Injectable compositions for accelerating the production of collagen-containing proteins and methods of treatment using injectable compositions

Abstract

A solution for injection into mammals to stimulate collagen growth includes solutes having amino acid tropocollagen factors including L-proline and L-lysine, in which the L- proline comprises at least 55% of the amino acid tropocollagen factors, and a solvent, wherein the solution contains the solutes in concentrations effective to accelerate the production of collagen proteins within a mammal when applied to the mammal via injection. In some embodiments L-glycine or L-glycine precursors may be part of the solute. Methods of treatment of mammalian joints to promote collagen growth, cartilage growth, and increased amounts of synovial fluid are also described.

Description

INJECTABLE COMPOSITIONS FOR ACCELERATING THE PRODUCTION OF COLLAGEN-CONTAINING PROTEINS AND METHOD OF TREATMENT USING INJECTABLE COMPOSITIONSFIELD OF THE INVENTION

[0001] This disclosure relates generally to injectable compositions formulated to accelerate the production of collagen proteins in mammals, and, more particularly, relates to compositions formulated to accelerate the production of collagen-containing tissues of mammals.BACKGROUND

[0002] Collagen, which is a structural protein, is the main component of connective tissue in mammals. It is also found in corneas, blood vessels, intervertebral discs, and even in teeth dentin. Type I collagen is the most abundant of the at least 28 different types of collagen currently identified to exist in mammals. Type I collagen forms a triple-helix structure that is the prototype for most of the other collagen types. Other types of collagen differ from Type I collagen by the length of their triple helix and the presence or absence of globular domains at their amino or carboxyl terminal ends. Collagen itself is made up of a unique Amino Acid (AA) and Imino Acid (IA) composition with approximately 33% of the total residues being glycine (Gly), 10% proline (Pro), 10% hydroxyproline (Hyp), which is derived from proline, 3% lysine, and about 1% hydroxylysine (Hyl), which is derived from lysine.

[0003] The basic structural unit of all types of collagen is tropocollagen, which is crosslinked to form large fibers of collagenous tissues. Tropocollagen is made of three polypeptide chains called alpha (a) chains, where each of the a chains is wound around the other to form a triple helix structure. Two of the a chains, referred to as alpha-1 (al) chains, are chemically identical, while the third a chain is referred to as an alpha-2 chain (a2) and has a slightly different chemical composition. Every third AA or IA in the a chain is a glycine, hence the value of 33% for the relative amount of glycine present in collagen.

[0004] Sixty percent of the a chains are made of either the sequence Gly-Pro-X or the sequence Gly-X-Hyp, where X may be any AA or IA. The remaining forty percent of the a chains are various sequences of AAs and lAs, with every third AA or IA being a glycine. The AAs and lAs that compose tropocollagen may be referred to as tropocollagen factors.

[0005] Since the presence of new collagen proteins encourages the production of new collagen-containing tissues, e.g., cartilage tissue, tendon tissue, ligament tissue, hair tissue, etc., it would be advantageous to develop compositions formulated to accelerate theproduction of new collagen proteins within a mammal. The inventor has surprisingly found that producing a formula to stimulate production of collagen proteins having amino acids in the approximate concentrations of collagen itself, i.e., Glycine at approximately 33%, Proline at approximately 20%, accounting for both the Proline and hydroxyproline, and Lysine at approximately 4%, accounting for both the Lysine and hydroxylysine, was not as effective at accelerating production of collagen proteins in mammals as are formulas according to embodiments of the disclosure.BRIEF DESCRIPTION OF THE FIGURES

[0006] Fig. 1A is a color-inverted Magnetic Resonance Image (MRI) showing an image of an injured knee of a female human subject prior to treatment.

[0007] Fig. IB is a color-inverted MRI showing an image of the subject of Fig. 1A showing cartilage growth following treatment according to embodiments of the disclosure.

[0008] Fig. 2A is an image of an Xray of a first equine subject taken prior to treatment.

[0009] Fig. 2B is an image of an Xray of the first equine subject of Fig. 2A showing cartilage growth following treatment according to embodiments of the disclosure.

[0010] Fig. 3A is an image of an Xray of a second equine subject taken prior to treatment.

[0011] Fig. 3B is an image of an Xray of the second equine subject of Fig. 3A showing cartilage growth following treatment according to embodiments of the disclosure.

[0012] Fig. 4A is an image of an Xray of a third equine subject taken prior to treatment.

[0013] Fig. 4B is an image of an Xray of the third equine subject of Fig. 4A showing cartilage growth following treatment according to embodiments of the disclosure.

[0014] Fig. 5A is an image of an Xray of a second human subject taken prior to treatment.

[0015] Fig. 5B is an image of an Xray of the second human subject of Fig. 5A showing cartilage growth following treatment according to embodiments of the disclosure.

[0016] Fig. 6A is an image of an Xray of a fourth equine subject taken prior to treatment showing a damaged tendon.

[0017] Fig. 6B is an image of an Xray of the fourth equine subject of Fig. 6A after treatment according to embodiments of the disclosure showing that the damaged area of the tendon has lessened in size and severity.

[0018] Fig. 7 is a flowchart illustrating an example set of operations, some of which are optional, used in conjunction with compounds for treating mammals, according to embodiments of the disclosure.DETAILED DESCRIPTION OF EMBODIMENTS

[0019] The inventor has recognized that the production of new collagen proteins may be advantageously accelerated by providing the tropocollagen factors proline, glycine, and lysine, in particular ratios in an injectable composition, which may be then injected, such as through intra-articular injection, into a mammal to promote collagen production proximal to the injection site. In some embodiments, the tropocollagen factors can be present as isolated amino- and / or imino-acids and not bound within one or more peptides.

[0020] Accordingly, aspects of the disclosure are directed to a composition of tropocollagen factors (e.g., proline, glycine, and lysine), in particular ratios. When amino acids such as proline, glycine, and lysine are described or referred to in this disclosure, it is to be assumed that such amino acids refer to the L-form of amino acids unless specifically referred to as the D-form. These ratios have unexpectedly shown to be particularly helpful in growing cartilage and reducing joint pain. As discussed herein, the ratios of proline, glycine, and lysine that comprise the disclosed composition differ from the ratios of these tropocollagen factors found in collagen itself. In other words, aspects of the disclosure are directed to a composition of matter having unexpected concentrations of tropocollagen factors compared with endogenous collagen naturally occurring in the body of a mammal. Additionally, aspects of the disclosure are directed to treatment methods utilizing the disclosed composition as an injectable composition. More specifically, aspects are directed to pre- and post-injection methods for improving the effectiveness of the injectable composition.

[0021] As mentioned, tropocollagen factors refer to amino- or imino-acids in their nonhydroxylated form. Within the body of a mammal, therefore, the tropocollagen factors are hydroxylated to produce collagen proteins.

