Note: Descriptions are shown in the official language in which they were submitted.
WO92/16181 ~ 7 3 PCT/US92/02~3
METHOD FOR PROMOTING
TISSUE REPAIR AND REGENERATIO~
Backqround of the Invention
Polypeptide growth factors are a class o~ natural
biological mediators that regulate the proliferation,
differentiation, motility and matrix synthesis of
S nearly all cell types. These properties, demonstrable
in vlvo, have led to the proposal that such factors
play important roles in soft and hard tissue repair.
Platelet-derived growth factor ( PDGF) is a well
characterized example of such a polypeptide growth
factor.
PDGF is a peptide hormone produced by blood
platelets which influences the regulation of a broad
array of biological systems including wound repair,
arteriosclerosis, neoplasia, embryogenesis and bone
marrow fibrosis. PDGF is a mitogen, that is, a
substance which induces mitosis of cells and thus
cellular proliferation. In wound repair, PDGF elicits
both chemotactic and mitogenic responses in
fibroblasts, smooth muscle, glial cells, etc. Injury
to the endothelium lining the vessel wall is believed
to cause platelets to adhere to exposed connective
tissue at the wound site, with the concomitant release
of PDGF. The released PDGF is thought to
chemotactically recruit many cell types including
fibroblasts, monocytes, glial and smooth muscle to
migrate into the site of the wound. Increased
proliferation of these cells leads to accelerated
tissue regeneration and wound healing.
It has been demonstrated that the mitogenic
properties of PDGF can be augmented by the addition of
growth factors. For example, Antonaides et al. in U.S.
Patent Nos. 4,861,757 and 4,874,746 showed that a
WO92/1618~ PC~/US9?/02~43
~ 0 7 3 -2- ~
combination of PDGF and insulin-like growth factor-l
(IGF-l) or transforming growth factor alpha (TGF-a) had
a greater effect on cell mitogenic activity than PDGF
alone.
The effect of combining PDGF with other compounds
is less clear. Levenson et al. in J. Biol. Chem.,
260:8056-63 (1985), showed that the synthetic
glucocorticoid, dexamethasone, acts synergistically
with cartilage-derived growth factor (CDGF) to enhance
the stimulation of DNA synthesis in quiescent Swiss 3T3
cells, while having only a neutral effect with PDGF.
In addition, Levenson et al. showed that the addition
of dexamethasone to PDGF-stimulated cultures had no
ef f ect on DNA synthesis over that observed with PDGF
alone.
WO92/16181 PCTtUS92/02~3
_3~ 3'(J ~)7 ~S
Summary of the Invention
The present invention relates to a method for
enhancing tissue regeneration and/or wound repair in a
mammal comprising applying to the tissue or wound an
effective amount of a composition comprising an anti-
inflammatory compound and PDGF. The method promotes
cellular activity at the site of the wound which
expedites healing of the wound.
More specifically, the combination of PDGF and
anti-inflammatory compound synergistically promotes the
proliferation of mammalian cells at the site. Either
natural-sourced or recombinant PDGF can be used in the
composition. It has been found that many anti-
inflammatory agents can synergistically enhance the
lS mitogenic effect of PDGF on cells. Anti-inflammatory
agents which are particularly ~seful are a class of
compounds known as glucocorticoids. Glucocorticoids
include cortisone, hydrocortisone (cortisol),
dexamethasone, and pharmacologically active derivatives
thereof, for example.
The method of the present invention can be used to
promote tissue regeneration and/or wound healing in a
variety of tissues. The method is effective for
enhancing tissue regeneration and wound healing in
epithelial tissues, and for promoting regeneration of
bone and/or cartilage tissues. In general, applying
the composition to an area where epithelium, bone or
cartilage has been broken, torn or eroded due to injury
or disease, for example, stimulates the regeneration
and repair of the epithelium, bone or cartilage.
The method is particularly effective for treating
tissues affected by periodontal disease. The method is
carried out by applying a composition of PDGF and the
anti-inflammatory compound to the affected gum tissue
and periodontal ligament. The composition promotes
W092/1618~ PCT/USg2/02~3
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regeneration of the gum tissue, of tooth tissues such
as dentin and pulp, and of the connective tissue
holding the tooth in place in the gum.
The present method provides an effective
S therapeutic composition for treating external wounds,
including skin ulcers, burns and lesions and for
regenerating connective tissue and/or bone. The method
is particularly effective for treating dental tissue
affected by periodontal disease.
