CA1073383A - Cell proliferation and tissue invasion inhibitor - Google Patents
Cell proliferation and tissue invasion inhibitorInfo
- Publication number
- CA1073383A CA1073383A CA275,440A CA275440A CA1073383A CA 1073383 A CA1073383 A CA 1073383A CA 275440 A CA275440 A CA 275440A CA 1073383 A CA1073383 A CA 1073383A
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Abstract
ABSTRACT
A composition of matter having activity as an inhibitor of cell proliferation and tissue invasion is obtained by aqueous extraction of tissue having a high content of collagen and/or proteoglycans.
A composition of matter having activity as an inhibitor of cell proliferation and tissue invasion is obtained by aqueous extraction of tissue having a high content of collagen and/or proteoglycans.
Description
~733~3 The lnvention descrlbed hereln was made ln the course of work under a grant or award from tlle ~nl~ed States Department of Health Rducation and Welfare.
The present inventlon re]ates generally to a composltlon of matter havlng activlty as an lnhibitor of cell proliferation and an lnhlbitor of tissue lnvaslon. More particu]arly, it relates to methods of preparing such compositlon, and to methods of inhlbiting proliferation of cells utiliæing such co~position.
In accordance with the present invention, a substance having activity as an inhibitor of cell proliferation or, as it sometimes ref~rred to herein, an inhibitor of cell growth, is prepared by extracti~e methods from tissue having a high content of collagen and/or proteoglycans, and preferably from connective tissue. The extract may be treated so as to con-centrate the inhibiting substances. The resultant concentrate has utility in inhibiting proliferation of cells, and parti-cularly in inhibiting prollferation of fibroblasts and endothelial cells.
More particularly, in the preferred embodiment of the present invention, connective tissue is extracted with an aqueous extraction medium. A preferred extraction medium includes a solute which does not irreversibly denature proteins or proteoglycans. One such preferred extractlon medium com~
prlses a l.O-3.OM aqueous solution of auanidine hydrochloride.
Examples of connective tissue which may be used in the preferred embodiment of the present invention include cartllagenous and ligamentary ~issues, vascular tissues, corneal tlssues, dental tlssues and dermal tissuesO The connectlve tlssue ls placed in condltlon for extractlon by mincing, com-mlnutlng, or other wise treating lt to increase the surfacearea of the tlssue over that ln its naturally-occurring condition.
The condltioned tlssue i9 then exposed to the aqueous extraction ~L~733~3 medium, with or without agitation, For a period of time 6ufflcient to result in extraction of polypeptides and proteo~lycans in substantial yield, or until equilibrium conditions are ., o ' :
:'.' ~`,' ' .
~:~` 30 .
~ la- - ~
1GI~733~33 ~
attained. Extraction is desirably effected at temperatures below room temperature, and preferably at approximately 5C.
Following extraction, the aqueous extract is treated to concentxate the growth inhibiting substance contained there-in. It is believed that the gr~wth inhibiting substance ofthe present invention is present primarily in the fraction of the extract having a molecular wèight of 50,000 or below.
Accordingly, the extract may be treated so as to fractionate compounds having a molecular weight of 50,000 or less from 10 those of higher molecular weight, by molecular sieve or ultra- -filtration techniques, etc. or the like. The growth inhibiting substance of the present invention may be further concentrated by removal of water, as by lyophilization of the extract. ;
The growth inhibiting suhstance thus obtained has a marked inhibitory effect on the rate of proliferation of endo-thelial cells. It also inhibits proliferation of some fibro-1 blasts, although inhibition of fibroblast growth is less marked - than inhibition of growth of endothelial cells. In gen~ral ; the ~rowth inhibiting substance of the present invention is more ; 20 effective against the proliferation of immature cells than of mature cells.
The extraction medium employed in the practice of the present invention may be any aqueous extraction medium, except that the extraction medium should not irreversibly de-nature the proteinaceous matter extracted from the connective .
tissue. An aqueous extraction medium containing a relatively high salt content, i.e., 1.0-3.0 M, is desirably used. A
preferred extraction medium is a 1.0-3.0 M aqueous solution of quanidine hydrochloride.