[0022] It is known that ascorbic acid is a hydroxylation co-factor to the tropocollagen factors. In process, the ascorbic acid hydroxylates the tropocollagen factors within cells of the body, which, in turn, accelerates the body's production of collagen proteins and consequently encourages the increased production of collagen-containing tissues (e.g., cartilage). The inventor has discovered, however, that the ascorbic acid is not a necessary component of the injected solution, nor does it need to be exogenously administered in connection with the tropocollagen factors. Instead, any ascorbic acid used for hydroxylation may be already found within the body itself. In some embodiments no detectible amounts of ascorbic acid are present in the composition (e.g., the composition excludes or substantially excludes ascorbicacid). In other embodiments no more than a trace amount of ascorbic acid is present in the composition, where trace amount means an insignificant amount compared to other types of molecules in the composition. Thus, embodiments of the disclosure further involve unexpected compositions and methods compared with the compositions and processes that naturally occur in the body of a mammal.

[0023] When injected into a mammal, the components in the disclosed composition, for example embodiments, work in conjunction with precursors already present in the mammal's body to produce specific collagen proteins incorporated within collagen-containing tissues at specific locations within the body. In particular, embodiments according to the disclosure produce therapeutic benefit to mammals after injection into anatomical joint locations, such as, but not limited to, knee joints, backjoints, shoulder joints, ankle joints, joints of the digits, such as fingers and toes, and hip joints, as described in detail below.

[0024] In other words, embodiments of the disclosed composition may be injected into a mammal and cooperate with precursors present in the mammal's body to produce collagen proteins that assist in recovering from injury and building cartilage. For example, the disclosed composition may be injected into an injured joint or joint capsule of a mammal, aid in the mammal's recovery process and strengthen the joint. Further, fibroblast type-B cells are a type of spindle-shaped biological cell that synthesizes both extracellular matrix in synovial fluid. These cells are located at various depths within the joint. Fibroblast type-B is significant in maintaining joint lubrication. Fibroblast type-B may be stimulated by ligands binding to the cell surface receptors within the joint capsule. Ligands are classified according to the electron number with L ligands represented by the amine group. It is suspected that injecting the amino acids described herein, as well as their potential conversion into imino acid form, may further release amines and consequently stimulate the ligand binding to the fibroblast cell surface. This, in turn, potentially generates an increased amount of synovial fluid within the injected joint capsule.

[0025] The compositions described herein can be used by the mammalian body to accelerate or initiate the formation of any type of collagen produced within the body, e.g., including Type I, Type II, Type III, Type IV, Type V, Type VI, Type VII, Type VII, Type IX, Type X, Type XI, etc., depending on administration location. Stated another way, all specialized cells located at specific sites in the body that contribute to the formation of specific collagen types can use the components in the compositions exemplarily described herein to initiate orsustain synthesis of collagen-containing tissues such as cartilage tissue, tendon tissue, ligament tissue, vitreous humor tissue, connective tissue, and bone tissue. Compositions exemplary described herein can be administered to repair damaged collagen-containing tissue or to maintain healthy collagen-containing tissue.

[0026] Table 1 lists ratios of amino acids, by weight, included in an injectable composition according to some embodiments of the disclosure, as well as exemplary concentration ranges for those ingredients.Table 1

[0027] Noticeable in Table 1 is the high percentage of Proline in the composition relative to the other components Glycine and Lysine. This is unusual because, recall that as described above, in endogenous collagen itself, Glycine makes up approximately 33% of the final collagen material, while Proline, in either its standard (Proline) or hydroxylated form (hydroxyproline), makes only approximately 20% of collagen. And Lysine, in its standard (Lysine) or hydroxylated form (hydroxylysine), makes up only about 4% of collagen.

[0028] The IA proline is synthesized from glutamate, and in the modified form is recognized as hydroxyproline which is found in structural proteins. Of interest here is its role in tropocollagen. When activated by the enzyme prolyl-4-hydroxylase, the prolines in the sequence X-Pro-Gly are converted to 4-hydroxyproline. The enzyme Prolyl-3-hydroxylase converts prolines in the sequence Hyp-Pro-Gly to 3-hydroxyproline.

[0029] Hydroxyproline comprises roughly 4% of all amino acids found in animal tissue, an amount greater than seven other amino acids that are translationally incorporated into the tissue. Proline is important for both protein synthesis and structure, metabolism, wound healing and immune response. L-proline also supports digestive and cardiovascular health. The biosynthesis of L-proline occurs from glutamine within the mitochondria. Glutamine in mammals can be severely depressed due to stress, trauma, infection, or other factors. Therefore, it is believed that introducing the additional amount of proline in the compositionaccording to embodiments of the disclosure makes up for the depletion of the biosynthesis source within the body due to stress, trauma, and infection factors described above.

[0030] Since glycine is the simplest AA in the body and is of major importance in the synthesis of proteins, the sequencing requirement of this substance for the alpha chains becomes readily apparent. In other words, the body's capability of synthesizing adequate amounts of glycine under stress loading may be of concern, particularly in the field of collagen protein generation.

[0031] The essential AA lysine participates in the biosynthesis of proteins, and the residues that are not metabolized in the liver are transported to various tissues in the body, such as connective tissues. As was described above, the unique AA and IA composition of collagen includes hydroxylysine (Hyl), which is a modified form of lysine. To produce 5- hydroxylysine (Hyl) from the lysine residues in the sequence X-Lys-Gly through a hydroxylation process, the enzyme lysyl hydroxylase is required. Further prolyl-4-hydroxylase then converts prolines in the sequence X-Pro-Gly to 4-hydroxyproline. Prolyl-3-hydroxylase then converts prolines in the sequence Hyp-Pro-Gly to 3-hydroxyproline.

[0032] As mentioned above, through research, the inventor has determined that including a much higher ratio of proline to other amino or imino acids in the injected composition provides increased benefit that strengthens joints by potentially increasing the generation of collagen proteins and can reduce pain and improve joint performance in mammals. As demonstrated by the three enzymes, prolyl-4-hydroxylase, prolyl-3- hydroxylase, and lysl hydroxylase, which are used in procollagen hydroxylation, as described above, the inventor determined increased relative levels of proline relative to other amino acids produces superior results, including pain reduction and improved performance as well as new cartilage growth in joints. In this way, embodiments of the disclosed injectable composition utilize ratios of proline, glycine, and lysine that differ from naturally occurring precursors in the bodies of mammals, yielding a therapeutic benefit from an unexpected composition.

[0033] In general, the composition includes the amino or imino acids as set forth in Table 1, in the relative concentrations set forth in Table 1. The Proline, Glycine, and Lysine are referred to below as the solute. In one exemplary method, filtered or distilled water (the solvent) is placed in a sterilized vessel, and then the solute is added. Then, the entire solution (solute plus solvent) is brought to a boil for approximately three minutes. The resultingsolution is immediately placed into sterile syringes. The sterile syringes are kept sterile until injected into the subject. Solvents other than or in addition to filtered or distilled water may also be used in some embodiments, such as organic solvents and co-solvents. When the solute and the solvent are combined to form the injectable solution, the injectable solution may have a liquid form, i.e., a substance that flows freely but is of constant volume. The injectable solution may also be in gel form, i.e., neither a liquid nor a solid, by the introduction of non-active, gelling ingredients into the injectable solution. Such gelling ingredients may include gelatin, agar-agar, hyaluronic acid, nano-emulsions, hydrogels, chitosan, and alginate, for example, among others. In yet other embodiments the injectable solution may be produced in as a depot injection, which releases the active ingredients of the solute into the body over a longer period of time than liquids. Depot injections may be injected as a liquid or gel, but later form into a soft, semi-solid form that continue to deliver active ingredients longer than liquid injections when injected into the joint capsule or elsewhere in the body. Depot forming materials may include polymers, such as poly (lactic-co-glycolic acid) (PLGA), lipids, or other oil-based formulations.