W~92/161X1 PCT/US92/02~3
_5~
Brief Description of the Drawinqs
Figure l is a graph showing the influence of dex~methasone on
the mitogenic activity of both PDGF alone and PDGF
IGF-l.
Figure 2 is a graph showing the effect on cell population
density of various concentrations of dexamethasone and
PDGF + IGF-1.
Figure ~ is a graph showing the influence of dexamethasone on
the mitogenic activity of PDGF using PDGF ~ IGF-l as a
control reference.
5 Figure 4 is a graph comparing the mitogenic activity of PDGF-a
and PDGF-~.
W092~61~1 PCl/US92/02~3
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Detailed Description o~ the Invention
The present ~ethod comprises applying a combinatian
of PDGF and an anti-inflammatory compound to a tissue.
Nati~e PDGF is a dimeric molecule comprised of two
polypeptide chains, one or more of which may be
glycosylated. The two chains (referred to as
alpha (~), and beta (~)) are homologous but not
identical. The chain has a molecular weight of about
17,000 to 18,000 and the ~ chain has a molecular weight
of about 13,000 to 14,000. The a and ~ chains are
synthesized in vivo from larger molecules which are
subsequently processed at the amino and carboxyl-
termini. The mature human a chain is comprised of 110
or 125 amino acids and various N-linked sugar side
chains, and the length and amino acid sequence is
dependen'c on the tissue source. The fully processed
human ~ chain is encoded by the c-sis gene and is
comprised of 112 amino acids. Biologically active PDGF
can exist as a homodimer e.g., ~a, ~, or a
heterodiminer ~. The molecular weights of the ~
homodimer and ~ homodimer are about 35,000 and about
32,000, respectively.
PDGF useful in the present invention may be natural
sourced, recombinant or synthetic PDGF. Natural
sourced can be extracted from human platelets, ~or
example as described by Heidin et al. (1979) Proc.
Natl. Acad. Sci. USA, 76:3722-33726; Antoniades et
al.(l979) Proc. Natl. Acad. Sci. USA, 76:1809-1813;
Antoniades et al., U.S. Patent No. 4,479,896; and
Lipton et al., U.S. Patent No. 4,350,687. Recombinant
PDGF can be produced using transformed eucaryotic
cells, such as yeast, (See, EP Publication No.
0177957), or procaryotic cells such as E. coli. PDGF
also can b~ synthesized using art-recognized peptide
synthesis techniques. Biologically actiYe fragments,
U092/l618l PCT/US9~/02~3
2 1 ~
,
derivatives or mutant forms of PDGF can be used in the
present invention. PDGF which can be used is
commercially available, for example, from Amgen
Corporation, Thousand Oaks, California; PDGF Inc.,
Boston, Massachusetts; Collaborative Research Inc.,
Waltham, Massachusetts; and Creative BioMolecules,
Inc., Hopkinton, Massachusetts.
Anti-inflammatory compounds are compounds which
reduce inflammation by acting on body mechanics without
directly antagonizing the causative agent. A class of
anti-inflammatory compounds which is particularly
useful in the present method comprises glucocorticoids.
Glucocorticoids include, for example, cortisone,
hydrocortisone (cortisol), dexamethasone and
pharmacologically active derivatives of these drugs,
e.g., hydrocortisone acetate. Dexamethasone and
cortisol are commercially available from a number of
sources, for example, Sigma Chemical Co., Saint Louis,
Missouri.
Wound healing and tissue regeneration can be
promoted by directly, locally applying an effective
amount of a composition comprising PDGF and the
selected anti-inflammatory compound to the affected
tissue. The tissue can be external epithelial tissue,
internal epithelial tissue, bone, cartilage, or dental
tissue, including gum tissue, dentin, pulp, cementum or
` periodontal ligature.
The concentration of PDGF and of the anti-
inflammatory compound will depend in part upon the
compound selected, its potency and the tissue it is
applied to. The amount can be determined empirically
by applying a low dose and observing the effects and
incrementally increasing the dose until the desired
effect is obtained. A concentration of PDGF of from
about 0.1 ~g/ml to about 10 mg/ml is effective for most
lo K~ r,~ro I I JU~ 15~3
- 8 - P ~ T / U S 9 ~ / o ~ 0 4 3
applications. For many qlucocorticoids, a
concentration of from about 10 5 M to about 10 l2 M can
be used. For example, a concentration of from about
10- 5 M to about 10-l2M of dexamethasone has been shown
to significantly enhance the activity to PDGF. A
composition containing from about 3.92 ~g/ml (10 M) to
about 0.000392 mg/ml (10 IZM) of dexamethasone (which
has a molecular weight of about 392 g/mole) is
preferred for most applications. A concentration of
about 10- 5 to about 10 ~M is most preferred.
