous to liver tissue counterparts.

Compositions of the present invention include populations of matrix/hepatic cell clusters comprising co-cultures of hepatocytes and nonparenchymal cells bound to a matrix coated with at least one biologically active molecule that promotes cell adhesion, proliferation or survival. Further, the invention provides a three-dimensional hepatic cell matrix system comprising a three-dimensional support matrix containing a population of matrix/hepatic cell clusters comprising hepatocytes and nonparenchymal cells bound to a matrix coated with at least one biologically active molecule that promotes cell adhesion, proliferation or survival.

The compositions of the present invention may be used to form bio-artificial livers through which a host's blood is perfused. Alternatively, the three-dimensional hepatic cell matrix system may be transplanted to a recipient host for providing hepatic function in subjects with liver disorders. The three-dimensional matrix system is administered in an effective amount to provide restoration of liver function, thereby alleviating the symptoms associated with liver disorders. The present invention, by enabling methods for generating long-term cultures of hepatocytes, provides a safer alternative to whole liver transplantation in subjects having liver disorders including, but not limited to, cirrhosis of the liver, alcohol induced hepatitis, chronic hepatitis, primary sclerosing cholangitis and alpha,-antitrypsin deficiency.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a novel tissue culture system that provides for long term culture of hepatocytes that retain their capacity to proliferate and express hepatic function. The invention provides compositions and methods for generating long term cultures of hepatocytes that can be used as bio-artificial livers for perfusion purposes. Alternatively, the hepatic cell culture systems may be implanted into a subject having a hepatic disorder to restore or supplement liver function.

The method of the present invention comprises the co-culturing of hepatocytes and nonparenchymal cells, in the presence of growth factors and a matrix material coated with at least one biologically active capable of a molecule promoting cell adhesion, proliferation or survival, thereby, resulting in the formation of matrix/hepatic cell clusters. The method of the present invention may further comprise the mixing of the matrix/hepatic cell clusters with a second matrix material that provides a three-dimensional structural support to form structures analogous to liver tissue found in vivo.

The compositions of the present invention include matrix/ hepatic cell cultures comprising hepatocytes that retain their capacity to proliferate while expressing hepatic function. Further, the invention provides a three-dimensional hepatic cell culture system comprising hepatic cells that retain their capacity to proliferate and express hepatic function growing in a three-dimensional structure.

The hepatic cell system can be used for generating bio-artificial livers that function as perfusion devices for restoration of liver function. The three-dimensional matrix hepatic cell system can be administered to an individual for providing hepatic function in subjects with liver disorders. The matrix/hepatic cell system is administered in an effective amount necessary for restoration of liver function, thereby alleviating the symptoms associated with liver disorders.

5.1. Mixed Cultures of Hepatocytes and Nonparenchymal Cells

The present invention relates to methods for generating long term cultures of proliferating hepatocytes that retain their hepatic function. The method generally comprises co-culturing or propagating hepatocytes and nonparenchymal cells on a matrix coated with a biologically active molecule that promotes cell adhesion, in vitro. The cells are cultured under conditions effective and for a time sufficient to allow formation of a culture of proliferating hepatocytes that retain hepatic function. The cells are grown in the presence of growth factors that maintain hepatic cell differentiation and the capacity to proliferate.

Hepatocytes and nonparenchymal cells may be obtained from a variety of different donor sources. In a preferred embodiment, autologous cells are obtained from the subject who is to utilize the bio-artificial liver or receive the transplanted hepatic cells to avoid immunological rejection of foreign tissue. In yet another preferred embodiment of the invention, allogenic liver tissue for use in purifying cells may be obtained from donors who are genetically related to the recipient and share the same transplantation antigens on the surface of their hepatic cells. Alternatively, if a sibling is unavailable, tissue may be derived from antigenically matched (identified through a national registry) donors.