[0034] In one example embodiment, the solute is compounded or mixed in powder form prior to being added to the solvent. For example, in one embodiment, 800 mg of powdered proline is combined with 150 mg of powdered Glycine, and 50 mg of powdered Lysine. Other embodiments may have ratios of these amino acids as set forth in the options of Table 1. Other components may also be added to the powder mixture, such as Zinc Picolinate and Ascorbic acid, although these last two components do not form part of the tropocollagen factors. Then 3 ml of the resulting powder is dissolved in approximately 15 ml of solvent, such as purified or distilled water. The powder dissolves into the solvent, in this example water, which may be accelerated by shaking or stirring the solution. The powder does not need to completely dissolve into the solvent at this step. Next, the mixture is brought to a temperature of 212 degrees Fahrenheit for a further sterilization process, although all of the ingredients are sterile to begin with. The mixture is kept at 212 Fahrenheit until the total volume of the mixture reduces to approximately 3 ml by evaporation. In embodiments the mixture remains at approximately 212 degrees Fahrenheit for between 3-10 minutes, preferably approximately five minutes. Finally, after the mixture has cooled, the mixture is placed in sterile syringes, which are kept in a sterile environment until injected into thepatient. The syringes containing the sterilized mixture may be stored at room temperature less than 80 degrees Fahrenheit, or refrigerated, for example.

[0035] In other embodiments part or all of the Glycine in the injectable composition described above may be omitted from the composition in favor of substituting Glycine- synthesis precursors in the composition instead of the Glycine itself. Glycine may be synthesized in the body from serine, an alpha-amino acid through a catalytic reaction of an enzyme, serine hydroxymethyltransferase (SHMT), which is naturally found in mammals. SHMT, in turn, may be dependent on Pyridoxal Phosphate (P5P), the active ingredient in Vitamin B6, to help the SHMT perform the conversion of serine to Glycine. Thus, embodiments according to the disclosure may substitute some or all of the Glycine in the injectable composition with precursors to Glycine, and then the Glycine is synthesized within the body from the injected precursors. Then the synthesized Glycine becomes one of the tropocollagen factors as described above.

[0036] In more detail, Lysyl hydroxylase converts lysines in the sequence X-Lys-Gly to 5- hydroxylysine. Prolyl-4-hydroxylase converts prolines in the sequence X-Pro-Gly to 4 hydroxyproline. Prolyl-3-hydroxylase converts prolines in the sequence Hyp-Pro-Glycine.

[0037] The formation of disulfide bonds occurs via the oxidation of cysteine residues which form interchain disulfide bonds in the pro-peptides that take place prior to triple helix formation.

[0038] In embodiments, precursors to synthesize Glycine may be those as set out in the amounts in Table 2 below.Table 2(Total amount as a substitute for the equivalent amount of Glycine in the compound)

[0039] As described above, some embodiments of the disclosed composition may also include additional components, such as copper, zinc, and bioflavonoids. Accordingly, the solute referred to herein may include proline, glycine, lysine, and still other components. It follows that for some embodiments, preparing the injectable composition involves addingproline, glycine, lysine, and concentrations of any additional components to filtered or distilled water, then heating and placing the solution in sterile syringes as described.

[0040] Once syringes are prepared and sterilized, the injectable composition can be delivered to an affected joint via injection into the subject. In particular, the composition may be injected into the joints of mammals in the following amounts as listed in Table 3, where 1 unit = 1 ml = 1 cc. In the described embodiment above, each syringe includes approximately 3 ml, or approximately 3 units.Table 3

[0041] It will be understood by one skilled in the art that the amount listed in Table 3 are guidelines, and still other amounts of the injectable composition may be appropriate depending on the specific joint, the nature and size of the joint, and the severity of the injury to the joint. For instance, as is evidenced by the varying amounts of the composition listed in Table 3, larger regions of the human body, such as the back, are fit to receive larger quantities of the disclosed composition compared with smaller regions, such as the digits. Similarly, the severity of the injury or the size of the patient may inform the quantity of the disclosed composition appropriate for treating the injury.

[0042] A typical treatment method is one or more daily injections with the composition, injected into the joint with the number of units as set forth in Table 3. Multiple treatments may be combined. For instance, for a moderately injured joint, treatment may consist of one administration of injected composition every day for three consecutive days. Or treatment may consist of injecting the composition at least two times within seven days. Other injuries may have fewer or additional treatments. Evaluations of the affected joint may occur for a period of 6-12 months following treatment.

[0043] In implementation, a person experiencing a joint injury may first be assessed to determine the severity and scope of the injury. That is, before a treatment using any of theinjection locations or numbers of units— or any combinations thereof— is administered, a treatment plan may be created based on a pretreatment assessment. Factors such as history, range of mobility, physical condition, and the extent and regularity of pain may inform this pretreatment assessment. A person experiencing knee and ankle pain, for example, may first discuss these example factors with the administrator of the treatment. Following the assessment, it may be determined that the knee and ankle injury is moderate, and thus the subsequent treatment may appropriately consist of an administered injection in the knee and ankle, in the amounts shown in Table 3, every day for three consecutive days. Optionally, pretreatment assessment may further include review of prior medical imaging, such as x-rays and MRIs, to further understand the scope and severity of the injury.

[0044] Once a pretreatment assessment is completed and a treatment plan is chosen, one or more optional pretreatment preparation methods may be implemented before an injection of the disclosed composition is administered. For example, in some implementations, electrical nerve stimulation is used prior to injection to induce iontophoresis, as well as to prepare the site for injection. In such implementations, acupuncture needles or other suitable needles may be inserted into the skin near the joint to be treated with the disclosed composition. A standard transcutaneous electrical nerve stimulation (TENS) device may then be used to apply low-voltage electrical stimulation at or near the joint. This stimulation with a standard TENS device may, for example, be applied for fifteen minutes in some implementations. However, it will be understood to one skilled in the art that still other durations of TENS application are appropriate, depending on the size of the joint, the severity of the injury and pain, and the chosen treatment plan.

[0045] Still other pretreatment preparation methods may include the use of infrared light. In some implementations, for instance, an infrared lamp is shone at or near the joint that is to receive the injection. When implemented in this way, infrared light shone at or near the joint of interest aids in dilating the capillary bed and increasing circulation around the joint of interest, facilitating uptake of the injected solution and promoting increased healing rates.

[0046] Additionally, in some implementations, ultrasound may be used prior to injection to dilate the capillary bed. Specifically, ultrasonic waves are directed at the joint of interest using a standard ultrasonic device, with the frequency of the ultrasonic waves and powerdensity being chosen based on the joint location and size. Example settings for an ultrasonic device are provided below in Table 4, for application to various joints.Table 4

[0047] Application of ultrasonic waves based on the example settings presented in Table 4 may occur for ten minutes, in example implementations. In still other implementations, shorter or longer durations of ultrasound treatment will be appropriate, depending on the treatment need and factors considered during pretreatment assessment. As mentioned, applying ultrasonic waves in this way dilates the capillary bed near the joint of interest, improving circulation. Additionally, applying ultrasonic waves removes effusion, bringing down any swelling of the tissue surrounding the joint caused by excess fluid, especially as may be present following an injury. As discussed further below, ultrasound may also be used after administering an injection of the disclosed composition to enhance the effectiveness of the injection.

