AU606038B2 - Tissue growth regulation - Google Patents

Description

6 06i0 38 WORLD INTELLECTUAL PROPERTY ORGANIZATION International Bureau

PCT

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (IPCT) (51) International Patent Classification 4 International Publication Number: WO 87/ 12244 A61K 33/06, 31/70 (A61K 33/06 Al (3 nentoa ulcto ae 3Arl18 130.7 A61 K 31:70, 31:415)(4)ItrainlPbiainDt: 2Apl197(308) (21) International Application Number: PCT/GB86/00607 US.

(22) International F~iling Date: 8 October 1986 (08.10.86) Published With international search report.

(31) Priority Application Number: 8524807

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(32) Priorit), Date: 8 October 1985 (08,10.85) (33) Priority Country: GBti SECTIk 10 (fl, DIRECTION SEE FO LAI640 4/86 NAME DIRECTECD Th'Y( A U-l 40 4 8 i7I~-~ IEfP -1JUN 1987 (81) Designated States: AT (European patent), AU, BE (Eu- ASRLA ropean patent), CR (European patent), DE (European ASRLA patent), FR (European patent), GB (European patent), IT (European patent), JP, LU (European pa- M AY 1987 tent), NL (European patent), SE (European patent), SU, PATENT OF~FICE~ (54) Title: TISSUE GROWTH REGULATION (57) Abstract A preparation for tissue growth regulation comprises at least one ofan N- acetyl. D-gl ycosa mine or an oligomer thereof or a deacetylated derivative thereof or a substituted product of these compounds, at least one of bic tin or an analogue or derivative of biotin or biologically active residue thereof, and a divalent metal cation together Ni ith a ptnrmaceutically acceptable anion.

made n r 'SOctiOn 49 and is corret for prI rg SFP4 To: The Commissioner of Patents Sarans 1 Tissue Growth Regulation Field of the invention This invention relates to preparation of substances for tissue growth control in humans or animals. More particularly, the invention relates to a group of physiologically active substances, at least one member of which promotes, whilst the others inhibit, the growth of both normal and abnormal tissue. Thus the substances of this invention provide for the enhancement of normal tissue growth to accelerate normal repair funcTions and the suppression of undesirable tissue growth, whether benign as in the fibrosis associated with excessive scar tissue formation, or aberrant, as in cancerous or other malignant change.

Background Art Extensive work has been done on the control of growth in plants, none of which is applicable to animal i tissue. Investigation of the growth requirements of cells of animal origin (including human cells) have been principally by tissue culture, that is by a technique facilitating the study of cells grown in isolation under S* laboratory conditions. Various "growth factors" have been J used to encourage cellular proliferation, commonly using i extracts of a relatively crude nature derived from whole organs such as mouse salivary gland or rat liver. Such growth factors have no possible applicability for systemic use in intact animals or humans. For examp-' riboflavin is thought to be a growth factor for rats, lipoic acid serves as a growth factor for certain microorganisms and biotin is a growth factor for yeast and certain bacteria. Glycyl-histidyl-lysine and pituitary growth hormone are two chemically characterised o, u pure substances with an influence on growth, the former in the culture of isolated liver cells and the latter in bodily growth as a whole. None of these "growth factors" has been recorded as having any known usefulness in a 2 tissue repair, and are on the whole remote from the substances to be disclosed hereinafter.

In patients who are malnourished, the systemic administration of amino acids is known to be of value in assisting wound healing but only by remedying existing amino acid deficiencies.

Physical or physico-chemical techniques such as the y use of magnetic fields or pulse electrical currents for localised stimulation of a wound site have some current vogue.

The inhibition of aberrant or malignant cells by chemical means is now common practice, and is effective where there is a favourable ratio of susceptibility between the malignant cells of the tumour and the normal cells of the host. In the absence of any more selective means of control, these cytotoxic substances have wide currency at present; but their toxicity and consequent high incidence of side effects makes it extremely desirable that improved physiological or chemical control **3b should be developed.

Cortisone and its derivatives are effective in the control of excess fibrous tissue formation such as may occur in some types of injury, in many cases of burns, and after some inflammatory diseases of the joints.

However use of cortisone involves a considerable risk of side effects.

