Title:  Use of nuclease to reduce wrinkles and discolorations in humans

United States Patent:  6,524,578

Issued:  February 25, 2003

Inventors:  Tackett; Scott E. (1418 Inglenook Dr., Jefferson City, MO 65109)

Appl. No.:  677838

Filed:  July 10, 1996

A method for reducing wrinkles and discoloration on human tissue skin cells which involves forming a nuclease solution and applying the nuclease solution to portions of a subject's body where the subject desires to have wrinkles or discoloration reduced or removed. The nuclease solution is comprised of a nuclease or nucleases, water, and co-factor of nuclease.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is for a method for reducing skin wrinkles and skin discoloration, such as "age spots", on a human being which works by applying a solution containing a nuclease to the surface of a human subject's skin. The nuclease solution is comprised of water, at least one nuclease, and a co-factor capable of activating the nuclease.

Also incorporated herein as non-essential material for purposes of indicating the background of the invention and illustrating the state of the art is Canadian Patent No. 1,312,298, which was invented and is owned by the inventor of the present invention.

The method of the present invention is used to apply a nuclease solution to a subject's skin, with the method summarized as follows, a nuclease solution is prepared and the nuclease solution is then applied to a human subject's skin to reduce wrinkles and discoloration spots. The method is initiated by first preparing the nuclease solution and then placing the nuclease solution in a delivery device which will allow the nuclease solution to contact the skin of the subject being treated. Preferably, the delivery device allows the subject to accurately place the nuclease solution where the subject desires. Typically, the delivery device used is either a wash cloth or similar type device which allows the nuclease solution to contact the skin without dissipating away from the desired area of contact. Also, an ice cube may be used to deliver the nuclease solution to the desired area. Finally, a delivery device does not have to be used, instead the nuclease solution can be applied directly on the subject. Thus, the nuclease solution is prepared and then placed on the area where the subject desires to have wrinkles and discoloration reduced. The method for reducing wrinkles and discoloration may be used once or a number of times depending upon the size and frequency of the wrinkles and discoloration.

The nuclease solution discussed herein is formed by combining an amount of water capable of effectively covering a desired portion of skin, with an effective amount of nuclease of sufficient concentration to readily digest extra-cellular DNA and/or RNA, and an effective amount of nuclease co-factor sufficient to activate the nuclease. Once the nuclease, water, and co-factor are combined, they are mixed and placed on a body area on a human subject. Where the solution is placed will depend upon where wrinkles or discoloration exist on the human and which wrinkles or discolorations the individual prefers to have reduced.

The most preferred nuclease composition used in the present method contains approximately 500 milliliters of water, approximately 60 grams of co-factor, preferably magnesium sulfate, and approximately 15,000 Kunitz units of DNase and 10,000 Kunitz units of RNase. The 60 grams of magnesium sulfate will form a one molar solution of magnesium sulfate in water. As mentioned, the solution may be applied directly to a human subject by using either a wash cloth or similar device which holds the nuclease solution and can be placed directly onto a subject's skin, whereby the solution is in contact with the subject's skin. Any other method of applying the nuclease solution to the subject's skin may be used as long as the nuclease is not degraded to the point that it will not adequately digest DNA and/or RNA. The amount of nuclease solution applied to a subject's skin may range between about 0.01 milliliters of nuclease solution per square centimeter of skin to about 10 milliliters of nuclease solution per square centimeter of skin. Additionally, the nuclease solution will be applied such that between about 1 Kunitz unit and about 50,000 Kunitz units of nuclease will be applied to each square centimeter of skin contacted by the nuclease solution. It is important to remember, however, that the activity of the nuclease is important, because that activity of the nuclease will generally determine the concentration of the nuclease applied. Thus, if a nuclease has higher activity, a lesser concentration of nuclease may be applied to accomplish the same result.

Further, it is possible to apply the nuclease enzyme directly to the skin of a subject without blending the nuclease into solution. This would work because the skin already contains some co-factor and water. While the nuclease solution is the most preferred way of contacting the nuclease with the skin it may be possible to apply the nuclease separately without solution.