[0048] Other pretreatment preparation methods may include manual manipulation of the injection site, in some implementations. That is, a person administering treatment may provide manual pressure to the injection site and massage the tissue surrounding the injection site, using their thumbs or other portions of their hands to apply deep pressure. This manual pressure assists in increasing joint space prior to injection, allowing for more effective reception of the disclosed composition. In some implementations, manual pressure is applied for fifteen minutes. However, it should be understood that shorter or longer durations of manual pressure will be appropriate and necessary depending on the circumstances of the treatment— for instance, the severity of the injury and pain.

[0049] Other pretreatment preparation methods may include using stem cells in conjunction with the injectable solution. In such embodiments, stem cells are typically harvested from a patient's own bone marrow, which reduces the chance of rejection. The stem cells are extracted along with blood. A stem cell separator may be used to isolate thestem cells from the extracted blood and then the blood may be returned to the patient. Next the stem cells are mixed with the injectable composition described above and placed into sterile syringes. Then the composition, including the stem cells, is injected into the body as described above.

[0050] A similar process may also be used to combine platelet-rich plasma (PRP) with the injectable solution prior to injection. Similar to the process described above, blood plasma may be extracted from the patient then placed in a centrifuge to separate various components within the plasma. First, red blood cells and other components may be isolated and discarded. Then the remaining plasma is returned to the centrifuge and spun again, for example at a higher rotational speed than was used to extract the red blood cells. What remains is platelet-rich plasma, which may then be extracted and combined with the injectable composition as described above and placed into sterile syringes. Then, the composition, including the platelet-rich plasma is injected into the body as described above.

[0051] In implementation, any one of the pretreatment preparation methods described above— and any combination of pretreatment preparation methods— may be used based on the specifics of the injury being treated. Put differently, factors discussed during a pretreatment assessment will inform not only the injection regimen but also the choice of appropriate pretreatment preparation methods. A treatment provider may thus choose any of the described pretreatment preparation methods, or any combination of them, to ensure effective delivery of the disclosed composition to the affected joint or joints. For example, a person experiencing significant back pain may discuss the severity of their pain with a treatment provider, including how their back pain affects their mobility, their medical history with back pain, and their present condition. The treatment provider, as previously mentioned, may also review images, if available. Following this pretreatment assessment, the treatment provider may determine that several of the described pretreatment preparation methods are appropriate to address the severity of the pain and ensure effective treatment using the disclosed composition.

[0052] Accordingly, in one pre-treatment regimen, once pretreatment assessment is complete and an injection regimen is chosen, the treatment provider may begin by applying electrical stimulation to the injection site. The provider places acupuncture needles on the back of the person receiving treatment, specifically targeting areas of interest based on the pretreatment assessment. The provider then connects a standard TENS unit, as describedabove, to deliver electrical stimulation to the areas of interest for approximately fifteen minutes. The provider may then disconnect the TENS unit and remove the acupuncture needles, transitioning to infrared pretreatment. The provider then directs an infrared lamp to shine on the back of the person receiving treatment, dilating the capillary bed and increasing circulation to the back. Next, the provider applies ultrasound treatment to the back for approximately ten minutes, applying ultrasonic waves at a frequency of 1 MHz and power of 21.5 Watts, as shown in Table 4. Finally, once ultrasound treatment is complete, the treatment provider may then manually apply pressure to the back, massaging the areas of interest to increase joint space.

[0053] When pretreatment preparation methods are complete, a treatment provider sterilizes the injection site or sites with an alcohol pad to ensure the injection site is sufficiently prepped. Once prepped, the treatment provider then proceeds with the chosen injection regimen. For instance, in the present example of a person experiencing back pain, the treatment provider proceeds with injecting 1-2 units of the disclosed composition to each area of interest— that is, each affected disc. Injection of the disclosed composition is gradual, and in some implementations, the injection is administered using one syringe inserted at various sites around the affected joint. That is, one unit to be delivered to one affected joint may, in practice, be delivered in more than one location around that affected joint. Alternatively, one unit may be delivered all at once to the affected joint. In implementation, factors discussed during the pretreatment assessment, such as the severity and scope of the injury as well as the nature of the joint receiving the injection, will inform the appropriate administration of the injection.

[0054] Once the injection of the disclosed composition has been administered, the injection site is bandaged. As previously mentioned, in some situations, ultrasound treatment may be implemented after the injection. Put differently, for some injuries and injection sites, it may be appropriate to bandage the site and once again direct ultrasonic waves to the site. Ultrasound treatment after injection may be performed for ten minutes, in some implementations, although shorter or longer durations of post-injection ultrasound, similar to ultrasound applied before the injection. Because ultrasound treatment dilates the capillary bed and removes effusion, as discussed with regard to pretreatment ultrasound, postinjection ultrasound aids in evenly distributing the injected composition at the affected joint.

[0055] Embodiments according to this disclosure have been used effectively in mammals, specifically humans as well as horses, to treat knee injuries and knee degradation.

[0056] Fig. 1A is a Magnetic Resonance Image (MRI), of a knee of a human female prior to treatment, whereas Fig. IB is another MRI of the same subject taken approximately 1 month after treatment using embodiments of the disclosure. In Figs. 1A and IB, the colors of the MRI have been inverted from black to white for clarity. Note the cartilage degradation, circled in Fig. 1A, near the top of tibial plateau, which is the top portion of the lower bone illustrated in the figure. Fig. IB is an MRI taken after approximately one month post-treatment of the subject of Fig. 1A, which shows cartilage growth in the circled area, as well as increased spacing between the bones of the knee. The increased spacing may be due to cartilage growth, an increase in an amount of synovial fluid in the knee capsule of the subject, or due to both cartilage growth and an accompanying an increase in synovial fluid.

[0057] Figs. 2A, 3A, and 4A are Xrays of knees of horses before treatment. Each of these figures shows a different horse subject that was experiencing knee pain and limited mobility. Figs. 2B, 3B, and 4B are Xrays of the respective subjects of Figs. 2A, 3A, and 4A between 3 - 6 months post treatment. Each of the Xrays in Figs. 2B, 3B, and 4B indicate cartilage growth in the knee joint of each of the subjects. Increased synovial fluid may also be present. Greater mobility and improved joint function were observed (e.g., through increased agility) in the subjects following the injection treatments of the compound described above. As the injected composition was the only treatment given to these equine subjects, it follows that the injected composition caused each subject to grow cartilage and / or an increase of synovial fluid in the treated joint area.

[0058] Fig. 5A is an image of an Xray of a second human subject taken prior to treatment, which indicates cartilage degradation in a hip joint. Fig. 5B is an image of an Xray of the same human subject of Fig. 5A showing cartilage growth following treatment according to embodiments of the disclosure. Increased synovial fluid may also be present in the image of Fig. 5B. The Xrays in Figs 5A and 5B were taken from opposite planes of the subject, which results in the treated hip joint appearing on the left side of Fig. 5A and the right side of Fig. 5B.

