Patent Application
20070254853
While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.
To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as “a”, “an” and “the” are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.
The present invention provides preparations and methods of using the preparations for the enhancement of the quality and rate of wound healing in immunosuppressed patients. Methods for preparing the various formulations are also provided for the topical delivery of the compounds of the present invention. As a therapeutic agent, the invention is a concentration of nucleotides together in a pharmaceutical acceptable carrier sufficient to promote wound healing. The therapeutic agents of the invention may be delivered topically to an organism through any of a number of dressings with equal therapeutic efficiency. The methods of the present invention may vary by the wound dressing chosen, the type of wound, the organism treated, the type of immunosuppressive agent and the time-frame of the treatment relative to the time of the wound healing. There are also a variety of methods of preparing the preparations, formulations and/or dressings encompassed within the contemplated scope of the present invention.
As used herein, the terms “active agent,” “pharmacologically active agent” and “drug” are used interchangeably herein to refer to a chemical compound that induces a desired pharmacological, physiological effect, namely wound healing. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of those active agents specifically mentioned herein, including, but not limited to, salts, esters, amides, pro-drugs, active metabolites, analogs, and the like. When the terms “active agent,” “pharmacologically active agent” and “drug” are used, then, it is to be understood that applicant intends to include the active agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, pro-drugs, metabolites, analogs, etc.
As used herein, the terms “contact”, “contacted”, and “contacting”, are used to describe the process by which a pharmacological agent, e.g., any of the compositions disclosed in the present invention, comes in direct juxtaposition with the target cell(s), tissue or wound site.
As used herein, the term “sustained release” refers to a drug formulation that provides for gradual release of a drug over an extended period of time, and that preferably, although not necessarily, results in substantially constant drug levels at the desired site over an extended period of time.
As used herein, the terms “effective amount” or “therapeutically effective amount” refer to the amount of an agent in a sufficient amount of the agent to provide the desired therapeutic effect. Furthermore, an “effective angiogenesis-inhibiting amount” of an agent is a sufficient amount of the agent to at least partially inhibit angiogenesis. Of course, undesirable effects, e.g., side effects, are sometimes manifested along with the desired therapeutic effect; hence a practitioner balances the potential benefits against the potential risks in determining what is an appropriate “effective amount” at the site or target of delivery of the active agent. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, mode of administration, and the like. Thus, it is not possible to specify an exact “effective amount.” However, an appropriate “effective amount” or “effective wound healing amount” in any individual case may be determined by one of ordinary skill in the art using only routine experimentation.
As used herein, the term “pharmaceutically acceptable” carrier refers to a pharmaceutical vehicle comprised of a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the selected active agent without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical formulation in which it is contained. Carriers may include excipients and other additives such as diluents, detergents, coloring agents, wetting or emulsifying agents, pH buffering agents, preservatives, and the like.
Of course, the nucleotide active compounds of the present invention may be administered topically by any of the other numerous techniques known to those of skill in the art as taught by, e.g., Remington's Pharmaceutical Science 18th Edition, (1990), which is specifically incorporated herein in pertinent part for this purpose. Supplementary active ingredients can also be incorporated into the compositions, e.g., analgesics, antibiotics and the like.
As used herein, the term a “pharmacologically acceptable” salt, ester, amide, prodrug, or derivative of a compound refers to a salt, ester, amide, prodrug, or derivative that is not biologically or otherwise undesirable.
As used herein, the term “substrate,” or “solid support” and “wound dressing” refer broadly to any substrate when prepared for, and applied to, a wound for protection, absorbance, drainage, etc. The present invention may include any one of the numerous types of substrates and/or backings that are commercially available, including films (e.g., polyurethane films), hydrocolloids (hydrophilic colloidal particles bound to polyurethane foam), hydrogels (cross-linked polymers containing about at least 60% water), foams (hydrophilic or hydrophobic), calcium alginates (non-woven composites of fibers from calcium alginate), and cellophane (cellulose with a plasticizer). The shape and size of a wound may be determined and the wound dressing customized for the exact site based on the measurements provided for the wound. As wound sites can vary in terms of mechanical strength, thickness, sensitivity, etc., the substrate can be molded to specifically address the mechanical and/or other needs of the site. For example, the thickness of the substrate may be minimized for locations that are highly enervated, e.g., the finger tips. Other wound sites, e.g., fingers, ankles, knees, elbows and the like, may be exposed to higher mechanical stress and require multiple layers of the substrate.