Earlier work (Nature, 199, 4991; 392; 1963) by the .present inventor showed that the level of activity of the enzyme N-acetyl-D-glucosaminidase was closely related to what might be termed the "reactivity" of a variety of disease processes. This relationship existed under such a wide range of circumstances as to suggest a more important role than was then known for N-acetyl-Dglucosamine (NAG) and perhaps for other N-acetyl-Dglycosamines.

3 N-Acetyl-D-glucosamine (2-acetamido-2-deoxy-Dglucose) residues are widely present in bound form in nature as is D-galactnsamine.

In a text entitled "Processes in Pathology" (Taussig, published in 1979 by Blackwell Scientific Publications, at pp 267-270, in a discussion primarily concerned with the activity of lectins, reference is made to NAG in a study of cell growth. Experiments relating to Suse of NAG are mentioned. In one of these NAG is used as S 10 its dimer di-N-acetylchitobiose bound to a protein carrier in an attempt to preimmunise mice against the i lethal effects of a transplanted tumour with limited success. In another inhibition of tumour cell growth by treatment with monovalent lectin to cover the surface agglutination sites (NAG residues) was proved reversible by addition of excess of free NAG.

*i Other workers have been active in this field, for example in the early 1960s Levvy and his associates at the Rowett Research Institute showed that the conversion to a lactone of a glycoside which formed the substrate f for a glycosidase, would specifically inhibit the S° glycosidase concerned. Unfortunately, these lactones were stable only under strictly controlled laboratory conditions. Attempts to employ them for therapeutic purposes were therefore not pursued.

Sout The inventor arrived at this invention by carrying out investigations into the possibility that N-acetyl-Di' glycosamines and their oligomers and derivatives of both classes of compound might play important, and previously unrecognised parts in the processes of healing and repair, and in the invasiveness and proliferation of malignant cells. The concept of inhibition of the Nacetyl-D-glycosaminidases and of N-acetyl-D- glucosaminidase specifically, under physiological conditions, opens up new possibilities both for the I mr~- 4 regulation of healing and repair, and for the control of cancerous growth.

DISCLOSURE OF THE INVENTION Accordingly the present inventor provides a method of making a preparation for use in tissue growth regulation which comprises forming an aqueous solution of at least one N-acetyl-D-glycosamine or an oligomer thereof or a deacetylated form thereof or an obvious bioequivalent of these compounds and a divalent metal cation together with a pharmaceutically acceptable anion, maintaining said solution at an elevated temperature for st. an extended period of time, and then freeze-drying the liquid to recover an 10 agent for use in tissue growth regulation.

Preferably the aqueous solution formed during the preparation of this invention comprises biotin.

Preferably the glycosamine used in the preparation of this invention S in N-acetyl-D-glucosamine or its oligomers or completely or partially deacetylated derivatives thereof.

Preferably the preparation is prepared as an aqueous solution containing N-acetyl-D-glucosamine, biotin and a divalent cation such as Mg 2 The solution may include buffer salts. As a solution the S preparation is suitable for parenteral or oral administration.

'0 The preparation of this invention may be made in a solid form for oral or rectal administration by formulation of a tablet, lozenge or suppository. Alternatively it may be made as a powder to be taken, or formulated as an encapsulated solution or emulsion for oral administration S. or subsequent formulation and administration as an injection solution.

25 Further it can be formulated for topical application in a suitable neutral S ointment base.

The method of making the preparation affects the properties thereof.

For example mixtures may be activated by subjecting them to varying periods of, and conditions of, heating and of drying or partial drying, leading to the intermediate recovery of crystalline and associated insoluble materials, with other materials remaining in '1 1 ~f 1V I I,

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solution or in a syrupy form. Those materials accompanying precipitated biotin on the one hand, and those materials remaining in solution after such precipitation and thereafter isolated in the syrupy or dried form on the other, have the separate biological activities referred to hereinbelow. Biological activity can also be obtained when the concentration of biotin and the periods and conditions of heating and drying or partial drying are so devised as to avoid the precipitation referred to above. Activity is then associated with the material either remaining in solution or produced in syrupy or dry form from it. These various materials influence the activity of N-acetyl-Dglucosaminidase and its two principal isozymes A and B.