Other growth factors such as transforming growth
factor-a (TGF-a) and insulin-like growth factors
(IGF-l) can be added to the composition containing PDCF
and the anti-inflammatory compound to further enhance
healing or regeneration of injured tissue. TGF-~,
IGF-1 or other growth factor can be added to the PDGF
mixture in a weight-to-weight ratio, for example, of
about 1:4 and 25:1, preferably between about 1:2 and
10:1, and more preferably 1:1 or 2:1.
In a preferred embodiment of the present invention,
the composition of PDGF and the anti-inflammatory
compound is combined with a pharmaceutically acceptable
carrier substance for locai, topical administration.
Examples of phaxmaceutically acceptable carriers
include, for example, commercially available inert
gels, or liquids supplemented with albumin, methyl
cellulose or a collagen matrix. Typical of such
formulations are ointments, creams and gels. Ointments
are typically prepared using an oleaginous base, e.g.,
containing mixed oils or hydrocarbons, such as white
petrolatum or mineral oil, or an absorbent base, e.g.,
consisting o~ an absorbent anhydrous substance or
substances, for example, anhydrous lanolin. Following
formatinn of the base, the active ingredients are added
in the desired concentration. Creams generally
SUB~TITIJT~ SH~T
lP~JS
1~ Rec'd P~/PT~ I I JUN ~993
21 C ~ )7 ~, P~T / ~ S 9~ ~ o 21' 4 ~
comprise an oil phase (internal phase) containing
typically fixed oils, hydrocarbons, and the like, such
as waxes, petrolatum, mineral oil, and the like, and an
aqueous phase (continuous phase), comprising water and
any water-soluble substances, such as added salts. The
two phases are stabilized by use oE an emulsifying
agent, for example, a surface active agent, suc~l as
sodium lauryl sulfate; hydrophilic colloids, such as
acacia colloidal clays, beegum, and the like. Upon
formation of the emulsion, the active ingredients are
added in the desired concentration. Gels are comprised
of a base selected from an oleaginous base, water, or
an emulsion-suspension base, as previously described.
To the base is added a gelling agent which forms a
matrix in the base, increasing its viscosity to a
semisolid consistency. Examples of gelling agents are
hydroxypropyl cellulose, acrylic acid polymers, and the
like. The active ingredients are added to the
formulation at the desired concentration at a point
preceding addition of the gelling agent.
The amounts of PDGF and anti-inflammatory compound
incorporated into the formulation of the present
invention is not critical; the concentration should be
sufficient to permit ready application of the
formulation to the wound area in an amount which will
deliver the desired amount of PDGF and the anti-
inflammatory compound. A typical gel formulation
useful for the topical administration of PDGF and
dexamethasone, for example, comprises the following:
% by Weiqht
sterile distilled water92.38
sodium dibasic phosphate0.03
CarbapOlr H 0 5
glycerin 1.6
m-cresol 0.25
sodium hydroxide (lN) 0.5
à~ lTlJTE SHEE~
.... ~ ..
WO92/16181 PCTtUS92~02043
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2 ~ ;7 ~
A bone collagen ~atrix as described in U.S. Patent
No. 4,975,526, which is incorporated herein by
reference, can be used as the carrier for application
S to bone and/or cartilage. The collagen matrix
described in this patent is a biodegradable,
biocompatible, mineral-free, insoluble Type-I bone
collagen particles being depleted of non-collagenous
protein. The collagen matrix particles have a mean
diameter of about 70 ~m - 850 ~m, and an increased
intraparticle surface area relative to untreated
material. In this embodiment, PDGF and the anti-
inflammatory agent are first dissolved in a suitable
solvent such as buffered sterile saline and then added
to the collagen matrix. The mixture is vortexed, and
the matrix is lyophilized and shaped as desired or
implanted into an area of bone or cartilage by packing.
Other useful matrix materials include synthetic
homopolymers and copolymers of glycolic acid and lactic
acid, hydroxyapatite, tricalcium and other calcium
phosphates, and particulate demineralized guanidine
extracted species-specific (allogenic) bone. The
matrix containing the PDGF and steroid can be applied
into a shape spanning the bone or cartilage defect to
serve as a "temporary scaffold" and substratum as a
base for anchoring and proliferation of differentiated
tissue cells.