- 30 If a high salt aqueous extraction medium is used, -the salt should be removed from the extract, and this may be accomplished by dialysis, in accordance with known proceduxes~
~:373383 The concentration of the growth inhlblting substance of the present invention in the connectlve t~;ssue and in the ~queous extract is low. For example, it is neces~ary to extract 6everal hundred grams of connective tissue in order to obtain a few milligrams of the lyophillzed extract having a molecular weight of 50,000 or below.
EXAMPLE I
As-a specific example of the preparation of the growth inhibi~ing substance of the present invention, extracts were prepared from bovine connective tissues. Bovine connective tissues which were extracted were nasal septum carti.lage, scapular epiphyseal growth plate, and dermis. The tissues were minced and placed in five volumes of an extraction medium comprising a 1.0 M aqueous solution of guanadine hydrochloride, the solution having a pH of 6Ø The mixture of tissue and extraction medium was stirred for 24 hours at 5C. The resultant aqueous extract was separated from the tissue and dialyzed exhaustively against water. The dialyzed extract was then lyophilized.
Dialyzed and lyophilized extract from scapular epiphyseal ~ growth plate was redissolved in 4.0 M guanidine hydrochloride ;;- solution, and idalyzed through an Amicon~ filter membrane with a pore size which allowed compounds of molecular weight 50,000 or below to pass through the membrane. Following dialysis, the solutions on both sides of the membrane were dialyzed exhaustively against water and lyophilized.
; There were thereby obtained lyophilized extracts of bovine cartilage and of bovine dermis, as well as lyophilized extracts from bovine cartilage which had been fractionated into an extract having a molecular weight of 50,000 or below and an extract having a molecular weight higher than 50,000.
These lyophilized extracts were then used to demon-Jl/~ ~ ~ _3~
) ., ~
`` . 1 -, ~733~3 :.~
strate their activity as inhibitors of cell proliferation.
E~AMPLE II
Cultures of various tissue cell types were pre-pared for test purposes. Bovine aortic endothelial cells were cultured from aortas collected ~rom a local slaughter house. Fresh aortas were immediately transported to the labo-ratory. There, the two ends of each aorta were clamped and the branches ligated. The lumen was rinsed with about 50 ml of 0.9~ NaCl. The aorta was then slightly distended with 25-50 ml of complete tissue culture medium. After about 5 minutes, the fluid was removed with a syringe and needle and ; ~ 5 ml aliquots dispensed into E'alcon T-25 plastic flasks. The .. .
flasks were incubated at 37C in a humidified 5~ Co~-air at-mosphere. The cultures generally re~uired 8-12 days to reach confluency.
; Steer fibroblasts were isolated from the subcutaneous tissue of ears from freshly slaughtered animals. Tissues were ~ remo~ed aseptically and incubated in a solution of 2 mg col--~ lagenase/ml HEPES. Two hours of digestion at 37C was suffi-cient to yield 1 x 10 cells for planting into Falcon flasks.
Bovine e~ryo fibroblasts were purchased from GIBCO
Corp., Grand Island, N.Y. Second passage human foreskin fibro-blasts were also obtained.
The tissue culture medium used for all cell types was RPMI 1640, obtained from GIBCO Corp~, Grand Island, N.Y., supplemented with 20~ fetal calf serum which had been heated for 50 minutes at 56C to inactivate bovine infectious agents present in the serum. Increased buffering capacity was provided by adding 20 mM HEPES. Antibiotics were added to yield a final concentration of gen-tamycin, 50 ug/ml and amphotericin B, 5 ug/ml in the complete medium. The medium was made up from the commercially supplied powder as a 2X concentrate and ~733133 then passed through 0.22 u pore size millipore filters for sterilization.
When tissue extracts or other materials were to be added, the lyophilized extracts were hydrated in distilled water, sterilized by filtration, and then added to the concen- .
trated culture medium. The filtration clarified the cloudiness seen in the aqueous solution of the extract.
For biological assays of the tissue extractsl estab-lished cultures of secondary cells were subcultured in 35 X
~ . 10 10 mm Falcon tissue culture dishes at an initial density of .~; 2 x 104 cells/ml in tissue culture medium and an additional 1 ml of medium added. The next day, the medium was removed and replaced with 2 ml of tissue culture medium containing the materials to be tested or with an equal volume of control medium. Control cell counts were done at this time.
