CA1289885C - Tissue growth regulation - Google Patents
Tissue growth regulationInfo
- Publication number
- CA1289885C CA1289885C CA000520028A CA520028A CA1289885C CA 1289885 C CA1289885 C CA 1289885C CA 000520028 A CA000520028 A CA 000520028A CA 520028 A CA520028 A CA 520028A CA 1289885 C CA1289885 C CA 1289885C
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- Canada
- Prior art keywords
- solution
- temperature
- biotin
- acetyl
- tissue growth
- Prior art date
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- Expired - Lifetime
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7004—Monosaccharides having only carbon, hydrogen and oxygen atoms
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/415—1,2-Diazoles
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/06—Aluminium, calcium or magnesium; Compounds thereof, e.g. clay
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
- C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
- C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Inorganic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Engineering & Computer Science (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Abstract Preparation for Tissue Growth Regulation 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
F r ~ I r ~ o ~ 1 l r~ e, ~ i:, I f~ , Z;! F I I ~ E 1~ i 5 l.X~3~3~
Field of the Inveneion This invention relates to a preparation for tiss~e growth control in humans or animals. More particularly, the ir,vention relates to a ~roup of physiolo~ically 5 active preparat;ons, at Least one ~ember of which promotes, whilst the others inhibitr the growth of both normal and abnormal tissue. Thus, the preparat;ons of th;s invention prov;de for ~a) the enhancement of normal t;ssue growth to accelerate normal repair functions and ~b3 the suppression of undes;rable t;ssue growth, whether benign as in the f;brosis assoG;ated with excessive scar tissue for~a~;~n, or aberrant, as in cancerous or other malignant change.
~ackground Art Extensive work has been done on the control of ~rowth in plants~ none of wh;ch is applicable to animal tissueO Invest;gation of the growth requ;rements of cells o~ anima~ origin (incL~ding human cells) have been princip~lly ~y t;s~ue culture, ~hat is by a technique facilitating the study o~ ceLls grown in ;solation under laboratory conditions. Various "growth fact~rs" have ~een used to encoura~e ceLlular proliferation, commonly using extracts of a relat;veLy crude nature derived from whole organs such as mouse salivary gLand or rat l;ver.
~5 Such grow~h f~ctors have no possible applicab;lity for systemic use ;n ;ntact an;~als or humans. For exampLe, riboflavin is tho~ght t~ b~ a yrowth factor for rats, lipoic acid serves as a growth factor for certa;n micro-or~anisms and biotin is a growth factor for yeast and ~0 certain bacteria. Glycyl-histidyl-lysine and pituitary growth hormone arr t~o chemicalLy char~cter;~ed pure substances w;th an influence on ~rowth, the former in th~ culture of isolated ~iver cells and the latter ;n bodily gro~th ~5 a whole. None of these "growth factors"
has been resorded as ha~ing any known usefulness in F ~ f l ~`~ F I T ,~ r ~ ; L ~ U i D I I J I ~ . 13 ~ r~, ~ P R ~ E ~ / 1 5 12a~
tissue repair, and are on the whole remote from the preparations to be disclosed hereinafter.
In patients ~ho are malno~rished, the syste~ic a~ministration of amino ac;ds ~s known to be of value ;n 5 ass~st~ng wound heaL1ng, but onLy by remedyin~ existiny amtno acid deficiencies.
PhysicaL or phys;co-chemical techniques such as the use of magnetic field~ or pulse elec~rical currents for local;sed stimulat;on of a wound s;te have some carrent vogue.
The inhibition of a~errant or malign~nt cells by chemical means is now common practice, and is effective ~here there ;s a favourabLe rat;o of suscePtibil;ty between the malignant cells of the tumour and the normaL
1~ ~eLLs of the host. In the absence of any more selective means o~ control, these cytotoxic substances have w;de currency at present; but their ~oxicity and consequent hi~h incidenc~ of side effects makes it extremely desirabLe that improved physiolo~;cal or chem;cal control should be deveLoped~
Cort;sone and i~s derivatives are effective in the control of excess f;brous t;ssue format;on such as may occur in ~ome types of injury, ;n many cases o~ burns~
and after som~ inflammatory d;seases of the joints.