As used herein, the terms “treating” and “treatment” refer to reduction in severity and/or frequency of symptoms, elimination of symptoms and/or underlying cause, prevention of the occurrence of symptoms and/or their underlying cause, and improvement or remediation of damage. Thus, for example, “treating” a patient involves prevention of a particular disorder or adverse physiological event in a susceptible individual and treatment of a surgical, blunt or other trauma of the skin by increasing healing at a wound site. Thus, for example, the present method of “treating” a patient in need of wound healing therapy encompasses both prevention of a condition, disease, or disorder that is responsive to anti-rejection or immunosuppressive therapy (e.g., transplant rejection) and the treatment of a wound site that is in need of healing.
As used herein, the term “patient” refers to a mammalian, e.g., a human, which can benefit from the pharmaceutical formulations and methods of the present invention. There is no limitation on the type of mammal that could benefit from the presently described pharmaceutical formulations and methods.
As used herein, the term “wound healing” refers to trauma and injury of a cell, tissue or organ. Such sudden and external trauma injury requires intact and able host defense mechanisms.
The present example is provided to detail the proposed preparation of a nucleotide-enriched composition suitable for topical administration to humans. These compositions can contain any combination of nucleotides. However, the inventors have made preliminary observations suggesting that combinations that include, e.g., purines and pyrimidines or simply pyrimidines alone.
For example, a nucleotide-enriched topical serum may contain about 250 μM to 2.3 M RNA, dissolved in water or another suitable liquid. To make such a preparation, 2.5 g of RNA (from yeast or another source) are mixed with a diluent, e.g., water or saline. It may also be necessary to add additional carriers to place the liquid in a suitable form for providing the patient with a topical composition that does not irritate the skin, e.g., providing the RNA in conjunction with a gel, e.g., aloe vera gel, glycerol, glycerin, KY-like jellies, vitamin E or other carriers known to be compatible with topical applications.
The present example is provided to outline proposed methods for the nucleotide regimens of the invention may be used in the treatment of humans for the promotion/enhancement of wound healing. Examples of use of RNA or nucleotide sources as a topical substrate for enhancing wound healing would include some of the following clinical uses. Note that in the following example, RNA can be substituted for similar levels of purines and/or pyrimidines.
RNA or nucleosides can be administered topically via salve, an ointment, an impregnated dressing, a sustained release patch, or as a powder. In this application the substrate can be applied directly to the wound to induce enhanced healing. The dose will range from 0.250 mM to 2.5 M.
Balb/c mice were divided into three dietary groups (F=normal mouse chow; NF=nucleotide free mouse chow; and NFR=nucleotide free mouse chow supplemented with yeast RNA). Each group was fed before initiation of wounds. After 4 weeks of feeding, a 4 mm punch wound was created on the dorsal surface of each mouse, using a 4 mm biopsy punch. The wounds were coated with four different topical agents: (1) saline; (2) uracil (100 M concentration); (3) saline+CCM; and (4) Uracil+CCM (CCM=crosslinked collagen mesh). CCM (“Collagen Permeable Membrane”) from Accurate Chemical & Scientific Corporation, Catalog No. YIC 152299. Four mm size circles were soaked with appropriate nucleotide or saline solution (4 mm punch wounds) and used to dress the wounds.