The cultures were refed on the third day after test . materials were added. A 2.25% solution o~ disodium ethylene `~; diamine tetra-acetic acid was then prepared in Ca-Mg free phosphate buffered saline, pH 7.4. The cells were then re-moved from the dish by digesting with a 0.25% trypsin solu-tion. Cell counts of the trypsin dispersed cells were done with a hemocytometer after staining with a 1% filtered solu- .
tion of tripan blue. Two culture dishes were counted each day for each experimental treatment. Each set o~ experiments 25 was repeated at least four times. At the termination of the ;
experiments, some cultures were fixed in 70% methanol and stained with hematoxylin. For dose-response curve studies o~ cartilage-derived materials, counts were done only at the beginning of the experiment and three days after the test mat-erial was added. In such experiments, three dishes were coun-ted for each dose and the experiment was repeated three times.
~L~733~3 Since identification of endothelial cells depend~ ln large part on the presence of Weibel-Pfllade bodies, electron mlcroscopy was done on these cultured cells and these organ~
elles were found as expected. For this, the culture dishes were washed with 0.9% NaCl. The cells were then scraped off the culture dish with a rubber spatula; fixed in 2.5% cacodylate buffered glutaraldehyde, washed overnight in buffer, stained en bloc in uranyl acetate, dehydrated and embedded in Epon~ 812.
Sections were mounted on copper grids, stained with lead citrate ; 10 and studied in an electron microscope.
~ The effects of the lyophilized extract of bovine cartilages ; on the growth of endothelial cells were determined. 40,000 endothelial cells in 2 ml of culture medium were initially dispensed into 35 mm Petri dishes. The following day the .; .
extract to be tested ~as injected into the dish, three dishes for each èxtract. Three control dishes were also provided, which were refed only with culture medium the day after culturing was begun.
Three days after injection of the respective lyophilized extracts, cell counts were done on each dish. In the case of the control dishes, the-original population of 40,000 cells has increased to 700,000. Table I shows the cell counts of -the dishes containing lyophilized cartilage extract at three differen`t concentrations of lyophilized ex~ract.
TABLE I ~-EFFECT OF CARTILAGE EXTRACT ON
ENDOTHELIAL CELL GROWTH
; Dose, Cells/dish micrograms lyophilized extract per ml. of culture medium 5~0 52,000 100 285,000 20 510,000 0 700,000 '` .
~1)7331533 ::
It will be seen from Table I that at a dosage o 500 micro~rams of lyophilized extract per milliliter of cul- ~ :
ture medium, proliferation of endothelial cells was sub-stantially inhibited, the cell count increasingly only to 52,000 from -the ori~inal count of 40,000. At lower dosages, inhibition was less complete, but in each case the cell count was substantially less than the 700,000 cell count of the control after three days.
Similar testing was done utilizing the fraction of 10 lyophilized cartilage extract having a molecular weight greater ' than 50,000. There was no observed inhibition of prolifera-tion of endothelial cells using this fraction, the cell count at a dosage level of 500 micrograms/milliliter being 710,000 after three days, not substantially different from the cell count of 700,000 in the c~ntrol. ~ ^
However, the fraction of lyophili2ed cartilage ex-tract having a molecular weight of 50,000 and below was sub- .~ :
. stantially more potent as a growth inhibitor than the lyo-philized extract which was the subject of Table I. Table II
shows the results of this test, ~hich was carried out in the same manner as testing of the unfractionated extract.
TABLE II
EFFECT OF CARTILAGE EXTRACT HAVING A MOLECULAR
WEIG~T OF 50,000 AND BELOW ON ENDOT~ELIAL CELL GROWTH
. 25 Dose Cells/dish micrograms lyophilized extract per ml. of culture medium 500 22,000 100 65,000 260,000 300,000 0 700,000 It will be seen from Table II that the fraction of lyophilized extract having a molecular weight of 50,000 or , ~733~33 : -below was effective at inhibiting proliferation of endothelial cells at dosages as low as 5 micrograms per milliliter~
The tests of which the results are reported in Tables I and II were repeated three times, and gave virtually identical results.
The same lyophilized extracts of bovine cartilage used in the tests of which the results are shown in Tables I
` and II were evaluated for activity in inhibiting proliferation ;. of mature steer fibroblasts in the same culture medium. There was no significant growth inhibition, nor was there significant ; growth inhibition of steer fibroblasts by lyophilized extracts of dermis. However, lyophilized extract from bovine cartilage was found to inhibit prolifera-tion of fetal bovine fibroblasts.