However, use of cortisone involves a cons;derable risk of side effec~s.
E~rl;er work tNature, 199, 4991; 3~2; 1q63) by the present inventor showed tha~ the level of activity ~f the enzyme N-ace~yl-~-glucosamîn;dase was closely reLated to ~hat m;ght be termed the "reactivity' ~f a var;ety of d;sease pro~esses. This relationsh;p existed under such a ~de range of c;rcumstances as to suggest a more important role than was then kno~n -For N-acetyl-D-glucosam;ne tNAG) and perhaps for other N-acetyl-D-~lycosamines.
i F 1? ~ r ~ 1 ( M CI ~ ~ U ~1 . r~ ~. . ' ô f. I, : 3 1 ~ I I P Q i E ~ / 2 ~2898135 N-acetyl-D-~lucosamine ~2-acetamido-2-deoxy-D-glucose~ re~idues ~re ~idely preser,t in bound form ;n nature as ;s D-galactosam;nQ.
In a text entitled "~rocesses in Pathology" tTaussig, M ~ published ;n ~979 by Black~ell Sc;entif;c Publications, at pp 2~7-270, in a discussion p~imar;ly concerned with the activ;ty of lect;ns, reference i5 made ~o NAG in a study of cell growth. ExperimentS relat;ng to us~ of NAG are ment;oned. In one of these NAG is used as ;ts dimer di-~-acetylchitobiose bGund to a protein carrier in an attempt to pre;~mun;se m;~e against the lethal effects of a transplanted t~mour ~ith li~ited success. In another inh;b;t;on of tumour cell growth by treatment w;th monoY~Lent Lectin to çover the surface a~glutination s;tes ~NAG res;dues) w~s proved re~ers;ble by add;t;on of excess of free NAB.
Other ~orkers have been acti~e ;n this f;e~d, ~or example, in the early 1960s ~evvy and his asso~iates at the ~owett Research Institute showed that the conversion to a lactone of a glycoside which formed the substraee for a glycosidase, ~ould spec;f;cally ;nh;bit the glycosidase concerned. ~nfortunately, these lactones were stab(e only under str;rtly controlled l~boratory - con~itions. Atte~pts to employ them for therape~tic purposes were therefore not pursued.
The inventor arr;ived at th;s ;nvent;on by carry;ng out investigations into the possibility that N-acetyl-D-~lycosamines and their oligomers and derivatives of both classes of compo~nd might play important, and pre~io~sly unrecogn;sed parts ;n the processes of healing and repair, and in the invasi~enes~ an~ proliferation of ; mal;gnant cells. The concep~ of inhibition of the N
aeetyl-P-glycosamin1d~ses and of N-acetyl-P-gl~cosaminidase specifically, under phys;olog;cal conditionsf opens ~p ne~ possib;lit;es both for the i 1~8~
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 regul~tion which comprises forming a solution of (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, 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.
Preferably the glycosamine used in the preparation of this invention is 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 Mg2+. The solution may include buffer salts. As a solution the preparation is suitable for parenteral or oral administration.
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 or subsequent formulation and administration as an injection solution.
Further it can be formulated for topical application in a suitable neutral 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 lZ89885 - 5 - 00123-660/PA/LEH/fs remaining in 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-D-glucosaminidase 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. Thus, the preparation can influence the total level of N-acetyl-D-glucosaminidase activity in the body and also alter the proportional effect of the two main isoenzymes~ 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 workers in this fieldthat the methods of preparation are applicable to analogous compounds and the biological effects obtained will vary depending on the enzyme or cellular system under investigation or therapy.
- ~ ..
- .
.. .. ~, ~
- - , , .
- 5 (a) - 00123-660/PA/LEH/fs 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 /
: . . - ' . - . ' ', ' ' . -, ~ ' " ' -, .
~ F U ~ 1 U l ~ ! I IJ, 13 b . ~ t~ I r~ U . c~ t ~ ~ i J
provides a new and selective means o~ ;nfluenc;ng the body's immune mechan;sms. The act;ve ~omponents mar be administered by mouth or by inject;on, either alone, or incorporated in phys;ologically-benign fluids or solids or any other pharmaceutica~ly acceptable veh;cLes, carriers and adjuvants. Whilst not w;sh;ng to be bound by any particular theory, the role of the active components may be selectlveLy to blor~k or unbiock active sites ;n enzymes participatin~ in t;ssue or ~ell growth. There may be act;on on ceLl surface sites recponsible for functions such as cell specificity, or the trans~er of nutr;ents and hormones. Sites such as these are known to function abnormaLLy in cancero~s cell 3, and are affected also in viral and poss;b~e other ;nfect;ons.