In an embodiment of the invention, hepatic cells and nonparenchymal cells are isolated from a disaggregated liver tissue biopsy. This may be readily accomplished using techniques known to those skilled in the art. For example, the liver tissue can be disaggregated mechanically and/or treated with digestive enzymes and/or chelating agents that weaken the connections between neighboring cells, making it possible to disperse the tissue suspension of individual cells. Enzymatic dissociation can be carried out by mincing the liver tissue and treating the minced tissue with any of a number of digestive enzymes. Such enzymes include, but are not limited to, trypsin, chymotrypsin, collagenase, elastase and/or hylauronidase. A review of tissue disaggregation techniques is provided in, e.g., Freshney, Culture of Animal Cells, A Manual of Basic Technique, 2d Ed., A. R. Liss, Inc., New York, 1987, Ch. 9, pp.107-126. In addition to primary cell cultures, established hepatic cell lines may also be utilized in the methods and compositions of the invention.

The present methods and compositions can also employ hepatic cells genetically engineered to enable them to produce a wide range of functionally active biologically active proteins, including but not limited to growth factors, cytokines, hormones, inhibitors of cytokines, peptide growth and differentiation factors. Additionally, the cells may be genetically engineered to increase their proliferative capacity, i.e, the cells may be immortalized. Methods which are well known to those skilled in the art can be used to construct expression vectors containing a nucleic acid encoding the protein coding region of interest operatively linked to appropriate transcriptional/translational control signals. See, for example, the techniques described in Sambrook, et al., 1992, Molecular Cloning, A Laboratory Manuel, Cold Spring Harbor Laboratory, N.Y., and Ausebel et al., 1989, Current Protocols in Molecular Biology, Greene Publishing Associates & Wiley Interscience, N.Y., incorporated herein by reference.

Once isolated, the hepatic and nonparenchymal cells can be grown in any culture medium known to those skilled in the art to support the growth and proliferation of such cells. For example, the mixed cultures of cells can be grown in chemically defined hepatocyte growth medium (HGM) supplemented with specific growth factors and regulatory factors. Such factors can be added to the culture media to enhance, alter or modulate proliferation and/or differentiation of the cultured hepatocytes and nonparenchymal cells. In a preferred embodiment of the invention, the culture media may be supplemented with growth factors such as hepatocyte growth factor (HGF) and/or epidermal growth factor (EGF), or functional homologs thereof, to impart phenotypic stability in terms of differentiated hepatocyte gene expression and the ability to proliferate.

In addition, the co-cultures of cells are propagated in the presence of a natural or synthetic matrix that provides support for hepatic cell growth during in vitro culturing. The type of matrix that may be used in the practice of the invention is virtually limitlessness. The matrix will have all the features commonly associated with being "biocompatible", in that it is in a form that does not produce an adverse, or allergic reaction when administered to the recipient host. In a preferred embodiment of the invention, the matrix is in the form of a bead to which the cultured cells may adhere. The beads may be composed of variety of different substances including, but not limited to, synthetic materials or naturally derived materials. The type of matrix material to be used will depend on the desired use of the hepatocyte cultures. For example, when the matrices are to be transplanted into a subject it is preferred that a biodegradable matrix material be used. For purposes of forming bio-artificial livers, the matrix may be composed of any suitable material to which the hepatocytes and nonparenchymal cells will adhere and proliferate.

Further, to improve hepatic cell adhesion, proliferation or survival, the matrix is coated on its external surface with factors known in the art to promote cell adhesion, growth or survival. Such factors include cell adhesion molecules, extra-cellular matrix molecules and/or growth factors for hepatocytes and/or nonparenchymal cells. Matrices may also be designed to allow for sustained release of growth factors over prolonged periods of time. Thus, appropriate matrices will ideally provide factors known to promote hepatic cell adhesion, growth or survival, and also act as a support on which the cultured cells differentiate and proliferate. In a preferred embodiment of the invention, the hepatic cell cultures are propagated in media containing matrices coated with collagen type I protein for promotion of cell adhesion and proliferation of bound hepatocytes.

The method of the present invention involves the co-culturing of hepatic and nonparenchymal cells in the presence of the selected matrix material. Although the cells may be propagated under static conditions, it is preferred that the cells arc propagated under mixing or stirring conditions wherein a cell suspension is combined with matrix, and mixed or stirred, to enhance the number and frequency of cell contacts with the matrix to maximize cell adhesion to the matrix, but not disrupt adherence to cells. Such conditions may be generated in variety of different ways including, for example, the use of roller bottles to provide continuous stirring or mixing of the culture. Preferably, the stirring is continued throughout the culturing of the hepatic and nonparenchymal cells.