Depending upon the exact method of preparation the materials may enhance or inhibit the whole enzyme or its A and B isozymes in isolation or together. The inhibition can be made to be either competitive or non-competitive.

S Thus the preparation can influence the total level of N- .2S acetyl-D-glucosaminidase activity in the body and also alter the proportional effect of the two main iso- S enzymes. In doing this the preparation appears to mimic the reactions of the body to many disease processes as, for example, in its response to foreign tissue rejection, to inflammation and to malignancy.

For ease of understanding the invention it will be described hereinbelow with reference to one glycosamine, N-acetyl-D-glucosamine, but it will be appreciated by W*nrkers in this field that the methods of preparation are 30 applicable to analogous compounds and the biological effeuts obtained will vary depending on the enzyme or cellular system under investigation or therapy.

N-Acetyl-D-glucosamine itself has been shown to have a stimulating effect upon defence cells in the body, and it is believed that the preparation of this invention

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Sr 1 0P 4 .4 4 br 4*B 6 provides a new and selective means of influencing the body's immune mechanisms. The active components may be administered by mouth or by injection, either alone, or incorporated in physiologically-benign fluids or solids or any other pharmaceutically acceptable vehicles, carriers and adjuvants. Whilst not wishing to be bound by any particular theory the role of the active components may be selectively to block or unblock active sites in enzymes participating in tissue or cell growth. There may be action on cell surface sites responsible for functions such as cell specificity, or the transfer of nutrients and hormones. Sites such as these are known to function abnormally in cancerous cells, and are affected also in viral and possibly other infections.

Accordingly, the present invention finds a use in the control of the proliferation, growth and invasiveness of malignant cells; the regulation of the level of activity of cells involved in the repair of tissue after injury or inflammation, with the objectixa of encouraging healing whilst at the same time controlling any tendency for one aspect of healing to outscrip the other, as for example, the over-production of collagen by fibroblasts which results in hypertrophic scar formation, with consequent cosmetic or functional impairment; and the stimulation of healing where it is indolent as, for instance, in the elderly or in those others having suffered very severe infections.

The invention will now be further described by way of the following Examples and Data indicating preferred methods of preparation and biological effects observed with use of the substances ot this invention.

Examples Example 1 A method of preparing a preparation for use in the stimulation of healing and a preparation for the control of malignant cells.

3.23 g. of N-Acetyl-D-glucosamine, 200 mg.of biotin, I375 mg. potassium dihydrogen phosphate, and 1.8 g. of I magnesium sulphate were dissolved in water to give 100ml.

of solution, which was maintained at 55 0 C. in a closed vessel for 72 hours. The contents were then cooled and S 10 kept at just above freezing point. During the next 24 j hours a mainly crystalline deposit separated. This was j removed by centrifugation and the solid deposit was washed with water (or ethanol). The solid had no Ssignificant inhibitory power on N-acetyl-D- ;16 glucosaminidase, but had a powerful enhancing action on its B-iso-enzyme. The solid was insoluble in water but S* soluble in lipids. Following treatment of patients with areas of indolent healing using the solid, rapid regeneration of skin cells was observed. Therefore the 0 r.:S aforementioned steps provide a means of stimulating healing.

The supernatant remaining after separation of the solid material as described above was reti rned to a closed vessel and incubated. It was then freeze-dried.

The resulting material showed a specific non-competitive type of inhibition of both the A and B isozymes of Nacetyl-D-glucosaminidase. When added to tissue cultures of malignant cells such as Landschutz cells or Hela cells 0 it produced a series of dose-related effects, including the destruction of the cells' normal anti-adhesive mechanism, and the inhibition of growth. It also led to cell fragmenta-icn and to the production of multiple non- viabie sub-cellular fragments. When administered to patients with terminal malignancy it controlled the invasiveness of the malignant process and increased survival, without other therapy, beyond the prognosis by normal methods.

~a 7 I1 1 17-- Example 2 A method of preparing a preparation for regulating the level of activity of cells involved in the repair of tissues following iniurv or inflammation i J 3.2 g. of N-Acetyl-D-glucosamine, 10 mg. of biotin, 375 mg. of potassium dihydrogen phosphate and 1.8 g. of magnesium sulphate were dissolved in water to provide 100 ml. of solution which was placed in a closed vessel and maintained at a temperature of 70-80 0 C for 72 hours.