Accordingly, the present invention finds a use in ~a) the control of the proliferat;on, growth and invas;veness of mal;gnant cel~s; tb) the regwlation of the ~evel of activity of ceL~s involved in the repair of - tissue after injury or inflammation, with t~e objective ~0 of encourag;ng healing ~hilst, at the same time, contro~Lin~ any tendency for one aspect of heaLing to outstrip the other, as, for exampLe, the over-production of collagen by fibroblasts results in hypertroPh;c scar format;on, with consequent cosmet;c or funct;onal inpairm~nt; and (c~ the sti0uLatjon of healing where it is indoLent as, for instance, ;n the eLderLy or in those others having suffered very severe infections.
The ;nvent;on w;ll now be further described by way of the following Examples and Pata indicating preferred methods of preparation and biolo~ical e~fects observed with use of the preparat;ons of this invention.
ExampLes Example 1 ~ method of preparing ~a) a preparation for .. . ..
use ;n the stimuLation o~ heaLing and (b) a preparation for the contro~ of mai;gnant cells.
... ... ~
.
.
1 ~89885 ~ a) 3~23 g of N-acetyl-D-glucosamine, 200 mg of b;otin, 375 mg potassium dihydrogen phosphate, and 1.8 9 o~ magnesium suLphate were d;ssoLved in water to give 100 mL of solution, which ~as maintained at 5~C in a closed vesseL for 72 hours. The tontents were then cooled and kept ~t just above free~;ng point. 3uring the ~ z~ u ~ n~y crys~a~1ne aeposl~ separatea.
Th;s ~as removed by centr;fu~a~ion and the soLid deposit was ~ashed with water tor ethanol) The sol;d had no significant ;nhibitory power on N-acetyl-D-g~ucosam;nidase, but had a powerful enhancing action on its B-iso'-enzyme.
The sol;d was ;nsoLuble in water, but soLuble in lipids.
F~Llowing treatment of pat-ents with areas of ;ndolent healing using the so~id, rapid regenerat;on of skin cells was observed. Therefore, the aforementi~e~ steps prov;de a means of st;mulating healing.
~ b~ The supernatant remaining after separation of the sol;d mater;al as described above was returned to a cLosed vessel and incub~ted. It was then free~e-dr;ed.
The resulting material sho~ed a specific non-competitive type of ;nh;b;t;on of both the A and 3 ;so~ymes o~
N-acetyL-~-glucosaminidase. When added to tissue cultures Of m~;P~n~n~ O~ DUCh 04 L~nda~hut- o~-a Or ll~o ~eLLs~
;t produ~ed a series of dose-related effects~ ;ncLuding the destruction of the cells' normal anti-adhesive me~hanism, and the ;nh1b1tion of gro~th. It also led to cell fra~mehtation and to the production of ~ult;pLe - non-viable sub-ceLLuLar fragments. When 3dm;nistered to patients with terminaL malignancy, it controlled the invasiveness of the mal;gnant process and ;ncrease~
surviva~, w;thout other therapy, beyond the prognos;s given by nor~aL nethods.
Exa~ple 2 A method of prepar;ng a pr~parat;on for reg~lating the leveL of act;v;ty of ceLLs invoLved in the repa;r of tissues foLLo~ing injury or inflammation.
. . ,.__ , _ -, ,,, ,. ., ~ .. ~ ._ .. . - -.