The inhi~i~ory effect of lyophilized extract from bovine cart-ilage on fetal bovine fibroblasts was less marked than -~he inhibitory effect on bovine endothelial cells. There was also growth inhibition by extracts from bovine cartilage on infant human foreskin fibroblasts, but again the degree of inhibition was less marked than in the case of endothelial cells.
Lyophilized extracts of dermis did not inhibit growth of fetal bovine fibroblasts, although they did inhibit growth o~ endothelial cells as effectively as cartilage extracts.
Lyophilized extracts of bovine aorta and of canine cartilage also inhibited endothelial cell growth.
The inhibitory effect of extracts from connective tissue on cell proliferation is believed to provide an ex- ;
planation as to why some such tissues are relatively resis-~ant to invasion by either neoplasms or inflammatory processes.
It has been observed for many years that poGrly vascularized or avascular tissues such as cartilage are relatively resistant to invasion. Clinicians and pathologist have long known, for example, that most forms of cancers of the respiratory passages .. ; . . ~ ~ . . , :. .. ... . .
1 C9~3383 o~ten encase~ but se]dom invade, bronchlal or laryngeal cartll-ages. More recently, investi~ation of the reslstance of certain ~;
tissues to invasion by explantation on to the chick chorio-allantoic membrane, showed that tissues which normally have a blood supply are rapidly invaded by vascularlzed mesenchyme orlginating from the chick embryo. On the other hand, post-natal hyaline cartilage, which is virtually devoid of blood vessels, was substantially impenetrable under the sam,e condtiions.
American Journal of Pathology, Volume 73, No. 3 Pages 765-772 December 1973. Aithough applicants do not intend to be bound by theory, nor to restrict the scope of their invention by theory, it is believed that the presence of the cell proliferation inhibiting substance of the present invention at relatively high concentrations in poorly vascularized or avascular tissues such as cartilage and blood vessels is responsible for the resistance of such tissues to invasion.
EXAMPLE III
As a further specific example of the preparation of ,1 ,, . :
' the growth inhibiting substance of the present invention, an extract was prepared from bovine cartilage utilizing an alternate extraction method. Bovine nasal septum cartilage was ~`
minced and placed in five volumes of an extraction medium comprising a 1.0 M aqueous solution of guanidine hydrochloride, the solution having a pH of 6Ø The mixture of tissue and extraction medium was stirred for ~l8 hours at 5C.
The resultant aque~us extract was separated from the tissue, and guanidine hydrochloride was added to the aqueous extract in an amount sufficient to raise the concentration from `~
; 1.0 M to 3.0 M. The resultant 3.0 M aqueous extract was sub~ected to pressure dialysis, using a dialysis membrane which permitted passage through it of materials having a molecular weight below about 100,000. Dialysis was continued until equilibrium was attained. About 10~ of the substance extracted g _ !
'~ ` ;
~ , . '' ~L~73383 from the tissue passed through the dialysis membrane.
The resultant dialysate comprising extracted sub-stance having a molecular weight below about 100,000, was ` again subjected to pressure dialysis, usi.ng a membrane which ~ .
: 5 permitted passage through i~ of materials having a molecular weight of below about 50,000. The resultant dialysate was collected and dialysed exhaustively against water through . a membrane to deplete it of guanidine hydrochloride and other substances having a molecular weight below about 3,500. The dialyzed e~tract was lyophilized to provide a substance which also had activity as an inhibitor of cell proli~eration.
Thus,.there has been provided a composition of matter having activity.as an inhibi-tor of cell proliferation, and there has also been provided methods of preparing such a com- :
position and methods of inhibiting cell proliferation utiliz-ing such a compoSitiQn.
~ arious of the features of the present invention are set forth in the following claims.
The present inventlon re]ates generally to a composltlon of matter havlng activlty as an lnhibitor of cell proliferation and an lnhlbitor of tissue lnvaslon. More particu]arly, it relates to methods of preparing such compositlon, and to methods of inhlbiting proliferation of cells utiliæing such co~position.
In accordance with the present invention, a substance having activity as an inhibitor of cell proliferation or, as it sometimes ref~rred to herein, an inhibitor of cell growth, is prepared by extracti~e methods from tissue having a high content of collagen and/or proteoglycans, and preferably from connective tissue. The extract may be treated so as to con-centrate the inhibiting substances. The resultant concentrate has utility in inhibiting proliferation of cells, and parti-cularly in inhibiting prollferation of fibroblasts and endothelial cells.
