U.S. patent number RE30,239 [Application Number 05/912,861] was granted by the patent office on 1980-03-25 for cell proliferation and tissue invasion inhibitor.
Invention is credited to Reuben Eisenstein, Klaus E. Kuettner, Nino Sorgente.
United States Patent |
RE30,239 |
Kuettner , et al. |
March 25, 1980 |
Cell proliferation and tissue invasion inhibitor
Abstract
A composition of matter having activity as an inhibitor of cell
proliferation .Iadd.and tissue invasion.Iaddend.is obtained by
aqueous extraction of tissue having a high content of collagen
and/or proteoglycans.
Inventors: |
Kuettner; Klaus E. (Chicago,
IL), Eisenstein; Reuben (Lincolnwood, IL), Sorgente;
Nino (Los Angeles, CA) |
Family
ID: |
27091120 |
Appl.
No.: |
05/912,861 |
Filed: |
June 5, 1978 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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Reissue of: |
630275 |
Nov 10, 1975 |
04042457 |
Aug 16, 1977 |
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Current U.S.
Class: |
424/569; 424/548;
424/572; 514/17.1; 514/19.2; 530/356 |
Current CPC
Class: |
A61K
35/32 (20130101); A61K 35/44 (20130101) |
Current International
Class: |
A61K
35/44 (20060101); A61K 35/32 (20060101); A61K
035/32 () |
Field of
Search: |
;424/95 |
Other References
Willmer-Cells and Tissues in Culture, vol. 1 (1965), pp. 44 and
573..
|
Primary Examiner: Rosen; Sam
Attorney, Agent or Firm: Fitch, Even & Tabin
Government Interests
The invention described herein was made in the course of work under
a grant or award from the Department of Health, Education and
Welfare.
Claims
What is claimed is:
1. The method of preparing a composition of matter having activity
as an inhibitor of cell proliferation .Iadd.and tissue invasion
.Iaddend.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 .Iadd.and tissue invasion .Iaddend.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.
Description
The present invention relates generally to a composition of matter
having activity as an inhibitor of cell proliferation .Iadd.and an
inhibitor of tissue invasion. .Iaddend.More particularly, it
relates to methods of preparing such composition, and to methods of
inhibiting proliferation of cells utilizing such composition.
In accordance with the present invention, a substance having
activity as an inhibitor of cell proliferation or, as it is
sometimes referred to herein, an inhibitor of cell growth, is
prepared by extractive 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 concentrate the
inhibiting substance. The resultant concentrate has utility in
inhibiting proliferation of cells, and particularly in inhibiting
proliferation 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 extraction medium comprises a 1.0-3.0 M aqueous
solution of guanidine hydrochloride.
Examples of connective tissue which may be used in the preferred
embodiment of the present invention include cartilagenous and
ligamentary tissues, vascular tissues, corneal tissues, dental
tissues and dermal tissues. The connective tissue is placed in
condition for extraction by mincing, comminuting, or otherwise
treating it to increase the surface area of the tissue over that in
its naturally-occurring condition. The conditioned tissue is then
exposed to the aqueous extraction medium, with or without
agitation, for a period of time sufficient to result in extraction
of polypeptides and proteoglycans in substantial yield, or until
equilibrium conditions are attained. Extraction is desirably
effected at temperatures below room temperature, and preferably at
approximately 5.degree. C.
Following extraction, the aqueous extract is treated to concentrate
the growth inhibiting substance contained therein. It is believed
that the growth inhibiting substance of the present invention is
present primarily in the fraction of the extract having a molecular
weight 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 those of higher molecular weight, by molecular sieve
or ultrafiltration 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 substance thus obtained has a marked
inhibitory effect on the rate of proliferation of endothelial
cells. It also inhibits proliferation of some fibroblasts, although
inhibition of growth of endothelial cells. In general, the growth
inhibiting substance of the present invention is more 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 denature 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 guanidine
hydrochloride.
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 procedures.
The concentration of the growth inhibiting substance of the present
invention in the connective tissue and in the aqueous extract is
low. For example, it is necessary to extract several hundred grams
of connective tissue in order to obtain a few milligrams of the
lyophilized extract having a molecular weight of 50,000 or
below.
EXAMPLE I
As a specific example of the preparation of the growth inhibiting
substance of the present invention, extracts were prepared from
bovine connective tissues. Bovine connective tissues which were
extracted were nasal septum cartilage, 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 6.0. The
mixture of tissue and extraction medium was stirred for 24 hours at
5.degree. C. 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 dialyzed 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 demonstrate their activity as inhibitors of cell
proliferation.
EXAMPLE II
Cultures of various tissue cell types were prepared for test
purposes. Bovine aortic endothelial cells were cultured from aortas
collected from a local slaughter house. Fresh aortas were
immediately transported to the laboratory. 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 Falcon T-25 plastic flasks. The
flasks were incubated at 37.degree. C. in a humidified 5% Co.sub.2
-air atmosphere. The cultures generally required 8-12 days to reach
confluency.