The conditions of long-term matrix-cell culturing will preferably be maximized to enhance hepatocyte proliferation while maintaining hepatic function. Although certain variations in cell number, seeding techniques, culture media, incubation temperatures and incubation times, may be utilized, such variations would be routine to those skilled in the art and are encompassed by the present invention.

5.2. Preparation of Three-dimensional Culture Systems

The present invention further relates to the use of the matrix/hepatic/nonparenchymal cell clusters, produced as described in Section 5.1, for generation of three-dimensional hepatic cell culture systems to form structures analogous to liver tissue counterparts. The method of the invention comprises growing hepatic and nonparenchymal cells on a three-dimensional matrix in vitro under conditions effective and for a period of time sufficient to allow proliferation of the cells to form a three-dimensional structure.

The three-dimensional matrices to be used are structural matrices that provide a scaffold for the cells, to guide the process of tissue formation. Cells cultured on a three-dimensional matrix will grow in multiple layers to develop organotypic structures occurring in three dimensions such as ducts, plates, and spaces between plates that resemble sinusoidal areas, thereby forming new liver tissue. Thus, in preferred aspects, the present invention provides a three-dimensional, multi-layer cell and tissue culture system. The resulting liver tissue culture system survives for prolonged periods of time and performs liver-specific functions for use as a perfusion device or following transplantation into the recipient host.

A wide variety of structural matrices may be used in the context of the present invention for preparation of a three-dimensional hepatic cell culture system. In preferred embodiments, the matrices are bio-compatible matrices that provide a scaffold for the cells to guide the development of tissue. Preferred matrices are generally those that define a space for subsequent tissue development. Such matrices include hydrogels, biomatrix gels, or porous materials such as fiber based or sponge like matrices. The culture system described herein provides for the proliferation of cells to form structures analogous to liver tissue counterparts in vivo.

In certain embodiments, synthetic matrices, such as synthetic polymer matrices, may be used. Such matrices include, but are not limited to, nylon, dacron, polystyrene and homopolymers or heterpolymers such as polylactic acid (PLA) polymers, polyglycolic acid (PGA) polymers and polylactic acid-polyglycolic acid (PLGA) copolymer matrices. In other embodiments, matrices for use in the invention may be naturally-derived matrices extracted from or resembling extracellular matrix materials such as a collagen matrix, such as type I collagen. Other naturally derived matrix materials include laminin-rich gels, alginate, agarose and other polysaccharides, gelatin and hyaluronic acid derivatives. Certain matrix materials may not support efficient cellular attachment and, in such instances, it may be advantageous to coat the matrix with molecules that promote cell adhesion, such as extracellular matrix proteins or, specifically, collagen type I.

To generate the three-dimensional hepatic cell cultures, matrix/hepatic/nonparenchymal cell clusters generated as described above in Section 5.1 are isolated from cell culture suspensions. For example, the cell clusters may be isolated by low speed gravity sedimentation. The matrix/hepatic/nonparenchymal cell clusters are then exposed to a second structural matrix material in the presence of an appropriate culture media, thereby providing an environment for three-dimensional hepatic cell growth. Many commercially available culture media, supplemented in some instances with growth factors and the like, may be suitable for use. In addition, the culture media may be replenished periodically to provide a fresh supply of nutrients. The three-dimensional hepatic cell culture system is cultured for a sufficiently long period of time to allow the hepatic cells to replicate to form a three-dimensional cell or tissue structure.

Prior to use of three-dimensional hepatic cell cultures, the cultures may be contacted with a number of different growth factors that can regulate tissue regeneration by affecting cell proliferation, and gene expression. Such growth factors include those capable of stimulating the proliferation and/or differentiation of hepatic progenitor cells. For example, epidermal growth factor (EGF), transforming growth factor .alpha. (TGF-.alpha.) or hepatocyte growth factor (HGF) may be utilized. The hepatic cells may be stimulated in vitro prior to transplantation into the recipient subject, or alternatively, by injecting the recipient with growth factors following transplantation.