The resulting solution was cooled and freeze-dried. The material resulting from this showed non-competitive specific inhibition of the A isozyme of N-acetyl-Dglucosaminidase and a varying degree of competitive .16 inhibition only of the B isozyme. Addition of the material to cultures of fibroblasts produced a doserelated inhibition of fibroblast proliferation about ewithout causing cell death. When administered to competition horses with recent injuries it produced S.20 diminution of swelling, and facilitated the return to o0** soundness. In longer term therapy studies it has been administered on 47 occasions to 35 horses suffering mostly from injuries to the deep flexor tendon of the fore-limb, a condition frequently resulting in permanent disability, sometimes to a degree necessitating slaughter. All 35 horses returned to competition, usually at pre-injury level of soundness.

Systemic use in humans can be expected, but awaits further evidence of non-toxicity. However topical application of the active substance in a neutral ointment base under the supervision of a consultant plastic surgeon has shown evidence of a capacity to control excessive scar tissue formation.

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9 Example 3 The results in the table illustrate the activity of material derived from N-acetyl-D-glycosamine treated in accordance with the invention as compared to N-acetyl-D-glycosamine per se, in the inhibition of fibroblasts.

TABLE 1 Protein results (Lowry) from studies on fibroblasts comparing NAG (purified) and purified anti-fibrotic (NH016).

[Lg/100ul Rg/flask Control 1. 105,5 527.5 2. 116.2 580.9 N A G 1. 126.9 634.4 (Img/ml) 2. 104.1 520.5 1. 99.0 495.0 2. 102.8 513.9 15 NH 016 61.2 (200.1) 153.1 a (1mg/ml) These results are illustrated in the a cell culture plates of fibroblasts.

Figure 1 depicts a control plate.

Figure 2 depicts fibroblasts treated w Figure 3 depicts fibroblasts treated w accordance with the present invention.

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*r a a aa TM TAl, 57M 9a Example 4 12.9g of N-acetyl-D-glucosamine. 1.5g of potassium dihydrogen phosphate and 7.2g of magnesium sulphate were dissolved in water to provide 100ml of solution which was placed in a closed vessel and maintained at a temperature of 70-80C for 72 hours.

The solution was allowed to cool to room temperature: it was then transferred to a separating funnel containing a freshly prepared mixture of 250 ml chloroform and 290 ml isopropanol and shaken vigourously for two minutes. A phase separation then occurred, with a large volume yellow 10 upper phase and a small volume darker yellow/amber lower phase. Any turbidity in the upper phase was cleared by the addition of successive 10 ml aliquots of isopropanol and the phase separation was allowed to stabilize over a period of twenty four hours. The lower phase was then discarded; the upper phase was decanted and dried by rotary evaporation at a temperature of 45 C. The resulting dry material was taken up in a minimal volume of water and freeze dried, The addition of the material to "in vitro" cultures of various cancer cell lines produced a dose related killing as illustrated below.

Compound Cell Line OIN.OB.CHIL KHOS/NP PC3 WiDr Dose (mg/ml) Cell no. Cell no./ Cell no./ .0 control controtl control Control 12654 /100 9643 /100 13159 /100 0.2 13704 /108** 9530 /99 13359 /102 12473 /98 7416 8688 166* 5825 146** 5697 9987 /76** Stat. sig: P 0.01 P 0.001 KHOS/NP is human osteosarcoma.

PC3 Is human prostate adenocarcinoma.

WiDr is human colonadenocarcinoma.

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Claims (13)

1. A method of making a preparation for use in tissue growth regulation which comprises forming an aqueous solution of at least one N-acetyl-D-glycosamine or an oligomer thereof or a deacetylated form thereof or an obvious bioequivalent of these compounds and a divalent metal cation together with a pharmaceutically acceptable anion, maintaining said solution at an elevated temperature for an extended period of time, and then freeze-drying the liquid to recover an agent for use in tissue growth regulation. j 2. A method of making a preparation for use in tissue growth regulation according to claim 1 characterised in that the aqueous solution contains biotin. I 3, A method according to claims 1 or 2 wherein prior to freeze-drying the solution the method further comprises the steps of cooling the solution to a temperature just above the freezing point of the solution to cause formation of a crystalline deposit and recovering this I deposit for use as a further active agent for tissue growth regulation.