Steer fibroblasts were isolated from the subcutaneous tissue of
ears from freshly slaughtered animals. Tissues were removed
aseptically and incubated in a solution of 2 mg collagenase/ml
HEPES. Two hours of digestion at 37.degree. C. was sufficient to
yield 1.times.10.sup.6 cells for planting into Falcon flasks.
Bovine embryo fibroblasts were purchased from GIBCO Corp., Grand
Island, N.Y. Second passage human foreskin fibroblasts 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
56.degree. C. 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
gentamycin, 50 .mu.g/ml and amphotericin B, 5 .mu.g/ml in the
complete medium. The medium was made up from the commercially
supplied powder as a 2X concentrate and then passed through
0.22.mu. 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 concentrated culture medium.
The filtration clarified the cloudiness seen in the aqueous
solution of the extract.
For biological assays of the tissue extracts, established cultures
of secondary cells were subcultured in 35.times.10 mm Falcon tissue
culture dishes at an initial density of 2.times.10.sup.4 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 of disodium ethylene diamine tetra-acetic
acid was then prepared in Ca-Mg free phosphate buffered saline, pH
7.4. The cells were then removed from the dish by digesting with a
0.25% trypsin solution. Cell counts of the trypsin dispersed cells
were done with a hemocytometer after staining with a 1% filtered
solution of tripan blue. Two culture dishes were counted each day
for each experimental treatment. Each set of experiments 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 of
cartilage-derived materials, counts were done only at the beginning
of the experiment and 3 days after the test material was added. In
such experiments, three dishes were counted for each dose and the
experiment was repeated three times.
Since identification of endothelial cells depends in large part on
the Weibel-Palade bodies, electron microscopy was done on these
cultured cells and these organelles 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 .[.gluteraldehyde.].
.Iadd.glutaraldehyde.Iaddend., 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
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 was
injected into the dish, three dishes for each extract. 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 had increased to
700,000. Table I shows the cell counts of the dishes containing
lyophilized cartilage extract at three different concentrations of
lyophilized extract.
TABLE I ______________________________________ EFFECT OF CARTILAGE
EXTRACTION ENDOTHELIAL CELL GROWTH Dose, Cells/dish
______________________________________ micrograms lyophilized
extract per ml. of culture medium 500 52,000 100 285,000 20 510,000
0 700,000 ______________________________________
It will be seen from Table I that at a dosage of 500 micrograms of
lyophilized extract per milliliter of culture medium, proliferation
of endothelial cells was substantially inhibited, the cell count
increasing only to 52,000 from the original 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 3 days.
Similar testing was done utilizing the fraction of lyophilized
cartilage extract having a molecular weight greater than 50,000.
There was no observed inhibition of proliferation 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
control.
However, the fraction of lyophilized cartilage extract having a
molecular weight of 50,000 and below was substantially more potent
as a growth inhibitor than the lyophilized extract which was the
subject of Table I. Table II shows the results of this test, which
was carried out in the same manner as testing of the unfractionated
extract.
TABLE II ______________________________________ EFFECT OF CARTILAGE
EXTRACT HAVING A MOLECULAR WEIGHT OF 50,000 AND BELOW ON
ENDOTHELIAL CELL GROWTH Dose Cells/dish
______________________________________ micrograms lyophilized
extract per ml. of culture medium 500 22,000 100 65,000 20 260,000
5 300,000 0 700,000 ______________________________________
It will be seen from Table II that the fraction of lyopholized
extract having a molecular weight of 50,000 or 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, now was there significant growth inhibition of
steer fibroblasts by lyophilized extracts of dermis. However,
lyophilized extract from bovine cartilage was found to inhibit
proliferation of fetal bovine fibroblasts. The inhibitory effect of
lyophilized extract from bovine cartilage on fetal bovine
fibroblasts was less marked than the 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 of 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 explanation as to why some
such tissues are relatively resistant to invasion by either
neoplasms or inflammatory processes. It has been observed for many
years that poorly 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 often encase, but seldom
invade, bronchial or laryngeal cartilages. More recently,
investigation of the resistance of certain tissues to invasion by
explantation on to the chick chorioallantoic membrane showed that
tissues which normally have a blood supply are rapidly invaded by
vascularized mesenchyme originating from the chick embryo. On the
other hand, post-natal hyaline cartilage, which is virtually devoid
of blood vessels, was substantially impenetrable under the same
conditions. American Journal of Pathology, Volume 73, No. 3 Pages
765-772, December 1973. Although 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 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 6.0 The
mixture of tissue and extraction medium was stirred for 48 hours at
5.degree. C.
The resultant aqueous 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 subjected 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 from the tissue passed through the dialysis
membrane.
The resultant dialysate comprising extracted substance having a
molecular weight below about 100,000, was again subjected to
pressure dialysis, using a membrane which permitted passage through
it 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 extract was lyophilized to provide a
substance which also had activity as an inhibitor of cell
proliferation.
Thus, there has been provided a composition of matter having
activity as an inhibitor of cell proliferation, and there has also
been provided methods of preparing such a composition and methods
of inhibiting cell proliferation utilizing such a composition.
Various of the features of the present invention are set forth in
the following claims.
* * * * *