5.3. Use of the Hepatic Cell Cultures

The hepatic cell cultures of the invention can be used as bio-artificial livers for use by subjects having liver disorders that result in hepatic failure or insufficiency. The use of such bio-artificial livers involves the perfusion of the subject's blood through the bio-artificial liver. In the blood perfusion protocol, the subject's blood is withdrawn and passes into contact with the hepatocyte cell cultures. During such passage, molecules dissolved in the patient's blood, such as bilirubin, are taken up and metabolized by the hepatocyte cultures. In addition, the cultured hepatocytes provide factors normally supplied by liver tissue.

To form the bio-artificial liver the three-dimensional hepatocyte cell cultures of the invention are grown within a containment vessel containing an input and output outlet for passage of the subjects blood through the containment vessel. The bio-artificial liver further includes a blood input line which is operatively coupled to a conventional peristaltic pump. A blood output line is also included. Input and output lines are connected to appropriate arterial-venous fistulas which are implanted into, for example, the forearm of a subject. In addition, the containment vessel may contain input and output outlets for circulation of appropriate growth medium to the hepatocytes for continuous cell culture within the containment vessel.

In an embodiment of the invention, semipermeable membranes may be included in the bio-artificial livers to prevent direct contact of the subject's blood with the three-dimensional hepatocyte cultures. In such instances, the molecules dissolved in the subject's blood will diffuse through the semipermeable membrane and are taken up and metabolized by the hepatocycte cultures.

The use of the cultured hepatocyte systems of the invention to form bio-artificial livers provides a method which may be utilized to provide liver function to subjects suffering from hepatic failure or insufficiency.

The three-dimensional hepatic cell cultures can also be administered or transplanted to the recipient in an effective amount to achieve restoration of liver function, thereby alleviating the symptoms associated with liver disorders. When the hepatic cell cultures are to be administered to a recipient, it is desirable to form the hepatocyte cultures with hepatocytes and nonparenchymal cells derived from the recipient so as to avoid tissue rejection.

The number of cells needed to achieve the purposes of the present invention will vary depending on the degree of liver damage and the size, age and weight of the host. For example, the cells are administered in an amount effective to restore liver function. Determination of effective amounts is well within the capability of those skilled in the art. The effective dose may be determined by using a variety of different assays designed to detect restoration of liver function. The progress of the transplant recipient can be determined using assays that include blood tests known as liver function tests. Such liver function tests include assays for alkaline phosphatase, alanine transaminase, aspartate transaminase and bilirubin. In addition, recipients can be examined for presence or disappearance of features normally associated with liver disease such as, for example, jaundice, anemia, leukopenia, thrombocytopenia, increased heart rate, and high levels of insulin. Further, imaging tests such as ultrasound, computer assisted tomography (CAT) and magnetic resonance (MR) may be used to assay for liver function.

The three-dimensional hepatic cell system can be administered by conventional techniques such as injection of cells into the recipient host liver, injection into the portal vein, or surgical transplantation of cells into the recipient host liver. In some instances it may be necessary to administer the hepatic cell composition more than once to restore liver function. In addition, growth factors, such as G-CSF, or hormones, may be administered to the recipient prior to and following transplantation for the purpose of priming the recipients liver and blood to accept the transplanted cells and/or to generate an environment supportive of hepatic cell proliferation.

Claim 1 of 6 Claims

We claim:

1. A population of hepatocytes and nonparenchymal cells, derived using a method comprising:

co-culturing hepatocytes and nonparenchymal cells, derived from disaggregated liver tissue, in the presence of

(a) one or more growth factors that support the growth of hepatocytes comprising epidermal growth factor or hepatocyte growth factor and

(b) beads coated with extracellular matrix protein that promotes cell adhesion under conditions sufficient to allow for the proliferation of said hepatocytes while retaining hepatic faction of said hepatocytes.