[0059] Fig. 6A is an image of an Xray of a fourth equine subject taken prior to treatment according to embodiments of the disclosure. The image of Fig. 6A shows tendon degradation in the subject. Fig. 6B is an image of an Xray of the fourth equine subject of Fig. 6A showing ageneral decrease in the severity and size of tendon degradation, which is believed to be caused by increased collagen production following treatment according to embodiments of the disclosure.

[0060] Fig. 7 shows a flowchart of a treatment method 700, incorporating example treatment steps described above. The treatment method 700 begins with a pretreatment assessment at operation 702, where the scope and severity of a joint injury is assessed based on factors like the injured subject's medical history, range of mobility, physical condition, and any available medical imaging. Once the injury is assessed at operation 702, the treatment method 700 continues to operation 704, where an injection regimen is chosen to address the scope and severity of the injury. The injection regimen may be chosen at operation 704 based on the composition quantity guidelines listed above in Table 3.

[0061] When an injection regimen is chosen at operation 704, pretreatment preparation methods may be applied to the injection site, if indicated. For instance, at operation 706, the injection site is electrically stimulated with a transcutaneous electrical nerve stimulation (TENS) device to dilate induce electrophoresis, and infrared light is shone on the injection site with an infrared lamp at operation 708 to dilate the capillary bed and increase circulation to the injection site. Then, ultrasonic waves are directed at the injection site at operation 710 before the treatment provider manually applies pressure to massage the injection site at operation 712. Each of the operations 704, 706, and 708 are individually optional. This means that the subject may receive one, two, or three of the optional operations 704, 706, 708, in any combination, or none at all, if the provider determines to omit them. Once these pretreatment preparation operations have been completed, if performed, the treatment provider proceeds to operation 714 of the treatment method 700, wherein the treatment provider injects units of the disclosed composition according to the chosen injection regimen.

[0062] As previously mentioned, any combination of pretreatment preparation methods may be deemed appropriate and effective for delivering the injectable composition, and thus treatment method 700 is just one combination of pretreatment methods for preparing an injection site. Still other example preparation methods are possible, utilizing various combinations and orders of the described pretreatment methods.

[0063] Although the composition according to some embodiments of the disclosure include only (e.g., consist essentially of) amino acids and a solvent or are otherwise substantially free of other ingredients, as set forth above, other embodiments may includeone or more adjuncts or other ingredients as set forth below without departing from the teachings of the disclosure. For example, compositions for accelerated production of collagen proteins according to embodiments of the disclosure further include copper, which is a transitional metal. Copper may be added to total about 1.0 % of the composition. According to some embodiments, copper may be omitted from the injectable composition or may be included within the injectable composition depending on the site in mammal where the composition is to be injected.

[0064] In some embodiments, compositions for accelerated production of collagen proteins may further include bioflavonoid extracts. For example, the bioflavonoid extracts, when included, amount to approximately 1.0 % or less of the composition.

[0065] Bioflavonoid extracts demonstrate a wide range of pharmacological activities. In particular, one class of bioflavonoids, the proanthocyanidins (which include the extracts from pine bark and grape seed), are known for increasing intercellular vitamin C levels and inhibiting the destruction of collagen. Thus, bioflavonoids in general, and proanthocyanidins in particular, are well-suited for embodiments of the invention due to their beneficial interaction with ascorbic acid and collagen. Additionally, bioflavonoids exhibit a cyto- protective effect that reduces inflammation and protects tissues and can be used to reinforce the natural cross-linking that forms the collagen matrix.

[0066] In some embodiments, compositions for accelerated production of collagen proteins may further include beta-1, 3D-glucans in an amount of 0.1% or less of the composition.

[0067] In some embodiments, compositions for accelerated production of collagen proteins may further include zinc, in the form of zinc salts, such as zinc picolinate. Zinc may be included for its outstanding role in the formation of "zinc fingers." A zinc finger is a structure which is 30 amino acids long and is tightly bound with an atom of zinc. Zinc fingers are known for their beneficial interaction with DNA to regulate the activity of genes. Furthermore, it has long been recognized that zinc is capable of maintaining the integrity of biological membranes by protecting them against oxidative injury. The element zinc also supports the synthesis and formation of the copper chaperone metallothionein.

[0068] A person skilled in the art will be able to practice the embodiments exemplarily discussed herein, where numerous details have been set forth to provide a more thorough understanding of the inventive concepts exemplarily described herein. In other instances,well-known features have not been described in detail in order not to unnecessarily obscure embodiments of the invention.

[0069] Embodiments exemplarily described herein may be practiced in many different ways. For example, the act of preparing a composition including Amino and Imino acids in relative percentages according to Table 1 is considered to be an embodiment of the invention, as is the act of administering the compositions prepared according to Tables 1 to encourage the accelerated production of collagen proteins.

[0070] Compositions such as those prepared according to Table 1 may be injected into the joints of a mammal. Injecting such compositions into the joints of a mammal animal encourages the accelerated production of collagen proteins and accelerates or initiates the production of collagen-containing tissues (e.g., cartilage, tendons, ligaments, etc.) within the mammal, depending upon the location where the composition is injected. For example, production of cartilage within any joint may be accelerated by means of direct application via injection (e.g., intramuscular or subcutaneous) of a composition prepared according to Table 1 at or in proximity of (e.g., within about 20 mm of) the joint capsule, joint surface, or other anatomical placement (e.g., rib).EXAMPLES

[0071] The forgoing disclosure may be implemented in various examples in many combinations. While not intended to be exhaustive, the following examples are presented as particular examples of the disclosed subject matter. The following examples are presented forthe purposes of clarity and should not be considered as limiting to the scope of the present disclosure.

[0072] Example 1 includes a solution for injection into mammals to stimulate collagen growth, including solutes having amino acid tropocollagen factors including L-proline, L- glycine or one or more L-glycine precursors, and L-lysine in which the L-proline comprises at least 55% of the amino acid tropocollagen factors, and a solvent, wherein the solution contains the solutes in concentrations effective to promote the production of collagen proteins within a mammal when applied to the mammal via injection.

[0073] Example 2 is a solution according to Example 1, wherein the solution contains the solutes in concentrations effective to promote the production of collagen proteins within a mammal when applied to the mammal via injection into a joint capsule.

[0074] Example 3 is a solution according to Examples 1 or 2, in which the L-proline comprises at least 70% of the weight of the amino acid tropocollagen factors in the solute.

[0075] Example 4 is a solution according to Examples 1 - 3, in which the L-proline comprises at least 80% of the amino acid tropocollagen factors.

[0076] Example 5 is a solution according to any preceding Example, in which the solvent comprises water.

[0077] Example 6 is a solution according to any preceding Example, in which the solution is brought to a temperature in excess of 200 degrees Fahrenheit and cooled to less than 80 degrees Fahrenheit prior to injection.

[0078] Example 7 is a solution according to any preceding Example, in which the solution is stored in a syringe.

[0079] Example 8 is a solution according to any preceding Example, in which the solutes are in powdered form prior to being combined with the solvent.

[0080] Example 9 is a solution according to any preceding Example, in which the solution contains the solutes in concentrations effective to promote the production of cartilage within a mammal when applied to the mammal via injection.

[0081] Example 10 is a solution according to any preceding Example, in which the solution contains the solutes in concentrations effective to promote the production of synovial fluid within a mammal when applied to the mammal via injection.

