CS265224B2 - Process for preparing tissue grow regulator - Google Patents

Abstract

A preparation for tissue growth regulation comprises (a) at least one of, an N-acetyl-D-glycosamine or an oligomer thereof or a deacetylated derivative thereof or a substituted product of these compounds, (b) at least one of biotin or an analogue or derivative of biotin or biologically active residue thereof, and (c) a divalent metal cation together with a pharmaceutically acceptable anion.

Description

The invention relates to a method of preparing a composition for controlling tissue growth in humans and animals. In particular, the invention relates to a process for the preparation of a group of physiologically active formulations whose at least one component promotes, while the other component inhibits the growth of both normal and abnormal tissues. The formulations of the present invention thus (a) promote normal tissue growth to accelerate normal regenerative functions, and (b) suppress unwanted tissue growth, both benign, such as fibrosis associated with excessive scarring, and defective, for example, cancer or other malignant changes.

Much effort has been made to limit the growth of plants, but none of this is applicable to animal tissues. Research on the requirements for the growth of cells of animal origin (including human cells) focuses in principle on tissue growth by a technique facilitating the study of cell growth isolated under laboratory conditions. Various growth factors were used to promote cell proliferation routinely using extracts of relatively crude nature taken from whole organs, for example, from the salivary gland or rat liver. Such growth factors have no utility for systemic use in intact animals or in intact humans. For example, riboflavin is considered a growth factor for rats, lipoid acid serves as a growth factor for certain microorganisms, and biotin serves as a growth factor for yeast and for certain bacteria. Glycel-histidy1-lysine and pituitary growth hormone are two chemically characterized growth-enhancing pure substances, with glycyl-histidy1-lysine affecting growth in isolated liver cell culture and pituitary growth hormone affecting the growth of the body as a whole. None of these growth factors has been reported to be useful for tissue repair and regeneration and is completely different from the formulations prepared by the method of the invention.

In poorly fed patients, systemic administration of amino acids is known to have a supportive role in wound healing but only in the treatment of established amino acid deficiency.

Physical and physicochemical methods such as the use of magnetic fields or pulsating electric currents to locally stimulate wounds are of current fashionable significance.

Inhibition of altered or malignant cells by chemical means is now common practice and is effective, with a favorable ratio of sensitivity between malignant tumor cells and normal cells of the affected; in the absence of any more selective mode of control, these cytotoxic agents are currently widely used; however, their toxicity and the consequent high incidence of side effects lead to an extraordinary need to develop improved physiological or chemical control of unwanted cells.

Cortisone and its derivatives are effective in reducing excessive tissue formation, which can occur in some types of lesions, in many cases of burns and after some inflammatory joint diseases. However, the use of cortisone involves a considerable risk of side effects.

Previous work by the inventors (Nature, 199, p. 4,991; 392; 1963) has shown that the potency of the enzyme N-acetyl-D-glucosaminidase is in a close relationship, which may be referred to as reactivity to various disease processes. This relationship exists under such a wide range of conditions that it is considered to play a much more important role than is known for N-acetyl-D-glucosamine (NAG) and perhaps for other N-acetyl-D-glucosamines. N-acetyl-D-glucosamine (2-acetamido-2-deoxy-D-glucose) residues are more widely present in bound form in nature than D-galactosamine.

In Processes in Pathology (Taussig MJ) published in 1979 in Blaskwell Scientific Publications at pp. 267-270, in a discussion primarily focused on lectin activity, reference is made to N-acetyl-D-gluc.osamine in the study cell growth. Experiments relating to the use of N-acetyl-D-glucosamine in the study of cell growth are recalled. In one case, N-acetyl-D-glucosamine is used in the form of its protein carrier-bound dimer of di-N-acetylchitobiosis to pre-immunize the mouse against the lethal effect of a transplanted tumor with limited success. In another inhibition of tumor cell growth by treatment with a monovalent lectin to cover the surface of the agglutination site, N-acetyl-D-glucosamine residues) have proven reversibility by addition of excess free N-acetyl-D-glycosamine.