, ~289885 3.~ ~ of N-ac~tYl-~-glucosamine~ 10 mg of biotln, 375 mg o~ potassium d;hydrogen phosphate and 1.8 g of ~agnes;um su~phate ~ere dissolved in water to provide 100 ml of solution wh;ch w~s placed in a closed vessel and mainta;ned at a temperature of 70-80C for 7~ hours~
The resulting so(ution was cooled and freeze-dried.
the material result;ng from th;s showed non-competitive- -spec;fic ;nhibit;on of the A isozy~e of N-acetyl-D-~lucosaminid~se and a vary;n~ degree of competitive - 10 inhib;tion only of the ~ isozy~e. Addition of the mater;al to cu~tures of fibroblaqts produced a dose-related ;nhibition of fibroblast prol;ferat;on about 50%
~ithout causing cell death. When administered to competition horses with recent injur;es, ;t produced diminution of swelling, and facilitated the return to soundness. In lon~er term therapy studies it has been adm;nistered on 47 occasions to 35 horses suffering mostly from injur;es to the deep flexor tendon of the fore-~imb, a condition frequently resulting ;n per~3nent disab;Lity~ somet;mes to a degree necess;~ating s~aughter.
A~l ~5 horses returned to competition, usuaLLy at pre-iniury ~evel of soundness.
System;c use in humans ean be expected, but awa~ts further evidence of non-toxic;~y. However, top;~al ~5 applicat;on of the act;ve substance in a neutral ointment base under the superv;s;on of a consultant plastic surgeon has sho~n evidence of a capacity to contro~
e~cessive scar tissue formation.
.
, .
Field of the Inveneion This invention relates to a preparation for tiss~e growth control in humans or animals. More particularly, the ir,vention relates to a ~roup of physiolo~ically 5 active preparat;ons, at Least one ~ember of which promotes, whilst the others inhibitr the growth of both normal and abnormal tissue. Thus, the preparat;ons of th;s invention prov;de for ~a) the enhancement of normal t;ssue growth to accelerate normal repair functions and ~b3 the suppression of undes;rable t;ssue growth, whether benign as in the f;brosis assoG;ated with excessive scar tissue for~a~;~n, or aberrant, as in cancerous or other malignant change.
~ackground Art Extensive work has been done on the control of ~rowth in plants~ none of wh;ch is applicable to animal tissueO Invest;gation of the growth requ;rements of cells o~ anima~ origin (incL~ding human cells) have been princip~lly ~y t;s~ue culture, ~hat is by a technique facilitating the study o~ ceLls grown in ;solation under laboratory conditions. Various "growth fact~rs" have ~een used to encoura~e ceLlular proliferation, commonly using extracts of a relat;veLy crude nature derived from whole organs such as mouse salivary gLand or rat l;ver.
~5 Such grow~h f~ctors have no possible applicab;lity for systemic use ;n ;ntact an;~als or humans. For exampLe, riboflavin is tho~ght t~ b~ a yrowth factor for rats, lipoic acid serves as a growth factor for certa;n micro-or~anisms and biotin is a growth factor for yeast and ~0 certain bacteria. Glycyl-histidyl-lysine and pituitary growth hormone arr t~o chemicalLy char~cter;~ed pure substances w;th an influence on ~rowth, the former in th~ culture of isolated ~iver cells and the latter ;n bodily gro~th ~5 a whole. None of these "growth factors"
has been resorded as ha~ing any known usefulness in F ~ f l ~`~ F I T ,~ r ~ ; L ~ U i D I I J I ~ . 13 ~ r~, ~ P R ~ E ~ / 1 5 12a~
tissue repair, and are on the whole remote from the preparations to be disclosed hereinafter.
In patients ~ho are malno~rished, the syste~ic a~ministration of amino ac;ds ~s known to be of value ;n 5 ass~st~ng wound heaL1ng, but onLy by remedyin~ existiny amtno acid deficiencies.
PhysicaL or phys;co-chemical techniques such as the use of magnetic field~ or pulse elec~rical currents for local;sed stimulat;on of a wound s;te have some carrent vogue.
The inhibition of a~errant or malign~nt cells by chemical means is now common practice, and is effective ~here there ;s a favourabLe rat;o of suscePtibil;ty between the malignant cells of the tumour and the normaL
1~ ~eLLs of the host. In the absence of any more selective means o~ control, these cytotoxic substances have w;de currency at present; but their ~oxicity and consequent hi~h incidenc~ of side effects makes it extremely desirabLe that improved physiolo~;cal or chem;cal control should be deveLoped~
Cort;sone and i~s derivatives are effective in the control of excess f;brous t;ssue format;on such as may occur in ~ome types of injury, ;n many cases o~ burns~
and after som~ inflammatory d;seases of the joints.