[0082] Example 11 is a solution according to any preceding Example, in which the solutes have amino acid tropocollagen factors including L-proline, L-glycine and L-lysine in a ratio of between 5.5:2:1 and 95:3:1 of the amino acid tropocollagen factors.

[0083] Example 12 is a solution according to any preceding Example, in which the solution contains no more than a trace amount of ascorbic acid.

[0084] Example 13 is a solution according to any preceding Example, in which the one or more L-glycine precursors include serine and pyridoxal phosphate.

[0085] Example 14 is a solution according to Example 13, in which the solute contains no L-glycine.

[0086] Example 15 is a solution according to Examples 13 or 14, in which the solute contains less than a trace amount of L-glycine.

[0087] Example 16 is a method of promoting collagen growth in a joint of a mammal, including obtaining an injectable solution as recited in any one of Examples 1-15; and injecting the solution into a joint capsule of the mammal.

[0088] Example 17 is a method of promoting collagen growth in a joint of a mammal, including administering to the joint capsule via injection, a collagen growth promoting combination of L-proline, L-glycine, or one or more L-glycine precursors, and L-lysine, wherein the L-proline is present in an amount of at least about 55% by weight of the combination.

[0089] Example 18 is a method of growing collagen in a joint of a mammal including injecting into the joint capsule, a therapeutically effective amount of L-proline, L-glycine, one or more L-glycine precursors, and L-lysine, wherein the amount of L-proline is at least about 55% by weight of the total amount of L-proline, L-glycine, or one or more L-glycine precursors, and L-lysine injected.

[0090] Example 19 is a method according to any of Examples 16-18, in which the one or more glycine precursors comprises serine and pyridoxal phosphate.

[0091] Example 20 is a method according to Example 19, in which the solute further comprises cysteine.

[0092] Example 21 is a method according to any of Examples 16-18, in which injecting the solution into a joint capsule of the mammal comprises injecting the solution into the joint capsule of the mammal two or more times within seven days.

[0093] Example 22 is a method according to any of Examples 16-18, further comprising electrically stimulating a site of the injection with transcutaneous electrical nerve stimulation prior to injecting the solution into the joint capsule.

[0094] Example 23 is a method according to any of Examples 16-18, further comprising directing infrared light at a site of the injection prior to injecting the solution into the joint capsule.

[0095] Example 24 is a method according to any of Examples 16-18, further comprising performing ultrasound treatment at a site of the injection prior to injecting the solution into the joint capsule.

[0096] Example 25 is a method according to any of Examples 16-18, further comprising applying manual pressure to a site of the injection prior to injecting the solution into the joint capsule.

[0097] Example 26 is a method according to any of Examples 16-18, in which the solute further comprises stem cells harvested from the mammal.

[0098] Example 27 is a method according to any of Examples 16-18, in which the solute further comprises platelet-rich plasma harvested from the mammal.

[0099] Example 28 is a method according to any of Examples 16-18, in which each milliliter of the solution is one unit, and further comprising injecting at least two units of the solution into a joint capsule of the mammal.

[0100] Example 29 is a method according to Example 28, wherein the joint capsule is a knee joint capsule and wherein injecting at least two units of the solution into a knee joint capsule of the mammal comprises injecting two units of the solution at least two times within seven days.

[0101] Example 30 is a method according to Example 28, wherein the joint capsule is a hip joint capsule and wherein injecting at least two units of the solution into a hip joint capsule of the mammal comprises injecting three units of the solution at least two times within seven days.

[0102] Example 31 is a method according to Example 28, wherein the joint capsule is a knee joint capsule, a hip joint capsule, an ankle joint capsule, a shoulder joint capsule, an elbow joint capsule, a wrist joint capsule, or a back joint capsule.

[0103] Example 32 is a method according to Example 28, in which the mammal is a human being.

[0104] Example 33 is a method according to Example 28, in which the mammal is a horse.

[0105] Example 34 is a method according to any of Examples 16-18, wherein the combination of L-proline, L-glycine, or one or more L-glycine precursors, and L-lysine are administered concurrently.

[0106] Example 35 is a method according to any of Examples 16-18, wherein the combination of L-proline, L-glycine, or one or more L-glycine precursors, and L-lysine are administered are administered simultaneously from a single composition.

[0107] Example 36 is a method according to any of Examples 16-18, wherein the combination of L-proline, L-glycine, or one or more L-glycine precursors, and L-lysine are administered from separate compositions.

[0108] Example 37 is a system for promoting collagen growth in the joint of a mammal including providing one or more containers containing a therapeutically effective amount ofone or more of L-proline, L-glycine, or an L-glycine precursor, and L-lysine, wherein the amount of L-proline is at least about 55% by weight of the total amount of L-proline, L-glycine, and L-lysine provided in the one or more containers, and wherein when administered to the joint of a mammal via injection, the combination of L-proline, L-glycine, or an L-glycine precursor, and L-lysine, promotes collagen growth within the joint.

[0109] Example 38 is a system according to Example 37, wherein one or more of the L- proline, L-glycine, or an L-glycine precursor, and L-lysine, are contained within a single container.

[0110] Example 39 is a system according to Example 37, wherein each of the L-proline, L- glycine, or an L-glycine precursor, and L-lysine are contained in a single container.

[0111] Example 40 is a system according to Example 37, wherein each of the L-proline, L- glycine, or an L-glycine precursor, and L-lysine, are contained in separate containers.

[0112] Example 41 is a system according to any of Examples 37-40, wherein the containers are syringes.

[0113] Example 42 is a use of a combination of L-proline, L-glycine, or an L-glycine precursor, and L-lysine, wherein the L-proline is present in an amount of at least about 55% by weight of the combination in the manufacture of a medicament for promoting one or more of collagen growth in a mammal, cartilage growth in a mammal, or production of synovial fluid in a mammal.

[0114] Example 43 is a use according to Example 42, in which the combination is delivered to the mammal via injection into a joint capsule.

[0115] It should be appreciated that reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Therefore, it should be emphasized and appreciated that two or more references to "an embodiment" or "one embodiment" or "an alternative embodiment" in various portions of this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures or characteristics may be combined as suitable in one or more embodiments of the invention.

[0116] In this application, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean "includes," "including," and the like, and are generally interpreted to be open ended terms. The terms"consisting of" or "consists of" are closed terms, and include only the components, structures, steps, or the like specifically listed in conjunction with such terms, as well as that which is in accordance with U.S. Patent law. "Consisting essentially of" or "consists essentially of" have the meaning generally ascribed to them by U.S. Patent law. In particular, such terms are generally closed terms, with the exception of allowing inclusion of additional items, materials, components, steps, or elements, that do not materially affect the basic and novel characteristics or function of the item(s) used in connection therewith. For example, trace elements present in a composition, but not affecting the compositions nature or characteristics would be permissible if present under the "consisting essentially of" language, even though not expressly recited in a list of items following such terminology. When using an open-ended term, like "comprising" or "including," in this written description it is understood that direct or express support should be afforded also to "consisting essentially of" language as well as "consisting of" language as if stated explicitly and vice versa. Furthermore, in this written description, the recitation of open-ended or inclusive terms, such as "comprising," "including," "having," etc. provides direct or express support for close-ended or exclusive terms such as "excluding," "omitting," "not including," "without," "other than," etc. as if stated explicitly and vice versa.