Abstract

This invention relates to methods of increasing the efficacy of peroxides such as benzoyl peroxide in the treatment of skin conditions such as acne. In a preferred embodiment, the invention relates to methods of increasing radicals formed by peroxides on/in the skin, more specifically near/in the comedone, for topical use in dermatology. In a specific embodiment, the invention relates to the use of transitional metals such as Cu(1) and ferrous ions-to increase the efficacy of peroxides such as benzoyl peroxide. In another embodiment, the invention relates to a method by which a peroxide such as benzoyl peroxide and its activator are added to the skin surface at the same time. In another embodiment, the invention relates to the use of a more soluble form of peroxide such as benzoyl peroxide to increase its efficacy. In another embodiment, the invention relates to the addition of a side chain to a peroxide such as benzoyl peroxide so that it is activated by light. In a further embodiment, the invention relates to the addition of a tertiary amine to a peroxide such as benzoyl peroxide at the time of skin application, to improve the efficacy of the peroxide. In another embodiment, the invention relates to the addition of dapsone or other material to a peroxide such as benzoyl peroxide to improve its efficacy.

SUMMARY OF THE INVENTION

This invention relates to methods of increasing the efficacy of peroxides such as benzoyl peroxide in the treatment of skin conditions such as acne. In a preferred embodiment, the invention relates to methods of increasing radicals formed by peroxides on/in the skin, more specifically near/in the comedone, for topical use in dermatology.

In a specific embodiment, the invention relates to the use of transitional metals such as Cu(1) and ferrous ions to increase the formation of peroxide radicals such as benzoyl peroxide radical.

In another embodiment, the invention relates to a method by which a peroxide such as benzoyl peroxide and its activator (or adjunctive agent) are added to the skin surface at the same time (and not days or months before). This ensures that the ingredients are not inactivated or lost strength by being placed together prior to usage.

In another embodiment, the invention relates to the use of a more soluble form of peroxide such as benzoyl peroxide to increase its efficacy.

In another embodiment, the invention relates to the addition of a side chain to a peroxide such as benzoyl peroxide so that it is activated by light.

In a further embodiment, the invention relates to the addition of a tertiary amine to a peroxide such as benzoyl peroxide at the time of skin application, to improve the efficacy of the peroxide. This could include any tertiary amine structure except for an erythromycin structure.

In another embodiment, the invention relates to the addition of dapsone or other material to a peroxide such as benzoyl peroxide to improve its efficacy.

Various advantages of this invention will become apparent to those skilled in the art from the following detailed description of the preferred embodiments.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

This invention relates to methods of increasing the efficacy of peroxides such as benzoyl peroxide in the treatment of skin conditions such as acne. In a preferred embodiment, the invention relates to methods of increasing radicals formed by peroxides on/in the skin, more specifically near/in the comedone (but not limited thereto), for topical use in dermatology. The methods use the radicals formed by peroxides such as benzoyl peroxide, optimizing conditions such that the skin/comedone is the only place they are formed as opposed to in a storage container or wherever the benzoyl peroxide happens to be from the time of application to when the benzoyl peroxide breaks down into its radicals or is metabolized).

The methods of the invention may use the principles of photodynamic therapy directed at acne. Instead of forming radicals in cancer cells, the methods form radicals in/by the comedone (skin surface, sebum within P. acnes). Location and timing of formation of radicals is a very important part of the methods.

The methods use the assumption that radicals derived from BP or other peroxides are the most useful in acne therapy (as opposed to reactive oxygen intermediates used in photodynamic therapy).

In a specific embodiment, the invention relates to the use of transitional metals such as Cu(1) and ferrous ions to increase the efficacy of peroxides such as benzoyl peroxide. The use of transitional metals such as Cu(1) and ferrous ions (as alluded to in the text) to increase the efficacy of benzoyl peroxide. It is anticipated that such an addition to benzoyl peroxide would increase the generation of benzoyloxyl radicals.

The transitional metals include all the elements between Group IIA and IIIa in the periodic table. The list includes zinc, cadmiumn, mercury, scandium, titanium, vanadium, chromium, manganese, yttrium, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver, lanthanum, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, mercury, actinium, unnilquadium, unnilpentium, unnilhexium, and uniseptium.