However, use of cortisone involves a cons;derable risk of side effec~s.
E~rl;er work tNature, 199, 4991; 3~2; 1q63) by the present inventor showed tha~ the level of activity ~f the enzyme N-ace~yl-~-glucosamîn;dase was closely reLated to ~hat m;ght be termed the "reactivity' ~f a var;ety of d;sease pro~esses. This relationsh;p existed under such a ~de range of c;rcumstances as to suggest a more important role than was then kno~n -For N-acetyl-D-glucosam;ne tNAG) and perhaps for other N-acetyl-D-~lycosamines.
i F 1? ~ r ~ 1 ( M CI ~ ~ U ~1 . r~ ~. . ' ô f. I, : 3 1 ~ I I P Q i E ~ / 2 ~2898135 N-acetyl-D-~lucosamine ~2-acetamido-2-deoxy-D-glucose~ re~idues ~re ~idely preser,t in bound form ;n nature as ;s D-galactosam;nQ.
In a text entitled "~rocesses in Pathology" tTaussig, M ~ published ;n ~979 by Black~ell Sc;entif;c Publications, at pp 2~7-270, in a discussion p~imar;ly concerned with the activ;ty of lect;ns, reference i5 made ~o NAG in a study of cell growth. ExperimentS relat;ng to us~ of NAG are ment;oned. In one of these NAG is used as ;ts dimer di-~-acetylchitobiose bGund to a protein carrier in an attempt to pre;~mun;se m;~e against the lethal effects of a transplanted t~mour ~ith li~ited success. In another inh;b;t;on of tumour cell growth by treatment w;th monoY~Lent Lectin to çover the surface a~glutination s;tes ~NAG res;dues) w~s proved re~ers;ble by add;t;on of excess of free NAB.
Other ~orkers have been acti~e ;n this f;e~d, ~or example, in the early 1960s ~evvy and his asso~iates at the ~owett Research Institute showed that the conversion to a lactone of a glycoside which formed the substraee for a glycosidase, ~ould spec;f;cally ;nh;bit the glycosidase concerned. ~nfortunately, these lactones were stab(e only under str;rtly controlled l~boratory - con~itions. Atte~pts to employ them for therape~tic purposes were therefore not pursued.
The inventor arr;ived at th;s ;nvent;on by carry;ng out investigations into the possibility that N-acetyl-D-~lycosamines and their oligomers and derivatives of both classes of compo~nd might play important, and pre~io~sly unrecogn;sed parts ;n the processes of healing and repair, and in the invasi~enes~ an~ proliferation of ; mal;gnant cells. The concep~ of inhibition of the N
aeetyl-P-glycosamin1d~ses and of N-acetyl-P-gl~cosaminidase specifically, under phys;olog;cal conditionsf opens ~p ne~ possib;lit;es both for the i 1~8~
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 regul~tion which comprises forming a solution of (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, 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.
Preferably the glycosamine used in the preparation of this invention is 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 Mg2+. The solution may include buffer salts. As a solution the preparation is suitable for parenteral or oral administration.
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 or subsequent formulation and administration as an injection solution.
Further it can be formulated for topical application in a suitable neutral 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 lZ89885 - 5 - 00123-660/PA/LEH/fs remaining in 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-D-glucosaminidase 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. Thus, the preparation can influence the total level of N-acetyl-D-glucosaminidase activity in the body and also alter the proportional effect of the two main isoenzymes~ 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 workers in this fieldthat the methods of preparation are applicable to analogous compounds and the biological effects obtained will vary depending on the enzyme or cellular system under investigation or therapy.
- ~ ..
- .
.. .. ~, ~
- - , , .
- 5 (a) - 00123-660/PA/LEH/fs 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 /
: . . - ' . - . ' ', ' ' . -, ~ ' " ' -, .