[0117] As used herein, comparative terms such as "increased," "decreased," "better," "worse," "higher," "lower," "enhanced," "maximized," "minimized," and the like refer to a property of a device, component, composition, or activity that is measurably different from other devices, components, compositions or activities that are in a surrounding or adjacent area, that are similarly situated, that are in a single device or composition or in multiple comparable devices or compositions, that are in a group or class, that are in multiple groups or classes, or as compared to the known state of the art.

[0118] As used herein, the term "substantially" refers to the complete or nearly complete extent or degree of an action, characteristic, property, state, structure, item, or result. For example, an object that is "substantially" enclosed would mean that the object is either completely enclosed or nearly completely enclosed. The exact allowable degree of deviation from absolute completeness may in some cases depend on the specific context. However, generally speaking the nearness of completion will be so as to have the same overall result as if absolute and total completion were obtained. The use of "substantially" is equally applicable when used in a negative connotation to refer to the complete or near completelack of an action, characteristic, property, state, structure, item, or result. For example, a composition that is "substantially free of" particles would either completely lack particles, or so nearly completely lack particles that the effect would be the same as if it completely lacked particles. In other words, a composition that is "substantially free of" an ingredient or element may still actually contain such item as long as there is no measurable effect thereof. It is to be understood that in this written description, instances where the term "substantially" is used in connection with a given item (e.g. element, process, result, characteristic, etc.) also provide express support for the item as though the term "substantially" did not appear, and vice versa.

[0119] As used herein, the term "about" is used to provide flexibility to a numerical range endpoint by providing that a given value may be "a little above" or "a little below" the endpoint. Unless otherwise stated, use of the term "about" in accordance with a specific number or numerical range should also be understood to provide support for such numerical terms or range without the term "about". For example, for the sake of convenience and brevity, a numerical range of "about 50 ml to about 80 ml" should also be understood to provide support for the range of "50 ml to 80 ml." Furthermore, it is to be understood that in this specification support for actual numerical values is provided even when the term "about" is used therewith. For example, the recitation of "about" 30 should be construed as not only providing support for values a little above and a little below 30, but also for the actual numerical value of 30 as well.

[0120] As used herein, a plurality of items, structural elements, compositional elements, and / or materials may be presented in a common list for convenience. However, these lists should be construed as though each member of the list is individually identified as a separate and unique member. Thus, no individual member of such list should be construed as a de facto equivalent of any other member of the same list solely based on their presentation in a common group without indications to the contrary.

[0121] Concentrations, amounts, and other numerical data may be expressed or presented herein in a range format. It is to be understood that such a range format is used merely for convenience and brevity and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. As an illustration, a numerical range of "about 1 toabout 5" should be interpreted to include not only the explicitly recited values of about 1 to about 5, but also include individual values and sub-ranges within the indicated range. Thus, included in this numerical range are individual values such as 2, 3, and 4 and sub-ranges such as from 1-3, from 2-4, and from 3-5, etc., as well as 1, 2, 3, 4, and 5, individually, and further including decimal or fraction values such as 1.8, 2.3, 3.7, and 4.2. This same principle applies to ranges reciting only one numerical value as a minimum or a maximum. Furthermore, such an interpretation should apply regardless of the breadth of the range or the characteristics being described.

[0122] Formulation or compositional ingredients included or recited herein are to be presumed to be in wt% unless specifically stated otherwise. In addition, ingredient amounts presented in the form of ratios are to be presumed to be in wt% (e.g. %w / w) ratios. As such, a composition containing four ingredients at a 1:1:1:1 ratio would indicate that each ingredient is present in an amount of 25 wt%. Accordingly, in some aspects, the amount of an ingredient in a composition or formulation in terms of wt% can be derived from a numerical ratio value.

[0123] Reference in this specification may be made to devices, structures, systems, or methods that provide "improved" performance. It is to be understood that unless otherwise stated, such "improvement" is a measure of a benefit obtained based on a comparison to devices, structures, systems or methods in the prior art. Furthermore, it is to be understood that the degree of improved performance may vary between disclosed embodiments and that no equality or consistency in the amount, degree, or realization of improved performance is to be assumed as universally applicable.

[0124] The term, "subject," "subjects," or "subjects in need thereof" include humans as well as non-human subjects, particularly domesticated and farm animals. It will be understood that the subject to which a compound of the invention is administered need not suffer from a specific traumatic state. Indeed, the compounds of the invention may be administered prophylactically, prior to any development of symptoms. The term "therapeutic," "therapeutically," and the like are used to encompass therapeutic, palliative as well as prophylactic uses.

[0125] As used herein a "therapeutic agent" and an "active agent" can be used interchangeably and refer to an element, compound, molecule, or substance, including without limitation amino acids, and peptides, that can have a beneficial or positive effect ona subject when administered to the subject in an appropriate or effective amount. Nonlimiting examples of a therapeutic or active agent include L-proline, L-glycine, and L-lysine, or functional precursors thereof.

[0126] As used herein, the terms "treat," "treatment," or "treating" when used in conjunction with the administration of an active agent, such as L-proline, L-glycine, and L- lysine, and the like, including compositions and dosage forms thereof, refers to administration to subjects who are either asymptomatic or symptomatic. In other words, "treat," "treatment," or "treating" can be to reduce, ameliorate or eliminate symptoms associated with a condition present in a subject, or can be prophylactic, (i.e. to prevent or reduce the occurrence of the symptoms in a subject). Such prophylactic treatment can also be referred to as prevention of the condition.

[0127] As used herein, "concurrently" or "concurrent" when used in the context of administering more than one agent, (e.g. an active agent such as an amino or imino acid, such as L-proline, L-glycine, and L-lysine) refers to the timing of administration. Notably, "concurrent" administration or administration of two or more agents "concurrently" does not require that the agents be administered at the same time (e.g. simultaneously), but rather, simply within a time frame where each can play its designated role in achieving a desired effect, such as a therapeutic effect. For example, agents can be administered within seconds, minutes, hours, or days within one another as long as such administration results in the desired effect. Moreover, it is to be understood that in this written description, the terms "concurrent," "concurrently," and the like provide express support for "simultaneous," "simultaneously" and other such terms and vice versa.

[0128] As used herein, an "effective amount" of an agent is an amount sufficient to accomplish a specified task or function desired of the agent. A "therapeutically effective amount" of a composition, drug, or agent refers to a non-toxic, but sufficient amount of the composition, drug, or agent, to achieve therapeutic results in treating or preventing a condition for which the composition, drug, or agent is known to be effective. It is understood that various biological factors may affect the ability of a substance to perform its intended task. Therefore, an "effective amount" or a "therapeutically effective amount" may be dependent in some instances on such biological factors. Further, while the achievement of therapeutic effects may be measured by physician, veterinarian, or other qualified medical personnel, using evaluations known in the art, it is recognized that individual variation andresponse to treatments may make the achievement of therapeutic effects a somewhat subjective decision. The determination of an effective amount or therapeutically effective amount is well within the ordinary skill in the art of pharmaceutical sciences and medicine. See, for example, Meiner and Tonascia, "Clinical Trials: Design, Conduct, and Analysis," Monographs in Epidemiology and Biostatistics, Vol. 8 (1986).