Generally, any type of water is acceptable for use in the solution used in the process to reduce wrinkles. The water is important to the nuclease solution because not only does it provide a medium for forming a solution but, more importantly, the water provides a substance that oligonucleotides and polynucleotides can migrate into from the cytoplasm of a cell. When the skin cells are contacted by the nuclease solution, oligonucleotides and polynucleotides will migrate out of the cell's cytoplasm, through the cell wall, and into the nuclease solution. Also, the water will contact and mix with the extra-cellular fluid that is located between some of the skin cells.

The amount of water used is dependent upon the amount of skin that must be covered by the nuclease solution. Thus, the amount of water used is only limited by the amount of nuclease and co-factor available to the user of the solution, and the amount of skin to be contacted by the solution. Consequently, the amount of water used may vary from approximately a single drop or around one half milliliter to a bathtub full of water or around ten liters. Water may be added to a subject's treated skin to keep the skin damp and the reaction running.

Any nuclease, that is capable of degrading oligonucleotides and polynucleotides present in the cytoplasm of human skin cells, may be used. It is desirable to use a combination of nucleases so that both RNA and DNA oligonucleotides and polynucleotides present in the cytoplasm and extra-cellular fluid will be digested. The oligonucleotides and polynucleotides will migrate from the cytoplasm to the extra-cellular fluid and nuclease solution. The migration occurs as a result of diffusion under the gas law. Thus, a concentration gradient is created whereby the oligonucleotides and polynucleotides present in the cell's cytoplasm will diffuse into the extra-cellular fluid or nuclease solution because the extra-cellular fluid and nuclease solution contain a lesser concentration of oligonucleotides and polynucleotides. Also, the nucleotides will continue to migrate out of the cytoplasm as the concentration of oligonucleotides and polynucleotides drops in the extra-cellular fluid and nuclease solution. Thus, when choosing a nuclease it is important to use a nuclease that not only sufficiently degrades or breaks up large fragments of DNA and/or RNA, but also to choose a nuclease or combination of nucleases that will break-up both DNA and RNA oligonucleotides and polynucleotides.

The most preferred nuclease solution uses a combination of both Deoxyribonuclease and Ribonuclease so that fragments of both RNA and DNA are attacked and digested. If only DNase is used, the RNA which is hydrogen bonded to the chromosome may inhibit the removal of DNA from the chromosome. If only RNase is used, DNA may inhibit the removal of RNA from the chromosome.

The concentration of the nuclease used in the nuclease solution will generally range between a total concentration equal to from about 1 Kunitz unit to about 50,000 Kunitz units per human subject. However, there may be no upper limit to the amount of nuclease used, as using more nuclease may not significantly increase the rate of reaction. More preferably, the amount of nuclease used is a concentration of from about 1 Kunitz unit/milliliter of water to about 70 Kunitz units/milliliter of water. Furthermore, the nuclease solution may be applied to a subject in an amount equal to from about 0.5 milliliters of nuclease solution per square centimeter of skin to about 10 milliliters of nuclease solution per square centimeter of skin. A Kunitz unit is a unit of activity which results in the degradation of either DNA or RNA at a specified temperature and within a specified period of time. The more preferred concentration of nuclease in solution will be equal to an amount of Ribonuclease equal to from about 1 Kunitz unit to about 20,000 Kunitz units or between about 1 milligram and about 200 milligrams of Ribonuclease per liter of water and an amount of Deoxyribonuclease equal to a concentration of from about 1 Kunitz unit to about 30,000 Kunitz units or between about 1 milligram and about 50 milligrams of Deoxyribonuclease per liter of water. Some nucleases will have a higher concentration of activity or Kunitz units per milligram of protein than other nucleases. This means some nucleases degrade the DNA or RNA faster. Because the activity may be varied, the concentration of nuclease may differ from the above disclosed amounts. The most preferred activity is for a Ribonuclease to have an activity equal to about 100 Kunitz units per milligram of protein and for the Deoxyribonuclease to have an activity equal to about 1,500 Kunitz units per milligram of protein.