~ F U ~ 1 U l ~ ! I IJ, 13 b . ~ t~ I r~ U . c~ t ~ ~ i J
provides a new and selective means o~ ;nfluenc;ng the body's immune mechan;sms. The act;ve ~omponents mar be administered by mouth or by inject;on, either alone, or incorporated in phys;ologically-benign fluids or solids or any other pharmaceutica~ly acceptable veh;cLes, carriers and adjuvants. Whilst not w;sh;ng to be bound by any particular theory, the role of the active components may be selectlveLy to blor~k or unbiock active sites ;n enzymes participatin~ in t;ssue or ~ell growth. There may be act;on on ceLl surface sites recponsible for functions such as cell specificity, or the trans~er of nutr;ents and hormones. Sites such as these are known to function abnormaLLy in cancero~s cell 3, and are affected also in viral and poss;b~e other ;nfect;ons.
Accordingly, the present invention finds a use in ~a) the control of the proliferat;on, growth and invas;veness of mal;gnant cel~s; tb) the regwlation of the ~evel of activity of ceL~s involved in the repair of - tissue after injury or inflammation, with t~e objective ~0 of encourag;ng healing ~hilst, at the same time, contro~Lin~ any tendency for one aspect of heaLing to outstrip the other, as, for exampLe, the over-production of collagen by fibroblasts results in hypertroPh;c scar format;on, with consequent cosmet;c or funct;onal inpairm~nt; and (c~ the sti0uLatjon of healing where it is indoLent as, for instance, ;n the eLderLy or in those others having suffered very severe infections.
The ;nvent;on w;ll now be further described by way of the following Examples and Pata indicating preferred methods of preparation and biolo~ical e~fects observed with use of the preparat;ons of this invention.
ExampLes Example 1 ~ method of preparing ~a) a preparation for .. . ..
use ;n the stimuLation o~ heaLing and (b) a preparation for the contro~ of mai;gnant cells.
... ... ~
.
.
1 ~89885 ~ a) 3~23 g of N-acetyl-D-glucosamine, 200 mg of b;otin, 375 mg potassium dihydrogen phosphate, and 1.8 9 o~ magnesium suLphate were d;ssoLved in water to give 100 mL of solution, which ~as maintained at 5~C in a closed vesseL for 72 hours. The tontents were then cooled and kept ~t just above free~;ng point. 3uring the ~ z~ u ~ n~y crys~a~1ne aeposl~ separatea.
Th;s ~as removed by centr;fu~a~ion and the soLid deposit was ~ashed with water tor ethanol) The sol;d had no significant ;nhibitory power on N-acetyl-D-g~ucosam;nidase, but had a powerful enhancing action on its B-iso'-enzyme.
The sol;d was ;nsoLuble in water, but soLuble in lipids.
F~Llowing treatment of pat-ents with areas of ;ndolent healing using the so~id, rapid regenerat;on of skin cells was observed. Therefore, the aforementi~e~ steps prov;de a means of st;mulating healing.
~ b~ The supernatant remaining after separation of the sol;d mater;al as described above was returned to a cLosed vessel and incub~ted. It was then free~e-dr;ed.
The resulting material sho~ed a specific non-competitive type of ;nh;b;t;on of both the A and 3 ;so~ymes o~
N-acetyL-~-glucosaminidase. When added to tissue cultures Of m~;P~n~n~ O~ DUCh 04 L~nda~hut- o~-a Or ll~o ~eLLs~
;t produ~ed a series of dose-related effects~ ;ncLuding the destruction of the cells' normal anti-adhesive me~hanism, and the ;nh1b1tion of gro~th. It also led to cell fra~mehtation and to the production of ~ult;pLe - non-viable sub-ceLLuLar fragments. When 3dm;nistered to patients with terminaL malignancy, it controlled the invasiveness of the mal;gnant process and ;ncrease~
surviva~, w;thout other therapy, beyond the prognos;s given by nor~aL nethods.
Exa~ple 2 A method of prepar;ng a pr~parat;on for reg~lating the leveL of act;v;ty of ceLLs invoLved in the repa;r of tissues foLLo~ing injury or inflammation.
. . ,.__ , _ -, ,,, ,. ., ~ .. ~ ._ .. . - -.