[0129] Similarly, it should be appreciated that in the foregoing description of exemplary embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment or description thereof for the purpose of streamlining the disclosure aiding in the understanding of one or more of the various inventive aspects. This method of disclosure, however, is not to be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects may lie in less than all features of a single disclosed embodiment. Thus, the claims following this detailed description are hereby expressly incorporated into this detailed description, with each claim standing on its own as a separate embodiment of this invention.

Claims

CLAIMSWhat is claimed is:

1. A solution for injection into mammals to stimulate collagen growth, the solution comprising: solutes having amino acid tropocollagen factors including L-proline, L-glycine or one or more L-glycine precursors, and L-lysine in which the L-proline comprises at least 55% of the amino acid tropocollagen factors; a solvent, wherein the solution contains the solutes in concentrations effective to promote the production of collagen proteins within a mammal when applied to the mammal via injection.

2. The solution according to claim 1, wherein the solution contains the solutes in concentrations effective to promote the production of collagen proteins within a mammal when applied to the mammal via injection into a joint capsule.

3. The solution according to claim 1, in which the L-proline comprises at least 70% of the weight of the amino acid tropocollagen factors in the solute.

4. The solution according to claim 1, in which the L-proline comprises at least 80% of the amino acid tropocollagen factors.

5. The solution according to claim 1, in which the solvent comprises water.

6. The solution according to claim 1, in which the solution is brought to a temperature in excess of 200 degrees Fahrenheit and cooled to less than 80 degrees Fahrenheit prior to injection.

7. The solution according to claim 1, in which the solution is stored in a syringe.

8. The solution according to claim 1, in which the solutes are in powdered form prior to being combined with the solvent.

9. The solution according to claim 1, in which the solution contains the solutes in concentrations effective to promote the production of cartilage within a mammal when applied to the mammal via injection.

10. The solution according to claim 1, in which the solution contains the solutes in concentrations effective to promote the production of synovial fluid within a mammal when applied to the mammal via injection.

11. The solution according to claim 1, in which the solutes have amino acid tropocollagen factors including L-proline, L-glycine and L-lysine in a ratio of between 5.5:2:1 and 95:3:1 of the amino acid tropocollagen factors.

12. The solution according to claim 1, in which the solution contains no more than a trace amount of ascorbic acid.

13. The solution according to claim 1, in which the one or more L-glycine precursors include serine and pyridoxal phosphate.

14. The solution according to claim 13, in which the solute contains no L-glycine.

15. The solution according to claim 13, in which the solute contains less than a trace amount of L-glycine.

16. A method of promoting collagen growth in a joint of a mammal, comprising: obtaining an injectable solution as recited in any one of claims 1-15; and injecting the solution into a joint capsule of the mammal.

17. A method of promoting collagen growth in a joint of a mammal, comprising: administering to the joint capsule via injection, a collagen growth promoting combination of L-proline, L-glycine, or one or more L-glycine precursors, and L-lysine, wherein the L-proline is present in an amount of at least about 55% by weight of the combination.

18. A method of growing collagen in a joint of a mammal comprising: injecting into the joint capsule, a therapeutically effective amount of L-proline, L- glycine, one or more L-glycine precursors, and L-lysine, wherein the amount of L-proline is at least about 55% by weight of the total amount of L-proline, L-glycine, or one or more L- glycine precursors, and L-lysine injected.

19. The method according to any of claims 16-18, in which the one or more glycine precursors comprises serine and pyridoxal phosphate.

20. The method according to claim 19, in which the solute further comprises cysteine.

21. The method according to any of claims 16-18, in which injecting the solution into a joint capsule of the mammal comprises injecting the solution into the joint capsule of the mammal two or more times within seven days.

22. The method according to any of claims 16-18, further comprising electrically stimulating a site of the injection with transcutaneous electrical nerve stimulation prior to injecting the solution into the joint capsule.

23. The method according to any of claims 16-18, further comprising directing infrared light at a site of the injection prior to injecting the solution into the joint capsule.

24. The method according to any of claims 16-18, further comprising performing ultrasound treatment at a site of the injection prior to injecting the solution into the joint capsule.

25. The method according to any of claims 16-18, further comprising applying manual pressure to a site of the injection prior to injecting the solution into the joint capsule.

26. The method according to any of claims 16-18, in which the solute further comprises stem cells harvested from the mammal.

27. The method according to any of claims 16-18, in which the solute further comprises platelet-rich plasma harvested from the mammal.

28. The method according to any of claims 16-18, in which each milliliter of the solution is one unit; and further comprising injecting at least two units of the solution into a joint capsule of the mammal.

29. The method according to claim 28, wherein the joint capsule is a knee joint capsule and wherein injecting at least two units of the solution into a knee joint capsule of the mammal comprises injecting two units of the solution at least two times within seven days.

30. The method according to claim 28, wherein the joint capsule is a hip joint capsule and wherein injecting at least two units of the solution into a hip joint capsule of the mammal comprises injecting three units of the solution at least two times within seven days.

31. The method according to claim 28, wherein the joint capsule is a knee joint capsule, a hip joint capsule, an ankle joint capsule, a shoulder joint capsule, an elbow joint capsule, a wrist joint capsule, or a back joint capsule.

32. The method according to claim 28, in which the mammal is a human being.

33. The method according to claim 28, in which the mammal is a horse.

34. The method of any of claims 16-18, wherein the combination of L-proline, L-glycine, or one or more L-glycine precursors, and L-lysine are administered concurrently.

35. The method of any of claims 16-18, wherein the combination of L-proline, L-glycine, or one or more L-glycine precursors, and L-lysine are administered are administered simultaneously from a single composition.

36. The method of any of claims 16-18, wherein the combination of L-proline, L-glycine, or one or more L-glycine precursors, and L-lysine are administered from separate compositions.

37. A system for promoting collagen growth in the joint of a mammal comprising: providing one or more containers containing a therapeutically effective amount of one or more of L-proline, L-glycine, or an L-glycine precursor, and L-lysine, wherein the amount of L-proline is at least about 55% by weight of the total amount of L-proline, L- glycine, and L-lysine provided in the one or more containers, and wherein when administered to the joint of a mammal via injection, the combination of L-proline, L-glycine, or an L-glycine precursor, and L-lysine, promotes collagen growth within the joint.

38. The system of claim 37, wherein one or more of the L-proline, L-glycine, or an L- glycine precursor, and L-lysine, are contained within a single container.

39. The system of claim 37, wherein each of the L-proline, L-glycine, or an L-glycine precursor, and L-lysine are contained in a single container.

40. The system of claim 37, wherein each of the L-proline, L-glycine, or an L-glycine precursor, and L-lysine, are contained in separate containers.

41. The system of any of claims 37-40, wherein the containers are syringes.

42. Use of a combination of L-proline, L-glycine, or an L-glycine precursor, and L-lysine, wherein the L-proline is present in an amount of at least about 55% by weight of thecombination in the manufacture of a medicament for promoting one or more of collagen growth in a mammal, cartilage growth in a mammal, or production of synovial fluid in a mammal.43 The use according to claim 42, in which the combination is delivered to the mammal via injection into a joint capsule.

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