Other workers also worked actively in this field; for example, in the early 1960s, Levvy et al. at the Rowett Research Institute demonstrated that conversion to the lactone glycoside, which is a substrate for glycosidase, could specifically inhibit the glycosidase under consideration. Unfortunately, these lactones are stable only under strictly controlled laboratory conditions. Experiments for their therapeutic use have therefore not been observed.

It has now surprisingly been found according to the invention that N-acetyl-D-glucosamines and their oligomers and derivatives of both classes of compound can play an important and yet unknown role in the processes of treatment and healing and the invasion and proliferation of malignant cells. The concept of inhibition of N-acetyl-D-glucosaminoidase and N-acetyl-D-gososaminidase specifically under physiological conditions opens up new possibilities both for controlling treatment and healing and for limiting cancer growth.

According to the invention, a tissue growth regulating composition is produced by preparing a solution comprising N-acetyl-D-glucosamine or an oligomer thereof or a deacetylated derivative thereof, biotin 2+ 2+ 2 + 2 + and a divalent metal cation selected from the group consisting of Mg ' Ca and Mn, preferably Mg, together with a pharmaceutically acceptable anion, the prepared solution is maintained at an elevated temperature of 50 to 80 ° C for three days, and then the liquid is freeze-dried.

The solution may contain buffer salts. As a solution, the formulation is suitable for parenteral or oral administration.

The composition of the invention may be prepared in solid form for oral or rectal administration; in this case it is formulated as tablets, lozenges or suppositories. Alternatively, it may be manufactured in powder form or as encapsulated solutions or emulsions for oral administration or for further processing into formulations and for administration as an injection solution. They can also be used for topical application in suitable neutral ointment bases.

The method of manufacture affects the properties of the formulation. For example, the compositions may be activated by subjecting them to varying times and under different heating and drying conditions or partial drying resulting in immediate recovery of the crystalline and accompanying material insoluble while the other materials remain in solution or in syrup form. The materials accompanying biotin on the one hand and the materials remaining in solution after such precipitation and then isolated in the form of a syrup or in the dried form on the other hand have separate biological activities, as discussed below. Biological efficacy can also be achieved if the concentration of biotin and the period and the heating and drying or partial drying conditions are selected to avoid the above precipitation. The activity is then associated with the material either remaining in solution or produced in syrup or in dried form from such a solution. These different materials affect the efficacy of N-acetyl-D-glucosaminidase and their two major enzymes А а В. Depending on the precise method of preparation of the materials, the enzyme as a whole or its а isozymes individually or simultaneously may be promoted or inhibited. The inhibition may be performed to be either competitive or non-competitive. Therefore, the preparation process can affect the overall level of N-acetyl-D-glucosaminidase activity in the body and also change the proportional action to the two major isoenzymes. By doing so, the preparation appears to mimic the body's response to numerous disease processes, for example in response to rejection of foreign tissues, inflammation, and malignancy.

For ease of understanding, the invention is now described with respect to one glycosamine, N-acetyl-D-glucosamine, but it will be apparent to those skilled in the art that the methods of preparation are useful for analogous compounds and the biological activity obtained will vary depending on the enzyme or cellular system under investigation or therapy.

N-acetyl-D-glucosamine itself has a stimulating effect on protective cells in the body, and the formulation prepared by the method of the invention appears to provide novel and selective means for influencing the body's immune mechanism. The active ingredients may be administered orally or by injection, either alone or incorporated into physiologically acceptable liquids or solids, or into any other pharmaceutically acceptable carrier or adjuvant. While not intending to be bound by any theory, the role of the active ingredients may be to selectively block or not block the active sites from the involvement of enzymes in tissue or cell growth.

Thus, cell surface sites responsible for the functions of such cell specificities or for the transport of nutrients and hormones may occur. Such sites are known to function abnormally in cancer cells and are also affected by viruses and possibly other infections.

The composition prepared by the method of the invention is useful (a) for limiting the proliferation, growth and aggressiveness of malignant cells; (b) for regulating the level of cell activity involved in repairing tissues after injury-assisted or inflammatory inflammation, while controlling any tendency of one aspect of healing to suppress another, e.g. c) stimulates healing where it is inert, such as in old age in cases of very severe infection.

The invention will now be further elucidated by the following examples and values indicating the preferred methods of preparation and biological activity observed in the formulations prepared by the process of the invention.