4. A method according to claim 3 wherein the liquid remaining after recovery of said deposit is Incubated for a period of time prior to freeze- drying to recover an agent for use in tissue growth regulation.

5. A method according to claims 1 or 2 wherein the solution is heated to a temperature of from 50 to 60 0 C.

6. A method according to claims 1 or 2 wherein the solution Is heated to a temperature of from 70 to

7. A method according to any one of claims 1 to 6 wherein the heated solution is maintained at an elevated temperature for about three days.

8. A method of making an agent for use in tissue growth regulation comprising forming an aqueous solution containing at least one N-acetyl-D- glycosamine or a deacetylated derivative thereof or a bioequivalent thereof, a divalent metal cation together with a pharmaceutically acceptable anion, heating the solution to a temperature in the range of to 80°C for up to three days to provide a liquid containing at least ant agent for use in tissue growth regulation,

9. A method according to any one of claims 1 to t whqv solution is cooled and centrifuged to assist recovery 4c a deposit therein. -i i. ~ii L .Li ul----3 solution at an elevated temperature for an extended period of time, and then freeze-drying the liquid to recover an agent for use in tissue growth regulation. t. 1 1 J E n A method according to any one of claims 1 to 9 wherein the metal cation is selected from Mg, Ca, and Mn.

11. A method according to any one of claims 1 to 10 wherein the solution is formed using N-acetyl-D-g'lucosamine or D-glucosamine.

12. A method according to claim 10 wherein the selected cation is Mg 2 i 13. A pharmaceutical product comprising a product obtained according to any one of the preceding claims in a physiologically-absorbable form together with one or more additives, as required, selected from a Spharmaceutically acceptable vehicle, carrier, exciplent, diluent or adjuvant. *14. A pharmaceutl;al compoition for the treatment of indolent j healing comprising a product derived by forming a solution of N-acetyl i glucosamine, a soluble non-toxic salt of magnesium and a buffer, heating jI the solution to about 55*C and maintaining this temperature for about three i days, cooling the solution and recovering a crystalline deposit tharefrom for use in formulation of the composition. 15, A pharmaceutical composition for the treatment of Indolent healing according to Claim 14 characterised in that the solution from which the product is derived comprises biotin. 16, A pharmaceutical composition for the treatment of cell 4 malignancy comprising a product derived by forming a solution of N-acetyl glucosamine, a soluble non-toxic salt of magnesium and a buffer, heatitig S the solution to about 55°C and maintaining this temperature for about three days, cooling the solution and recovering a crystalline deposit therefrom, returning the solution to a closed vessel and incubating same for a period of time and thereafter freeze-drying same to recover material for use in formulating the composition. i 17. A pharmaceutical composition for the treatment of cell malignancy according to claim 16 characterised in that the solution from which the product is derived comprises biotin. 18, A pharmaceutical composition for the Listment of injured or inflamed tissues comprising a product derived by forming a solution of N-acetyl glucosamine a soluble non-toxic salt of magnesium and a buffer, heating the solution to a temperature of fro, 70 to 80*C and maintaining this temperature for about three days, cooling the solution and freeze- drying to recover material for use in formulating the composition. 1 0884c I .A rcyp;~L~~L---I I LI~LVII owes 9. 0 age* a 0 *to* I I I,. .0 S CO C I S. 12

19. A pharmaceutical composition for the treatment of injured or inflamed tissues according to claim 18 characterised in that the solution from which the product is derived comprises biotin. A method of making a preparation for tissue growth control substantially as hereinbefore described with reference to Example 1 or Example 2.

21. A method of treating indolent healing in a patient requiring said treatment which method comprises administering to the patient an effective amount of a composition according to claim 14 or

22. A method of treating cell malignancy in a patient requiring said treatment which method comprises administering to the patient an effective amount of a composition according to claim 16 or 17.

23. A method of treating injured or inflamed tissues in a patient requiring said treatment which method comprises administering to the patient an effective amount of a composition according to claim 18 or 19. DATED this TNENTY-FIFTH day of OCTOBER 1990 Bestarx Limited Patent Attorneys for the Applicant SPRUSON FERGUSON iMS/QS84C