, ~289885 3.~ ~ of N-ac~tYl-~-glucosamine~ 10 mg of biotln, 375 mg o~ potassium d;hydrogen phosphate and 1.8 g of ~agnes;um su~phate ~ere dissolved in water to provide 100 ml of solution wh;ch w~s placed in a closed vessel and mainta;ned at a temperature of 70-80C for 7~ hours~
The resulting so(ution was cooled and freeze-dried.
the material result;ng from th;s showed non-competitive- -spec;fic ;nhibit;on of the A isozy~e of N-acetyl-D-~lucosaminid~se and a vary;n~ degree of competitive - 10 inhib;tion only of the ~ isozy~e. Addition of the mater;al to cu~tures of fibroblaqts produced a dose-related ;nhibition of fibroblast prol;ferat;on about 50%
~ithout causing cell death. When administered to competition horses with recent injur;es, ;t produced diminution of swelling, and facilitated the return to soundness. In lon~er term therapy studies it has been adm;nistered on 47 occasions to 35 horses suffering mostly from injur;es to the deep flexor tendon of the fore-~imb, a condition frequently resulting ;n per~3nent disab;Lity~ somet;mes to a degree necess;~ating s~aughter.
A~l ~5 horses returned to competition, usuaLLy at pre-iniury ~evel of soundness.
System;c use in humans ean be expected, but awa~ts further evidence of non-toxic;~y. However, top;~al ~5 applicat;on of the act;ve substance in a neutral ointment base under the superv;s;on of a consultant plastic surgeon has sho~n evidence of a capacity to contro~
e~cessive scar tissue formation.
.
, .
Claims (14)
1. A method of making a preparation for use in tissue growth regulation which comprises forming a solution of (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, 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.
2. A method according to claim 1 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, recovering this deposit for use as a further active agent for tissue growth regulation.
3. A method according to claim 2 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.
4. A method according to claim 1 wherein the solution is heated to a temperature of from 50 to 60°C.
5. A method according to claim 1 wherein the solution is heated to a temperature of from 70 to 80°C.
6. A method according to claim 1 wherein the heated solution is maintained at an elevated temperature for about three days.
7. A method according to claim 1 wherein the cooled solution is centrifuged to assist recovery of a crystalline deposit therein.
8. A method according to claim 1 wherein the solution is formed using N-acetyl-D-glucosamine or D-glucosamine.
9. A method according to claim 1 wherein the metal cation is selected from Mg, Ca, and Mn.
10. A method according to claim 9 wherein the selected cation is Mg2+.
11. A pharmaceutical product comprising a preparation obtained by a method according to claim 1 in a physiologically-absorbable form together with one or more additives, as required, selected from a pharmaceutically acceptable vehicle, carrier, excipient, diluent or adjuvant.
12. A pharmaceutical composition for the treatment of indolent healing comprising a product derived by forming a solution of N-acetyl glucosamine, biotin, a soluble non-toxic salt of magnesium and a buffer, heating the solution to about 55°C and maintaining this temperature for about three days, cooling the solution and recovering a crystalline deposit therefrom for use in formulation of the composition.
13. A pharmaceutical composition for the treatment of cell malignancy comprising a product derived by forming a solution of N-acetyl glucosamine, biotin, a soluble non-toxic salt of magnesium and a buffer, heating 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.