The method is particularly useful for treating
tissues affected by periodontal disease. Periodontal
disease is characterized by gingivitis, destruction of
alveolar bone and periodontal ligament, apical
migration of the epithelial attachment resulting in the
formation of periodontal pockets. Thus, a number of
different tissues are involved, including epithelium,
cartilage and bone. The method of the invention
W~92~16181 PCTtUS92tO2~3
--1 1-- 2 ~ 3
promotes healing and regeneration of the gum tissues
(epithial tissue) the periodontal ligament (cartilage)
and the jaw bone (bone). Pulp and dentin tissue within
the tooth which was eroded or attacked by periodontal
disease can be regenerated using the present method.
The most preferred composition for this purpose is a
combination af PDGF and dexamethasone.
The data shown in the following Examples all
demonstrate that PDGF mitoyenesis is enhanced in the
presence of a glucocorticoid. Current methodologies
described in the literature disclose the use of PDGF as
an agent to enhance tissue repair or reduce
regeneration ln vivo utilizing another human growth
factor, IGF-l in combination with PDGF. This i5
lS substantially more expensive and less efective than
using the small amounts (e.g., less than lO-5M) of
glucocorticoid drugs, as described herein. In
addition, the glucocorticoids have anti-inflammatory
properties, which are beneficial where inflammation is
present, such as with many periodontal diseases. As
demonstrated herein, the addition of a glucocorticoid
drug in a preferred concentration of from about lO-7M
to about 10-9M was consistently more effective than
adding l~g/ml IFG-l for enhancing the cell
proliferation which is the basis of tissue regeneration
and repair.
The invention will be more readily understood by
the following specific non-limiting examples which are
included for purposes of illustration.
EXAMPLES
Example l: Potentiation of mitogenic effect of PDGF
and IGF-l by Dexamethasone
WO92~16181 PCT/VS92/02043
~ 7 ~ -12-
The following experiments were all performed on low
passage, human diploid fi~roblasts obtained from the
periodontal ligaments and dental pulps of extracted
teeth. The cells were cultured under standard culture
conditions and stocks were propagated with 10% fetal
bovine serum (FBS) as the source of growth factors.
For the experiments detailed here, the cells were
plated at lO,000 to lS,000 cells per 1~88 cm2 of
surface area in 24 well culture plates and conditioned
in medium containing 0.1% FBS ~or 24-48 hours prior to
treatment. The cells were then exposed once to the
indicated concentrations of PDGF, IGF-l and/or
dexamethasone in culture media at time zero. The cells
were quantitatively harvested from each well and the
total cell population densities were determined using a
Coulter counter by standard methods. The PDGF-~ and
PDGF-aa used in these studies were recombinant human
analogs of PDGF produced in E. coli which were provided
by Creative BioMolecules, Hopkinton, Massachusetts.
The dexamethasone was purchased from Sigma Chemical
Company, St. Louis, Missouri.
Cell cultures in 24 well plates prepared as
described above were treated with the following
materials and the extent of cell growth determined.
Plate l contained 0.1% FBS
Plate 2 contained 0.1% FBS
Plate 3 contained PDGF-~ 200 ng/ml + lGF-l 200
ng/ml
Plate 4 contained PDGF-~ 200 ng/ml + lGF-l 200
ng/ml + lO sM Dexamethasone
Plate 5 contained PDGF-~ 200 ng/ml + lGF-l 200
ng/ml + lO- 6 M Dexamethasone
Plate 6 contained PDGF-~ 200 ng/ml + lGF-l 200
ng/ml + lO 7 M Dexamethasone
Plate 7 contained PDGF-~ 200 ng/ml + lGF-l 200
W092/16183 PCT/US92/02~43
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ng/ml + 10 ~M Dexamethasone
Plate ~ contained PDGF-~ 200 ng/ml + lGF-l ~00
ng/ml ~ 10 M Dexamethasone
Plate 9 contained PDGF-~ 200 ng/ml + lGF-l 200
S ng/ml + 10 1lM Dexamethasone
Plate 10 contained PDGF-~ 200 ng/ml + lGF-l 200
ng/ml + 10 M Dexamethasone
Plate 11 contained PDGF-~ 200 ng/ml ~ 10 7M
Dexamethasone
Plate 12 contained PDGF-~ 200 ng/ml
All of the plates except for plate 1 were incubated
for 96 hours. Plate 1 was incubated for 30 minutes.
the cells then were removed from the plates and
counted. The efficacy of the agents on cell mitogenic
lS activity was measured by the number of cells per square
centimeter after the incubation period.