More particularly, in the preferred embodiment of the present invention, connective tissue is extracted with an aqueous extraction medium. A preferred extraction medium includes a solute which does not irreversibly denature proteins or proteoglycans. One such preferred extractlon medium com~
prlses a l.O-3.OM aqueous solution of auanidine hydrochloride.
Examples of connective tissue which may be used in the preferred embodiment of the present invention include cartllagenous and ligamentary ~issues, vascular tissues, corneal tlssues, dental tlssues and dermal tissuesO The connectlve tlssue ls placed in condltlon for extractlon by mincing, com-mlnutlng, or other wise treating lt to increase the surfacearea of the tlssue over that ln its naturally-occurring condition.
The condltioned tlssue i9 then exposed to the aqueous extraction ~L~733~3 medium, with or without agitation, For a period of time 6ufflcient to result in extraction of polypeptides and proteo~lycans in substantial yield, or until equilibrium conditions are ., o ' :
:'.' ~`,' ' .
~:~` 30 .
~ la- - ~
1GI~733~33 ~
attained. Extraction is desirably effected at temperatures below room temperature, and preferably at approximately 5C.
Following extraction, the aqueous extract is treated to concentxate the growth inhibiting substance contained there-in. It is believed that the gr~wth inhibiting substance ofthe present invention is present primarily in the fraction of the extract having a molecular wèight of 50,000 or below.
Accordingly, the extract may be treated so as to fractionate compounds having a molecular weight of 50,000 or less from 10 those of higher molecular weight, by molecular sieve or ultra- -filtration techniques, etc. or the like. The growth inhibiting substance of the present invention may be further concentrated by removal of water, as by lyophilization of the extract. ;
The growth inhibiting suhstance thus obtained has a marked inhibitory effect on the rate of proliferation of endo-thelial cells. It also inhibits proliferation of some fibro-1 blasts, although inhibition of fibroblast growth is less marked - than inhibition of growth of endothelial cells. In gen~ral ; the ~rowth inhibiting substance of the present invention is more ; 20 effective against the proliferation of immature cells than of mature cells.
The extraction medium employed in the practice of the present invention may be any aqueous extraction medium, except that the extraction medium should not irreversibly de-nature the proteinaceous matter extracted from the connective .
tissue. An aqueous extraction medium containing a relatively high salt content, i.e., 1.0-3.0 M, is desirably used. A
preferred extraction medium is a 1.0-3.0 M aqueous solution of quanidine hydrochloride.
- 30 If a high salt aqueous extraction medium is used, -the salt should be removed from the extract, and this may be accomplished by dialysis, in accordance with known proceduxes~
~:373383 The concentration of the growth inhlblting substance of the present invention in the connectlve t~;ssue and in the ~queous extract is low. For example, it is neces~ary to extract 6everal hundred grams of connective tissue in order to obtain a few milligrams of the lyophillzed extract having a molecular weight of 50,000 or below.
EXAMPLE I
As-a specific example of the preparation of the growth inhibi~ing substance of the present invention, extracts were prepared from bovine connective tissues. Bovine connective tissues which were extracted were nasal septum carti.lage, scapular epiphyseal growth plate, and dermis. The tissues were minced and placed in five volumes of an extraction medium comprising a 1.0 M aqueous solution of guanadine hydrochloride, the solution having a pH of 6Ø The mixture of tissue and extraction medium was stirred for 24 hours at 5C. The resultant aqueous extract was separated from the tissue and dialyzed exhaustively against water. The dialyzed extract was then lyophilized.
Dialyzed and lyophilized extract from scapular epiphyseal ~ growth plate was redissolved in 4.0 M guanidine hydrochloride ;;- solution, and idalyzed through an Amicon~ filter membrane with a pore size which allowed compounds of molecular weight 50,000 or below to pass through the membrane. Following dialysis, the solutions on both sides of the membrane were dialyzed exhaustively against water and lyophilized.
; There were thereby obtained lyophilized extracts of bovine cartilage and of bovine dermis, as well as lyophilized extracts from bovine cartilage which had been fractionated into an extract having a molecular weight of 50,000 or below and an extract having a molecular weight higher than 50,000.