Twelve groups were formed from these combinations (n=3). Mice were continued on their respective diets. Wounds were observed visually and photographed at four different times during the period of two weeks. These studies were repeated using a 100 μM concentration of inosine in place of uracil as the topical agent. The results yielded no significant differences from that which was seen with the uracil.
Sirolimus and everolimus are two macrolide immunosuppressants increasingly used in organ and cell transplantation because they effectively suppress allograft rejection responses while not impairing renal function, unlike calcineurin inhibitors. The mechanism of action of these agents appears to be blocking of signal transduction from cell surface membrane receptors to the nucleus.
A powerful side effect observed for immunosuppressive agents include decreased wound healing, presumably secondary to the observed suppression of vascular endothelial growth factor (VEGF), a critical protein in wound angiogenesis. For example, Sirolimus also decreases response to platelet derived growth factor (PDGF), a protein which also enhances angiogenesis.
Sirolimus is an effective immunosuppressant that has been associated with delayed wound healing. On a full thickness skin murine model of immunosuppression and decreased wound healing, the Sirolimus dose that delayed wound healing was first determined. Next, an effective dose of topical administration of mixed nucleotides was determined that demonstrated enhanced wound healing despite Sirolimus treatment.
Methods: For 2 weeks Balb/c mice were gavage-fed saline, 4 mg/kg, 8 mg/kg or 12 mg/kg Sirolimus (10 per group). After drug loading, an 8 mm in diameter full thickness dorsal skin punch wound was made in each mouse. Every second day digital pictures of the wound were taken and analyzed. After determining the effective Sirolimus dose that causes decreased wound healing and immunosuppression, 40 Balb/c mice were gavaged with 8 mg/kg of Sirolimus every other day. After establishing the punch wounds, the mono and dinucleotides were applied topically daily. The topical treatment was applied using the following formulations (10 mice per group): a mixture of mono and dinucleotides derived from RNA in 0.025%, 0.25% and 2.5% concentrations and one group with KY jelly as control. Every second day a digital picture was taken and wound size measured. P2Y2 receptor expression was qualitatively assessed using immunohistology and receptor specific antibodies. T-test was used to compare the groups.
Results. It was found that at 4 mg/kg Sirolimus dose showed no effect on wound healing. An 8 mg/kg and 12 mg/kg doses on day 4 and after delayed wound healing in a significant manner (p=0.01). A significant difference was found during the whole healing period.
Following this dose response test, an effective dose of 8 mg/kg was established for Sirolimus. It was found that at: 0.25% and 2.5% mixed RNA, the wound size of animals treated topically decreased faster after day 2 and this decrease turned to be significant on day 6 (p=0.02 and p=0.025). This statistically significant enhanced wound healing was observed through day 10. On day 14, all wounds were healed.
These studies demonstrate that mixed RNA applied topically was capable of overcoming the effect of an immunosuppressive agent on wound healing. It was found that Sirolimus impaired wound healing in this well-known model system was overcome, when mixed RNA was applied topically. Furthermore, it was found that mixed RNA accelerates wound healing in these immunosuppressive drug-treated animals. The qualitative assessment of the P2Y2 receptors suggests that binding to these receptors may be the mechanism by which the positive influence of the nucleotides applied topically are mediated. This effective treatment offers an opportunity to mitigate a complication of Sirolimus therapy.
Therefore, RNA derived nucleotides, nucleosides or nucleobases when applied topically to wounds in mice treated with an immunosuppressive dose of sirolimus (8 mg/kg/every other day) significantly enhance wound healing in mice despite sirolimus therapy. The effective dose ranges from 250 μM to 2.3 M as a topical treatment of nucleosides, nucleotides, or nucleobases. At least one mechanism of this effect of the topical compounds appears to be via enhancing angiogenesis in the wound.
It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.
All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
In the claims, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of,” respectively, shall be closed or semi-closed transitional phrases.
All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
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This application claims priority to U.S. Provisional Patent Application Ser. No. 60/796,489, filed May 1, 2006, the entire contents of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 60796489 | May 2006 | US |