It is preferable to include a co-factor in the solution, as the co-factor is required to activate the DNase and may be required to activate the RNase. Without the co-factor the DNase will be unable to digest strands of DNA and/or RNA. Any co-factor may be used that is capable of activating the DNase. Typically, the preferred co-factor is a magnesium salt selected from the group consisting of magnesium chloride, magnesium phosphate, magnesium sulfate, and magnesium nitrate. The amount of co-factor used generally ranges between about 4.2 microMoles and about 2 Moles, with the most preferred concentration of co-factor equal to about 1 Mole.

Thus, the inventive process comprises the step of treating skin cells with a nuclease solution comprised of an exogenous nuclease added to a water solution. When the nuclease solution is placed on the surface of the skin it is in fluid communication with the skin cells, so that the exogenous nuclease migrates into the extra-cellular fluid or remains in the nuclease solution under suitable conditions and for an adequate period of time to allow the exogenous nuclease to digest extra-cellular oligonucleotides and polynucleotides which migrate into the extra-cellular fluid or nuclease solution. The exogenous nuclease will digest extra-cellular polynucleotides and oligonucleotides into nucleotides or oligonucleotides which are too short to bind with substantial avidity to the chromosomal DNA. This effectively alters the diffusion and uptake of extracellular oligonucleotides and polynucleotides into the cells, thereby altering the concentration and distribution of oligonucleotides and polynucleotides inside the cells which are bound to the chromosomes of the cells by hydrogen bonding.

The process of this invention does not lead to the alteration of the DNA in the main strands of the chromosomal double helices. Instead, this process can be roughly summarized by the phrase, "cleaning the chromosomes." The nuclease treatment of this invention alters the distribution of oligonucleotides and polynucleotides in the extra-cellular fluid and cytoplasm of the cell, which are generated by various processes such as digestion, cell death and lysis, etc. Extra-cellular oligonucleotides and polynucleotides can diffuse or be actively transported into cells, since they normally serve as nutrients once they enter a cell. However, the inventor has discovered that such oligonucleotides and polynucleotides, if they remain in relatively long strands, apparently can diffuse or be transported through the cytoplasm and into the nuclei of eukaryotic cells, including skin cells. After they reach the nuclei, the exogenous strands can anneal to single strands of DNA that become exposed when a segment of DNA temporarily opens up to allow for transcription, replication, or other processes.

When an exogenous polynucleotide or oligonucleotide anneals to an exposed single strand of chromosomal DNA, the semi-complementary polynucleotide or oligonucleotide binds to the chromosomal strand less tightly than the fully-complementary chromosomal strand would binds but still tightly enough to remain attached for some period of time. In a sense, the exogenous polynucleotide creates a "pseudo-strand" that binds to and entangles one of the strands of the chromosomal DNA, preventing that strand from returning to the double helical configuration with its fully complementary true chromosomal strand. In that manner, the pseudo-strand interferes with the proper functioning of any genes that are contained within or affected by the region of DNA that is entangled by the exogenous polynucleotide.

The problem of pseudo-strand interference remains unless and until the exogenous polynucleotide or oligonucleotide is removed somehow; however, the process of removal might never happen in a particular cell. A gene might remain entangled and encumbered by the exogenous polynucleotide until the cell or the organism that contains the cell eventually dies.

Alternatively, an exogenous polynucleotide or oligonucleotide might be substituted for the original DNA sequence. This process would involve several steps, including (1) the annealing of an exogenous strand to an exposed semi-complementary single strand of chromosomal DNA, followed by (2) the mistaken excision of the proper strand by a chromosomal repair mechanism which cannot properly distinguish between the proper strand and the pseudo-strand, followed by (3) the generation of a second strand of DNA which is incorporated within the chromosome, to match the exogenous strand. In this process, the exogenous strand would displace and eliminate the proper gene.

Even if an exogenous pseudo-strand is recognized as exogenous and is removed and digested into nucleotides by some chromosomal repair mechanism, the proper expression of the affected gene(s) will be inhibited for as long as it takes for the repair mechanism to recognize and get rid of the exogenous strand.