These lyophilized extracts were then used to demon-Jl/~ ~ ~ _3~
) ., ~
`` . 1 -, ~733~3 :.~
strate their activity as inhibitors of cell proliferation.
E~AMPLE II
Cultures of various tissue cell types were pre-pared for test purposes. Bovine aortic endothelial cells were cultured from aortas collected ~rom a local slaughter house. Fresh aortas were immediately transported to the labo-ratory. There, the two ends of each aorta were clamped and the branches ligated. The lumen was rinsed with about 50 ml of 0.9~ NaCl. The aorta was then slightly distended with 25-50 ml of complete tissue culture medium. After about 5 minutes, the fluid was removed with a syringe and needle and ; ~ 5 ml aliquots dispensed into E'alcon T-25 plastic flasks. The .. .
flasks were incubated at 37C in a humidified 5~ Co~-air at-mosphere. The cultures generally re~uired 8-12 days to reach confluency.
; Steer fibroblasts were isolated from the subcutaneous tissue of ears from freshly slaughtered animals. Tissues were ~ remo~ed aseptically and incubated in a solution of 2 mg col--~ lagenase/ml HEPES. Two hours of digestion at 37C was suffi-cient to yield 1 x 10 cells for planting into Falcon flasks.
Bovine e~ryo fibroblasts were purchased from GIBCO
Corp., Grand Island, N.Y. Second passage human foreskin fibro-blasts were also obtained.
The tissue culture medium used for all cell types was RPMI 1640, obtained from GIBCO Corp~, Grand Island, N.Y., supplemented with 20~ fetal calf serum which had been heated for 50 minutes at 56C to inactivate bovine infectious agents present in the serum. Increased buffering capacity was provided by adding 20 mM HEPES. Antibiotics were added to yield a final concentration of gen-tamycin, 50 ug/ml and amphotericin B, 5 ug/ml in the complete medium. The medium was made up from the commercially supplied powder as a 2X concentrate and ~733133 then passed through 0.22 u pore size millipore filters for sterilization.
When tissue extracts or other materials were to be added, the lyophilized extracts were hydrated in distilled water, sterilized by filtration, and then added to the concen- .
trated culture medium. The filtration clarified the cloudiness seen in the aqueous solution of the extract.
For biological assays of the tissue extractsl estab-lished cultures of secondary cells were subcultured in 35 X
~ . 10 10 mm Falcon tissue culture dishes at an initial density of .~; 2 x 104 cells/ml in tissue culture medium and an additional 1 ml of medium added. The next day, the medium was removed and replaced with 2 ml of tissue culture medium containing the materials to be tested or with an equal volume of control medium. Control cell counts were done at this time.
The cultures were refed on the third day after test . materials were added. A 2.25% solution o~ disodium ethylene `~; diamine tetra-acetic acid was then prepared in Ca-Mg free phosphate buffered saline, pH 7.4. The cells were then re-moved from the dish by digesting with a 0.25% trypsin solu-tion. Cell counts of the trypsin dispersed cells were done with a hemocytometer after staining with a 1% filtered solu- .
tion of tripan blue. Two culture dishes were counted each day for each experimental treatment. Each set o~ experiments 25 was repeated at least four times. At the termination of the ;
experiments, some cultures were fixed in 70% methanol and stained with hematoxylin. For dose-response curve studies o~ cartilage-derived materials, counts were done only at the beginning of the experiment and three days after the test mat-erial was added. In such experiments, three dishes were coun-ted for each dose and the experiment was repeated three times.
~L~733~3 Since identification of endothelial cells depend~ ln large part on the presence of Weibel-Pfllade bodies, electron mlcroscopy was done on these cultured cells and these organ~
elles were found as expected. For this, the culture dishes were washed with 0.9% NaCl. The cells were then scraped off the culture dish with a rubber spatula; fixed in 2.5% cacodylate buffered glutaraldehyde, washed overnight in buffer, stained en bloc in uranyl acetate, dehydrated and embedded in Epon~ 812.
Sections were mounted on copper grids, stained with lead citrate ; 10 and studied in an electron microscope.
~ The effects of the lyophilized extract of bovine cartilages ; on the growth of endothelial cells were determined. 40,000 endothelial cells in 2 ml of culture medium were initially dispensed into 35 mm Petri dishes. The following day the .; .
extract to be tested ~as injected into the dish, three dishes for each èxtract. Three control dishes were also provided, which were refed only with culture medium the day after culturing was begun.