14. A pharmaceutical composition for the treatment of injured or inflamed tissues comprising a product derived by forming a solution of N-acetyl glucosamine, biotin, a soluble non-toxic salt of magnesium and a buffer, heating the solution to a temperature of from 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.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB8524807 | 1985-10-08 | ||
| GB858524807A GB8524807D0 (en) | 1985-10-08 | 1985-10-08 | Tissue growth regulation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1289885C true CA1289885C (en) | 1991-10-01 |
Family
ID=10586368
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000520028A Expired - Lifetime CA1289885C (en) | 1985-10-08 | 1986-10-07 | Tissue growth regulation |
Country Status (10)
| Country | Link |
|---|---|
| EP (1) | EP0239607A1 (en) |
| JP (1) | JPS63501077A (en) |
| AU (1) | AU606038B2 (en) |
| CA (1) | CA1289885C (en) |
| CS (1) | CS265224B2 (en) |
| DD (1) | DD272034A5 (en) |
| GB (1) | GB8524807D0 (en) |
| IN (1) | IN168295B (en) |
| WO (1) | WO1987002244A1 (en) |
| ZA (1) | ZA867664B (en) |
Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5273900A (en) * | 1987-04-28 | 1993-12-28 | The Regents Of The University Of California | Method and apparatus for preparing composite skin replacement |
| CA1318592C (en) * | 1988-11-18 | 1993-06-01 | University Of British Columbia | N-acetyl glucosamine as a cytoprotective agent |
| US5229374A (en) * | 1992-01-28 | 1993-07-20 | Burton Albert F | Method for treatment of lower gastrointestinal tract disorders |
| US5192750A (en) * | 1992-01-28 | 1993-03-09 | The University Of British Columbia | Method and composition for treatment of food allergy |
| US5217962A (en) * | 1992-01-28 | 1993-06-08 | Burton Albert F | Method and composition for treating psoriasis |
| US6046179A (en) * | 1998-04-17 | 2000-04-04 | Murch; Simon | Composition for and treatment of inflammatory bowel disease by colon administration of N-acetylglucosamine |
| US6159485A (en) | 1999-01-08 | 2000-12-12 | Yugenic Limited Partnership | N-acetyl aldosamines, n-acetylamino acids and related n-acetyl compounds and their topical use |
| CN1173706C (en) | 2001-02-28 | 2004-11-03 | 中国人民解放军第三军医大学 | Application of N-acetyl-D-aminoglucose in preparing medicines to treat cervical erosion |
| CN1199645C (en) | 2002-08-13 | 2005-05-04 | 中国人民解放军第三军医大学 | Application of N-acetyl-D-aminoglucose in the preparation of medicine for treating urogenital system infection |
| JP2007506645A (en) * | 2003-09-17 | 2007-03-22 | 中国人民解放▲軍▼第三▲軍▼医大学 | Use of N-acetyl-D-glucosamine in the manufacture of formulations for antitumor and antimetastasis |
| WO2005112948A1 (en) * | 2004-05-21 | 2005-12-01 | Tottori University | Drug for remedy or treatment of wound |
| ITBS20070178A1 (en) * | 2007-11-15 | 2009-05-16 | Paoli Ambrosi Gianfranco De | COMPOSITION FOR PHARMACEUTICAL AND / OR COSMETIC AND / OR IN THE FORM OF A MEDICAL DEVICE TO ENCOURAGE SCARING PROCESSES, FOR THE TREATMENT OF HYPERTROPHIC SCARS AND TO IMPROVE THE BIOMECHANICAL PROPERTIES OF THE SKIN |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3232836A (en) * | 1959-08-24 | 1966-02-01 | Pfizer & Co C | Facilitating healing of body surface wounds by intravenous administration of n-acetyl glucosamine, glucosamine, or pharmaceutically acceptable acid salts of glucosamine |
-
1985
- 1985-10-08 GB GB858524807A patent/GB8524807D0/en active Pending
-
1986
- 1986-10-07 CA CA000520028A patent/CA1289885C/en not_active Expired - Lifetime
- 1986-10-08 AU AU64094/86A patent/AU606038B2/en not_active Ceased
- 1986-10-08 CS CS867292A patent/CS265224B2/en unknown
- 1986-10-08 IN IN896/DEL/86A patent/IN168295B/en unknown
- 1986-10-08 WO PCT/GB1986/000607 patent/WO1987002244A1/en not_active Application Discontinuation
- 1986-10-08 ZA ZA867664A patent/ZA867664B/en unknown
- 1986-10-08 EP EP86905906A patent/EP0239607A1/en not_active Withdrawn
- 1986-10-08 DD DD86295099A patent/DD272034A5/en not_active IP Right Cessation
- 1986-10-08 JP JP61505290A patent/JPS63501077A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| ZA867664B (en) | 1987-05-27 |
| CS265224B2 (en) | 1989-10-13 |
| AU606038B2 (en) | 1991-01-31 |
| EP0239607A1 (en) | 1987-10-07 |
| JPS63501077A (en) | 1988-04-21 |
| IN168295B (en) | 1991-03-09 |
| WO1987002244A1 (en) | 1987-04-23 |
| GB8524807D0 (en) | 1985-11-13 |
| CS729286A2 (en) | 1989-01-12 |
| AU6409486A (en) | 1987-05-05 |
| DD272034A5 (en) | 1989-09-27 |
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