The subject inventive method centers around the following discovery: by digesting exogenous oligonucleotides and polynucleotides while they are in the extra-cellular fluid or after they have migrated into a nuclease solution from a cell's cytoplasm, it is possible to alter the presence and distribution of exogenous oligonucleotides and polynucleotides inside the cells. That process, in turn, alters the rate or quantity of hydrogen binding of exogenous oligonucleotides and polynucleotides to the chromosomes of the cells. The process is in essence a two part process whereby the exogenous nuclease degrades extra-cellular polynucleotides and oligonucleotides, which leads to a change in equilibrium between the extra-cellular polynucleotides/oligionucleotides and the intra-cellular polynucleotides/oligonucleotides, resulting in a passage of nucleotides from the cytoplasm of the cell to the extra-cellular fluid. Once this occurs there is a change in equilibrium between the non-chromosomal nucleotides, attached to the chromosome in the nucleus, and the nucleotides present in the cell's cytoplasm, which results in the passage of the non-chromosomal DNA and RNA from the nucleus, where it was hydrogen bonded to the chromosome, to the cytoplasm of the cell. Therefore, treatment with exogenous nucleases causes an alteration in the amount and type of protein produced in the "cleaned" cells. By "cleaning the chromosomes" (or, more precisely, by preventing them from becoming entangled by unwanted exogenous polynucleotides and oligonucleotides), proper gene expression is protected and enhanced. By preventing improper gene expression the amount of improper protein produced is limited. Because the amount of protein improperly produced is reduced it is theorized that this reduces the aging of cells. By reducing the aging of cells this prevents the breakdown of the skin tissue and thus prevents the formation of wrinkles.

As used herein, the term "nuclease" refers to an enzyme or enzymes capable of degrading extra-cellular DNA and/or RNA. An "exogenous" nuclease refers to a nuclease originally produced by cells other than the cells being contacted, and to nuclease produced by artificial means such as peptide synthesis or chemical alteration of a different enzyme.

The phrase, "altering the gene expression" refers to changes which are observable using available methodology. Such changes may involve phenotypic alternations such as cell or culture morphology, pH of cell metabolites or cell culture fluids, expression of one or more observable polypeptides, etc., or they may involve genotypic alternations observable by means of DNA or RNA hybridization using Southern or Northern blots or similar methods.

The phrase, "contacting the cells with an exogenous nuclease for the purpose of digesting extra-cellular nucleic acids" does not include cell and nuclease contacts which are merely incidental to other intended purposes. For example, whenever a cell dies, it releases nucleases which had been digesting mRNA into nucleotides within that cell's own cytoplasm. Such release is merely incidental, and is not included within the cited phrase.

The inventive process disclosed herein is not gene-specific genetic engineering, instead the process is used to remove unwanted oligonucleotides and polynucleotides from the cell's chromosome. The treatment can be used on any culture of human tissue skin cells, and the actual effects can be determined by visually observing the treated cells, as is shown in the accompanying photographs. It has been observed that use of the present process will not only reduce wrinkles and discoloration on human subjects, but increase hair growth in those areas treated with the process disclosed herein. The process in essence results in the cells being more efficient and prevents them from producing unneeded and undesirable proteins. This in turn results in less degradation of surrounding tissues, more efficient growth of surrounding tissues, and in essence a reversal of the aging process, which is hypothesized to be the result of the accumulation of unwanted proteins within the cells.

Claim 1 of 1 Claim

What is claimed is:

1. A method for reducing wrinkles and discoloration in humans which comprises the step of applying to human tissue skin cells an effective amount of a nuclease solution, said nuclease solution comprised of:

(a) water;

(b) an amount of nuclease equal to from about 1 Kunitz unit per millimeter of water to about 70 Kunitz units per milliliter of water, and

(c) a co-factor capable of activating the nuclease,

wherein said nuclease solution is applied on a subject's skin in an amount equal to from about 0.01 milliliters of said nuclease solution per 1 square centimeter of skin to about 1 milliliter of said nuclease solution per 1 centimeter of skin, and

wherein after application of said nuclease solution wrinkles and discolorations are reduced in humans.

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