Three days after injection of the respective lyophilized extracts, cell counts were done on each dish. In the case of the control dishes, the-original population of 40,000 cells has increased to 700,000. Table I shows the cell counts of -the dishes containing lyophilized cartilage extract at three differen`t concentrations of lyophilized ex~ract.
TABLE I ~-EFFECT OF CARTILAGE EXTRACT ON
ENDOTHELIAL CELL GROWTH
; Dose, Cells/dish micrograms lyophilized extract per ml. of culture medium 5~0 52,000 100 285,000 20 510,000 0 700,000 '` .
~1)7331533 ::
It will be seen from Table I that at a dosage o 500 micro~rams of lyophilized extract per milliliter of cul- ~ :
ture medium, proliferation of endothelial cells was sub-stantially inhibited, the cell count increasingly only to 52,000 from -the ori~inal count of 40,000. At lower dosages, inhibition was less complete, but in each case the cell count was substantially less than the 700,000 cell count of the control after three days.
Similar testing was done utilizing the fraction of 10 lyophilized cartilage extract having a molecular weight greater ' than 50,000. There was no observed inhibition of prolifera-tion of endothelial cells using this fraction, the cell count at a dosage level of 500 micrograms/milliliter being 710,000 after three days, not substantially different from the cell count of 700,000 in the c~ntrol. ~ ^
However, the fraction of lyophili2ed cartilage ex-tract having a molecular weight of 50,000 and below was sub- .~ :
. stantially more potent as a growth inhibitor than the lyo-philized extract which was the subject of Table I. Table II
shows the results of this test, ~hich was carried out in the same manner as testing of the unfractionated extract.
TABLE II
EFFECT OF CARTILAGE EXTRACT HAVING A MOLECULAR
WEIG~T OF 50,000 AND BELOW ON ENDOT~ELIAL CELL GROWTH
. 25 Dose Cells/dish micrograms lyophilized extract per ml. of culture medium 500 22,000 100 65,000 260,000 300,000 0 700,000 It will be seen from Table II that the fraction of lyophilized extract having a molecular weight of 50,000 or , ~733~33 : -below was effective at inhibiting proliferation of endothelial cells at dosages as low as 5 micrograms per milliliter~
The tests of which the results are reported in Tables I and II were repeated three times, and gave virtually identical results.
The same lyophilized extracts of bovine cartilage used in the tests of which the results are shown in Tables I
` and II were evaluated for activity in inhibiting proliferation ;. of mature steer fibroblasts in the same culture medium. There was no significant growth inhibition, nor was there significant ; growth inhibition of steer fibroblasts by lyophilized extracts of dermis. However, lyophilized extract from bovine cartilage was found to inhibit prolifera-tion of fetal bovine fibroblasts.
The inhi~i~ory effect of lyophilized extract from bovine cart-ilage on fetal bovine fibroblasts was less marked than -~he inhibitory effect on bovine endothelial cells. There was also growth inhibition by extracts from bovine cartilage on infant human foreskin fibroblasts, but again the degree of inhibition was less marked than in the case of endothelial cells.
Lyophilized extracts of dermis did not inhibit growth of fetal bovine fibroblasts, although they did inhibit growth o~ endothelial cells as effectively as cartilage extracts.
Lyophilized extracts of bovine aorta and of canine cartilage also inhibited endothelial cell growth.
The inhibitory effect of extracts from connective tissue on cell proliferation is believed to provide an ex- ;
planation as to why some such tissues are relatively resis-~ant to invasion by either neoplasms or inflammatory processes.
It has been observed for many years that poGrly vascularized or avascular tissues such as cartilage are relatively resistant to invasion. Clinicians and pathologist have long known, for example, that most forms of cancers of the respiratory passages .. ; . . ~ ~ . . , :. .. ... . .
1 C9~3383 o~ten encase~ but se]dom invade, bronchlal or laryngeal cartll-ages. More recently, investi~ation of the reslstance of certain ~;
tissues to invasion by explantation on to the chick chorio-allantoic membrane, showed that tissues which normally have a blood supply are rapidly invaded by vascularlzed mesenchyme orlginating from the chick embryo. On the other hand, post-natal hyaline cartilage, which is virtually devoid of blood vessels, was substantially impenetrable under the sam,e condtiions.