The results are illustrated in Figures 1-2.
Figure 1 shows that cultures treated with dexamethasone
in combination with PDGF proliferated faster than
cultures treated with PDGF alone and PDGF + IGF-l.
Figure 2 shows that dexamethasone is effective in
enhancing all proliferation over concentrations ranging
from 10 5 to 10 1lM. The concentration of
dexamethasone to optimally potentiate the mitogenic
activity of PDGF at 200 ng/ml is about 10 M~ The
mitogenic activity of the cells was increased by the
combination of dexamethasone and PDGF more than by just
PDGF alone or PDGF ~ IGF-l. However, there was no
~urther enhancement of mitogenic activities by the
addition of IGF-1 to PDGF and dexamethasone.
Dexamethasone alone has no effect on cell mitogenic
activity, as shown in Figure 4.
Example 2: Comparison of the effects on mitogenic
activity of dexamethasone with IGF-l over
timeO
WO92/161B1 PCT/US92/02043
2 1 0 5 ~ 7 ;~ -14-
To determine the time course of the effects of
dexamethasone on PDGF-~ mitogenic activity, cultures
were treated with PDGF + IGF-l and PDGF with and
without dexamethasone at lO- and lO 12M, harvested and
counted at the time indicated over a period of
160 hours. Data from the same experiment are plotted
against the controls in two separate plots for clarity.
The results, shown in Figure 3, revealed that a
single exposure of the cultured cells at time zero to
PDGF-~ plus dexamethasone resulted in final increased
cell population densities after 160 hours that were
similar to those obtained by exposure of the cultured
cells to PDGF-~ plus IGF-l. The final total cell
number was similar for treatment with lO ng/ml and
500 ng/ml PDGF-~. The PDGF-~ ~ IGF-l treated
cultures reached maximal cell population densities at
96 hours, while those exposed to PDGF-~ +
dexamethasone had not plateaued by 160 hours,
demonstrating the prolonged effect of a single dose of
PDGF ~ dexamethasone on cell proliferation~
Example 3: Comparison of the Mitogenic Activity of
PDGF-~ and PDGF-~.
Plates prepared as described above were treated
with PDGF-aa or PDGF-~ and dexamethasone or IGF-l.
The extent of cell growth determined.
.
PDGF-~ Studies
Plate l contained 0.1% FBS + lO 7 M dexamethasone.
Plate 2 contained PDGF-~ 500 ng/ml.
Plate 3 contained PDGF-~ 500 ng/ml + IGF-l 500
ng/ml.
Plate 4 contained PDGF-~ 500 ng/ml + lO M
dexamethasone.
WO92/16181 PCT/US92/02043
--1 ~; 2 ~ ë ;~ r
PDGF-a Studies
Plate 5 contained 0.1% FRS _ l0-7 dexamethasone.
Plate 6 contained PDGF-aa 500 ng/ml.
Plate 7 contained PDGF-aa 500 ng~ml ~ IGF-l 500
ng/ml.
Plate 8 contained PDGF-a 500 ng/ml + l0 7M
dexamethasone.
The results of the present experiment, shown in
Figure 4, which demonstrate that dexamethasone
potentiates the mitogenic activity of both PDGF-aa and
PDG~ . As shown in Figure 4, after a single
application of PDGF plus dexamethasone, the rate of
cell proliferation at 168 hours did not appear to have
diminished. The data shown in Table l suggest that
PDG~ is a more potent mitogen for these cells than
PDGF-aa, and that PDGF-~ treated cultures may be
slightly more responsive to dexamethasone than PDGF-aa
treated cultures.
TABLE l
Ratios of Cell Population Densities
PDGF-~ DEX/PDGF-~ PDGF-a DEX + PDGF-aa PDGF-~ + PDGF-aa
48 HOURS 1.24 0.92 1.08
96 HOURS 1.97 1.44 1.32
168 HOURS 2.73 2.l9 l.57
Data are ratios of means of cells/cm from replicate experiments.
Equivalents
One skllled in the art will be able to ascertain,
with no more than routine experimentation, many
equivalents to the specific embodiments descri~ed
herein. Such equivalents are intended to be
encompassed by the following claims.