A few characteristics of transitional metals include:

most are harder and more brittle with higher melting points, boiling points, and heats of vaporization than the non-transitional metals.

their ions and compounds are usually colored.

they form many complex ions.

most exhibit multiple oxidation states.

many of them are paramagnetic, as are many of their compounds.

many of the metals and associated compounds are effective catalysts.

In another embodiment, the invention relates to a method by which a peroxide such as benzoyl peroxide and its activator (or adjunctive agent) are added to the skin surface at the same time (and not days or months before). An example of such would be a better package system in which the various ingredients that would be added to benzoyl peroxide would be put into a dispenser with two or three chamber (depending upon the number of items combined) to separate the product's ingredients so they do not interact until the instant you apply them to one's acne. This separation would ensure that the ingredients are not inactivated or lost strength by being placed together prior to usage.

Another example of such a system would be benzoyl peroxide (bp) dissolved in a hydrophobic solvent and the activator in a polar solvent. The BP and activator wouldn't meet until applied onto the skin surface. Lipophilic carriers are well known in the art. For an example of the activator in a hydrophilic solvent, both protic and aprotic solvents are included. Protic solvents such as methanol, ethanol, forinamide, N-methylformamide, and water, a hydrogen is attached to the electronegative part of the reagent. The hydrogen has a proton-like character and strongly reacts with anionic nucleophiles. Aprotic solvents do not contain positively polarized hydrogens. These include acetone, acetonnitrile, N,N-dimethylformarnide, DMSO, hexamaethylphophoric triamide--the aprotic solvents increase the reactivity of nucleophiles in SN2 reactions (the possible mechanism of radical formation by the BP tertiary amine combination).

Retin A micro is an example of a product released by a polymer. The retin A is stored in a small polymer bead. After application of these beads onto the skin, retin A slowly diffuses out of the polymer and into the skin. The invention would have the activator of benzoyl peroxide radical formation contained in a similar polymer. The activator would be slowly released (by diffusion or breakdown of the polymer) into the skin allowing it to react with BP. Alternatively, the BP could be stored in and released from the polymer. Or, both the activator and BP could be released from their own individual polymers to react when the meet (in the environment of the skin/comedone).

In another embodiment, the invention relates to the use of a more soluble form of peroxide such as benzoyl peroxide to increase its efficacy. The use of a more soluble form of benzoyl peroxide. The present-day products actually use benzoyl peroxide in the form of crystals. We are able to solubilize benzoyl peroxide either by altering its hydric solvents, or by adding a side chain to its structure.

In another embodiment, the invention relates to the addition of a side chain to a peroxide such as benzoyl peroxide so that it is activated by light. We could also add a side chain to benzoyl peroxide so that it is activated by light.

In a further embodiment, the invention relates to the addition of a tertiary amine to a peroxide such as benzoyl peroxide at the time of skin application, to improve the efficacy of the peroxide. This could include any tertiary amine structure except for an erythromycin structure. We believe that benzoyl peroxide efficacy can be improved by adding a tertiary amine at the time of skin application. Therefore, we would be including all substances (and chemicals) which have a tertiary amine within the provisional patent, be they antibiotics or whatever. The invention would include all tertiary amine structures, save for the erythromycin structure that is presently used in a commercial product named benzymycin.