American Journal of Pathology, Volume 73, No. 3 Pages 765-772 December 1973. Aithough applicants do not intend to be bound by theory, nor to restrict the scope of their invention by theory, it is believed that the presence of the cell proliferation inhibiting substance of the present invention at relatively high concentrations in poorly vascularized or avascular tissues such as cartilage and blood vessels is responsible for the resistance of such tissues to invasion.
EXAMPLE III
As a further specific example of the preparation of ,1 ,, . :
' the growth inhibiting substance of the present invention, an extract was prepared from bovine cartilage utilizing an alternate extraction method. Bovine nasal septum cartilage was ~`
minced and placed in five volumes of an extraction medium comprising a 1.0 M aqueous solution of guanidine hydrochloride, the solution having a pH of 6Ø The mixture of tissue and extraction medium was stirred for ~l8 hours at 5C.
The resultant aque~us extract was separated from the tissue, and guanidine hydrochloride was added to the aqueous extract in an amount sufficient to raise the concentration from `~
; 1.0 M to 3.0 M. The resultant 3.0 M aqueous extract was sub~ected to pressure dialysis, using a dialysis membrane which permitted passage through it of materials having a molecular weight below about 100,000. Dialysis was continued until equilibrium was attained. About 10~ of the substance extracted g _ !
'~ ` ;
~ , . '' ~L~73383 from the tissue passed through the dialysis membrane.
The resultant dialysate comprising extracted sub-stance having a molecular weight below about 100,000, was ` again subjected to pressure dialysis, usi.ng a membrane which ~ .
: 5 permitted passage through i~ of materials having a molecular weight of below about 50,000. The resultant dialysate was collected and dialysed exhaustively against water through . a membrane to deplete it of guanidine hydrochloride and other substances having a molecular weight below about 3,500. The dialyzed e~tract was lyophilized to provide a substance which also had activity as an inhibitor of cell proli~eration.
Thus,.there has been provided a composition of matter having activity.as an inhibi-tor of cell proliferation, and there has also been provided methods of preparing such a com- :
position and methods of inhibiting cell proliferation utiliz-ing such a compoSitiQn.
~ arious of the features of the present invention are set forth in the following claims.
Claims (10)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. The method of preparing a composition of matter having activity as an inhibitor of cell proliferation and tissue invasion comprising the steps of providing connective tissue having a high content of collagen or proteoglycans in condition for extraction, extracting said inhibitor from said tissue with an aqueous extraction medium which includes a solute which does not irreversibly denature the proteinaceous matter to be extracted, separating the resultant aqueous extract from the tissue, recovering from the aqueous extract substances having a molecular weight below about 50,000, treating the fraction of aqueous extract having a molecular weight below about 50,000 to remove salts therefrom, and dehydrating the resultant material.
2. The method of Claim 1 wherein the connective tissue comprises tissue normally devoid of an intimate capillary blood supply.
3. The method of Claim 2 wherein the tissue comprises cartilage.
4. The method of Claim 2 wherein the tissue comprises blood vessels.
5. A composition of matter having activity as an inhibitor of cell proliferation and tissue invasion comprising the product of the method of Claim 1.
6. A composition of matter having activity as an inhibitor of cell proliferation comprising the product of the method of Claim 2.
7. A method of inhibiting proliferation of cells comprising treating the cells with the product of Claim 1.
8. A method of inhibiting proliferation of cells comprising treating the cells with the product of Claim 2.
9. A method of inhibiting proliferation of cells comprising treating the cells with the product of Claim 3.
10. A method of inhibiting proliferation of cells comprising treating the cells with the product of Claim 4.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA275,440A CA1073383A (en) | 1977-04-04 | 1977-04-04 | Cell proliferation and tissue invasion inhibitor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA275,440A CA1073383A (en) | 1977-04-04 | 1977-04-04 | Cell proliferation and tissue invasion inhibitor |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1073383A true CA1073383A (en) | 1980-03-11 |
Family
ID=4108312
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA275,440A Expired CA1073383A (en) | 1977-04-04 | 1977-04-04 | Cell proliferation and tissue invasion inhibitor |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1073383A (en) |
-
1977
- 1977-04-04 CA CA275,440A patent/CA1073383A/en not_active Expired
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