Example 1

Process for preparing (a) a preparation for use in stimulating healing and (b) a malignant cell suppressant (a) Dissolve in water 3.23 g of N-acetyl-D-glucosamine, 200 mg of biotin, 375 mg of potassium dihydrogen phosphate and 1.8 g of magnesium sulfate to obtain 100 ml of a solution which is maintained at 55 ° C in a sealed vessel for 72 hours. The contents are then cooled and kept just above the freezing point. Within a further 24 hours, the main crystalline deposit is separated. Remove by centrifugation and wash the solid with water or ethanol. The solid has no significant inhibitory action on N-acetyl-D-glucosaminidase, but has a significant effect, promoting the action on its B-isoenzyme. The solid is insoluble in water, but is soluble in lipids. Rapid treatment of skin cells is observed after treatment of patients with areas resistant to solid wound healing. Thus, the above-described process provides healing stimulants.

(b) The supernatant remaining after separation of the solid material as described above is returned to a sealed container and incubated. It is then freeze-dried. The resulting material exhibits a specifically non-competitive type of inhibition of both the A and N isozymes of N-acetyl-D-glucosaminidase. When added to tissue cultures of malignant cells, such as Landschutz cells or Hela cells, it produces a number of dose-dependent events, including destruction of the cellular normal anti-adhesion mechanism and growth inhibition. It also leads to cell fragmentation and thus to the formation of many inanimate subcellular fragments. When administered to patients with terminal malignancy, it reduces the aggressiveness of the malignant process and increases survival without other therapy having a prognosis given by normal methods.

Example 2

A method of preparing a composition for regulating the level of activity of cells involved in tissue recovery after damage or inflammation

Dissolve 3.2 g of N-acetyl-D-glucosamine, 10 mg of biotin, 375 mg of potassium dihydrogen phosphate and 1.8 g of magnesium sulfate in water to obtain a 100 ml solution which is placed in a sealed vessel and maintained at 70-80 ° C. ° C for 72 hours. The resulting solution was cooled and freeze-dried. The material obtained exhibits a non-competitive specific inhibition of the A-isozyme N-acetyl-D-glucosaminidase and a different degree of competitive inhibition only in the isozyme.

Addition of the material to fibroblast cultures induces a dose-dependent approximately 50% inhibition of fibroblast proliferation without cell killing. When administered to racing horses with a fresh wound, it reduces swelling and makes it easier to return to a healthy state. In the study of long-term therapy, the composition was administered in 47 cases to 35 horses suffering mainly from damage to the flexor tendon of the foreleg, which often leads to permanent disability, sometimes to the extent that slaughter is required. All 35 horses returned to the racetrack, usually in a prone state as before injury.

Systemic use in humans can be expected, but additional evidence of non-toxicity is required. However, the topical use of the active ingredient in a neutral butter base under the control of a plastic surgery consultant has shown the ability to control excessive scar tissue formation.

Claims (7)

SUBJECT OF THE INVENTION A process for the preparation of a tissue growth regulating composition comprising preparing a solution comprising N-acetyl-D-glucosamine or an oligomer thereof or a deacetylated derivative thereof, 2+ 2+ 2+ biotin and a divalent metal cation selected from the group consisting of Mg, Ca and Mn together with a pharmaceutically acceptable anion, the prepared solution is maintained at an elevated temperature of 50-80 ° C for three days, and then the liquid is freeze-dried. 2. A process according to claim 1, characterized in that the solution is cooled to a temperature just above the freezing point of the solution prior to freeze-drying and the crystalline fraction formed is separated as another active substance for controlling tissue growth. 3. A method according to claim 2, characterized in that the liquid remaining after separation of the crystalline fraction is incubated prior to freeze-drying to obtain a tissue growth regulating agent. 4. The method of claim 1, wherein the solution is maintained at a temperature of 50-60 ° C. 5. The method of claim 1, wherein the solution is maintained at a temperature of 70-80 ° C. 6. The process of claim 1, wherein the cooled solution is centrifuged after standing in the cooled state for up to 24 hours to obtain a crystalline fraction. 7. The process of claim 1, wherein the solution contains Mg2 ⁺ as a divalent metal cation.