Some nonlimiting examples of tertiary amines include Alfuzosin, Alimemazine, Analgesic drug (Reference 97), Atropine, alpha,alpha-bis [3-(N-benzyl-N-methyl-carbamoyl)-piperidino]-p-xylene dihydrobromide, Bupivacaine, cis-trans-Cavinton, Cloperastine, Cyamemeazine, Cyclopentolate, 2-(4,5-dihydro-1H-imidazol-2-yl)-2-propyl-1,2,3,4-tetrahydropyrrolo]3,2,1- hi[-indole, 1-decyl-3-(N,N-diethylcarbamoyl)piperidine hydrobromide, Diltiazem, Dimethindene, Diperodone, Disopyramide, Disopyamide, semipreparative, Dixyrazine, Doxazosin, Dropropizine, Hydroxychloroquine and metabolites, Ketoconazole, Laudanosine, Marcaine, Medetomidine, Mepivacaine, Mepivacaine (micro column), Meptazinol, Methadon, Nefopam, Nicotine, Omeprazole, Oxybutynin, Oxyphencyclimide, Pheniramine, 3-PPP, Procyclidine, Promethazine, Proxyphylline, Remoxipride, Tetrahydrozoline, Tetramisole, Tetramisole (micro column), Thioridazine ring-sulphoxide, Tolperisone, Trihexyphenidyl, Trimipramine, Tropicamide, Vamicamide, Verapamil, and Vinca alcaloids. The structures and other characteristics of these tertiary amines can be found on the internet at www.chromtech.se/tertiary.htm. The listed amines are all drugs, but the methods of the invention are not limited to just drugs--any tertiary amine would work.

Along with transition metals, tertiary amines potentiate radical formation by BP. A possible mechanism involves reaction of the amine and BP by a S_N 2 mechanism. The intermediate thus formed thermally decomposes to benzoyloxy radicals and an amine radical cation. The benzoyloxy radicals may further decompose into phenyl radicals. All of these radicals can react with biological molecules possibly causing some biological effect.

In another embodiment, the invention relates to the addition of dapsone to a peroxide such as benzoyl peroxide to improve its efficacy. Heme is a protoporphyrin. P. acnes actually produces protoporphyrins. 5-aminolevulinlc acid (ALA) increases protoporphyrin production by P. acnes. ALA is the same stuff used in photodynamic chemotherapy and photodynamic antimicrobial chemotherapy. Methylene blue, toluidine blue O, phthalocyanine, and haematoporphyrin derivative could also be used. Phenothiazinium dyes could also be used. These materials might work by depleting the antioxidant levels in/around the comedone allowing the BP derived radicals to reach the comedone or spread further throughout the comedone.

Viagra (sildenafil) increases NO production by blood vessels (and maybe the skin). It is an example of a molecule inducing the skin to produce a benzoyl peroxide activator.

Testing and Discussion

Objective: The purpose was to compare radical activity of BP alone and with various antibiotics to determine whether BP and antibiotics may be chemically synergistic.

Methods: Polymerization of tetra ethylene glycol dimethacrylate was used as a test of BP radical activity. Solutions of BP, antibiotics, and BP and antibiotics were made at 3% w/w in tetraethylene glycol dimethacrylate. All of the antibiotics except erythromycin (ERY) were obtained from prescription pills, which were crushed in a crucible. The portion of the pills that disolved in tetraethylene glycol dimethacrylate were used in the experiment. ERY was obtained in powdered form from Benzamycin.RTM. acne treatments. Aliquots of ten drops of these solutions were placed in an eight well plastic plate. The samples were heated in an oven that maintained a temperature range between 90 to 100 degrees Celsius. After various amounts of time the samples were taken out of the oven and tested for gel formation. Polymerization of tetraethylene glycol dimethacrylate was detected visually by swirling a spatula in the solutions. Gelling constituted an indicator of BP radical activity.

Results: The results suggest that radical activity increases upon addition of certain antibiotics, such as erythromycin, to a solution of BP. ERY, minocycline (Vectrin.RTM.), and levofloxacin (Levaquin.RTM.) in combination with BP caused the tetraethylene glycol dimethacrylate to polymerize the fastest. This is assumed to be due to elevated BP radical formation. Agents that did not augmnent BP radical activity included doxycycline (Monodox.RTM.), and trovofloxacin (Trovan.RTM.). Upon storage in a dark room at room temperature, the ERY-BP combination gelled within an hour. The Vectring.RTM.-BP, Diflucang.RTM.-BP, Trovan.RTM.-BP, Monodox.RTM.-BP, and Levaquin.RTM.-BP combinations did not gel within six hours. Zithromycin.RTM. (a prescription drug containing a macrolide similar to ERY) in combination with BP also gelled within an hour when stored in a dark room at room temperature. Furthermore, Zithromycin.RTM.-BP and ERY.RTM.-BP solutions gelled within an hour when stored in a refrigerator. Zithromycin.RTM. has not been tested yet at higher temperatures.

Discussion: BP induces a variety of biological effects. BP can inhibit metabolic cooperation, alter protein synthesis, induce omithine decarboxylase activity, cause DNA strand breaks, suppress DNA synthesis, and may interfere with mitochondrial respiration. Several of these effects, such as DNA strand breaks, may be caused by BP-derived radicals. Thus, acne treatments that increase the radical activity of BP may be more effective.

Tertiary amines potentiate radical formation by BP. A possible mechanism involves reaction of the amine and BP by a S_N 2 mechanism. The intermediate thus formed thermally decomposes to benzoyloxy radicals and an amine radical cation. The benzoyloxy radicals may further decompose into phenyl radicals. All of these radicals can react with biological molecules possibly causing some biological effect. Of the antibiotics tested, ERY, doxycycline (Monodox.RTM.), minocycline (Vectrin.RTM.), levofloxacin (Levaquin.RTM.), and trovofloxacin (Trovan.RTM.) contain tertiary amines. ERY-BP, Levaquin.RTM.-BP, and Vectrin.RTM.-BP combinations all behaved as would be expected as they demonstrated faster kinetics for radical formation than BP alone.

Contaminants and solubility may have caused the unexpected results from the Monodox.RTM.-BP and Trovan.RTM.-BP combinations. The extra chemicals contained in the pills may have dissolved in the tetraethylene glycol dimethacrylate and acted as plastisizers or radical scavengers, thus, hiding any enhanced radical formation by the antibiotic-BP combination. On the other hand, the contaminants may have accelerated the formation of BP-derived radicals. The contaminants may have affected the results for the Levaquin.RTM.-BP and Vectrin.RTM.-BP combinations as well. Furthermore, some of the antibiotics may not have dissolved in the tetraethylene glycol dimethacrylate, thus, preventing them from being involved in the experiment as only dissolved material was transferred to the plastic plate for testing.

The most impressive result was the speed that the ERY-BP and Zithromycin.RTM.-BP solutions gelled at room temperature and below. The speed of reaction between the macrolides and BP insinuates that all the BP in Benzamycin.RTM. may be completely depleted by the time a patient picks up his/her prescription to the time it is applied to his/her body. As Benzamycin.RTM. is a very effective drug for the treatment of acne, a novel drug may be formed as a product of reactions of BP and ERY with each other and/or other components in Benzamycin.RTM. that is very effective against acne. Finding this chemical may result in the discovery of improved acne treatments that do not require BP. As Zithromycin.RTM. similarly increased BP radical formation, it is probable that many macrolides mixed with BP are effective drugs for the treatment of acne.

It may be true that the BP is protected from ERY while stored in its container. For example, much of BP is in a less reactive crystalline form while in acne creams, where as it was fully dissolved in these experiments. Upon application to the skin these crystals of BP may dissolve and react with ERY producing radicals. Depending on where these radicals are formed DNA strand breaks, lipid peroxidation, or other effects may occur.

Conclusion: Radical activity of BP in tetraetylene glycol dimethacrylate is of increased when tested in consort with several antibiotics, such as the macrolides. We propose that the tertiary amines contained on certain antibiotics are responsible for catalysis of BP radical formation. If increased radical formation correlates with enhanced biological effect, then these data reveal the possibility of biological synergism in mixtures of BP and antibiotics. An understanding of the mechanism of catalysis of BP radical formation by antibiotics may lead to the discovery of improved treatments for acne.

In accordance with the provisions of the patent statutes, the principle and mode of operation of this invention have been explained and illustrated in its preferred embodiment. However, it must be understood that this invention may be practiced otherwise than as specifically explained and illustrated without departing from its spirit or scope.

Claim 1 of 17 Claims

What is claimed is:

1. A method of topically treating a skin condition comprising applying to the skin a combination of a peroxide and a tertiary amine, with the exclusion of erythromycin, the tertiary amine increasing radicals formed by the peroxide on/in the skin to thereby increase the efficacy of the peroxide in the treatment of the skin condition.

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