U.S. patent application number 11/120154 was filed with the patent office on 2005-11-10 for combination pdgf, kgf, igf, and igfbp for wound healing.
This patent application is currently assigned to Chiron Corporation. Invention is credited to Williams, Lewis T..
Application Number | 20050250695 11/120154 |
Document ID | / |
Family ID | 27357798 |
Filed Date | 2005-11-10 |
United States Patent
Application |
20050250695 |
Kind Code |
A1 |
Williams, Lewis T. |
November 10, 2005 |
Combination PDGF, KGF, IGF, and IGFBP for wound healing
Abstract
The invention provides a therapeutic composition for epithelial
wound repair that is a combination of PDGF and KGF. Further, the
invention provides a composition for epithelial wound repair that
is a therapeutic combination of PDGF, KGF, and IGF. Additionally,
the invention provides a therapeutic composition of PDGF, KGF, IGF
and IGFBP for epithelial wound repair.
Inventors: |
Williams, Lewis T.;
(Tiburon, CA) |
Correspondence
Address: |
Chiron Corporation
Intellectual Property - R440
P.O. Box 8097
Emeryville
CA
94662-8097
US
|
Assignee: |
Chiron Corporation
Emeryville
CA
|
Family ID: |
27357798 |
Appl. No.: |
11/120154 |
Filed: |
May 2, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11120154 |
May 2, 2005 |
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09723449 |
Nov 27, 2000 |
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6903078 |
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09723449 |
Nov 27, 2000 |
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08719742 |
Sep 25, 1996 |
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60021540 |
Jul 11, 1996 |
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60005075 |
Oct 11, 1995 |
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Current U.S.
Class: |
514/44R ;
435/320.1; 435/325; 435/69.1; 514/8.2; 514/8.5; 514/8.7; 514/9.2;
514/9.4; 530/399; 536/23.5 |
Current CPC
Class: |
A61K 9/06 20130101; C07K
14/49 20130101; A61K 9/4825 20130101; A61K 9/127 20130101; A61K
38/00 20130101; A61P 17/00 20180101; A61K 2300/00 20130101; A61K
2300/00 20130101; A61K 2300/00 20130101; A61P 17/02 20180101; A61K
9/7015 20130101; A61K 48/00 20130101; A61K 38/30 20130101; A61K
38/1754 20130101; A61K 38/30 20130101; A61K 38/1825 20130101; A61K
38/1825 20130101; A61L 26/0047 20130101; A61K 38/1754 20130101 |
Class at
Publication: |
514/012 ;
435/069.1; 435/320.1; 435/325; 530/399; 536/023.5 |
International
Class: |
C07H 021/04; A61K
038/18 |
Claims
1. A pharmaceutical composition for repair of epithelial tissues
comprising a first polypeptide having the biological activity of a
platelet derived growth factor (PDGF) and a second polypeptide
having the biological activity of keratinocyte growth factor
(KGF).
2. The pharmaceutical composition of claim 1, wherein the first
polypeptide comprises a full-length PDGF polypeptide.
3. The pharmaceutical composition of claim 1, wherein the first
polypeptide comprises a biologically active fragment of a
full-length PDGF polypeptide.
4. The pharmaceutical composition of claim 1, wherein the first
polypeptide comprises one selected from the group consisting of
PDGF A chain and PDGF B chain.
5. The pharmaceutical composition of claim 1, wherein the first
polypeptide is produced by expression of a DNA molecule that
encodes PDGF in a host cell, wherein the host cell comprises one
selected from the group consisting of a bacterial cell, a yeast
cell, a mammalian cell, and an insect cell.
6. The pharmaceutical composition of claim 1, wherein the second
polypeptide compriese a full-length KGF.
7. The pharmaceutical composition of claim 1, wherein the second
polypeptide comprises a biologically active fragment of a
full-length KGF polypeptide.
8. The pharmaceutical composition of claim 1, wherein the second
polypeptide is produced by expression of a DNA molecule that
encodes KGF in a host cell, wherein the host cell comprises one
selected from the group consisting of a bacterial cell, a yeast
cell, a mammalian cell, and an insect cell.
9. The pharmaceutical composition of claim 1, further comprising a
pharmaceutically acceptable carrier.
10. A method of repairing epithelial tissues comprising applying to
the tissue to be repaired the pharmaceutical composition of claim
1.
11. The method of claim 10, wherein the epithelial tissue is
selected from the group consisting of skin, gastric lining, and
intestinal lining.
12. The method of claim 10, wherein the pharmaceutical composition
is applied in the manner selected from the group consisting of
locally, orally, intradermally, subcutaneously, intraluminally,
intragastrically, and intraperitoneally.
13. A method of repairing or preventing epithelial cell damage
comprising applying to the cells to be protected or repaired a
pharmaceutical composition comprising PDGF and a composition
comprising KGF.
14. The method of claim 13, wherein the pharmaceutical composition
comprising PDGF and the pharmaceutical composition KGF are the same
pharmaceutical composition.
15. The method of claim 13, wherein application of PDGF and KGF is
contemporaneous.
16-24. (canceled)
25. The pharmaceutical composition of claim 1, also comprising a
third polynucleotide having the biological activity of insulin-like
growth factor (IGF).
26. The pharmaceutical composition of claim 25, wherein IGF
comprises one selected from the group consisting of IGF-1 and
IGF-2.
27. The pharmaceutical composition of claim 25, also comprising a
fourth polynucleotide having the biological activity of
insulin-like growth factor binding protein (IGFBP).
28. The pharmaceutical composition of claim 27, wherein the IGFBP
comprises one selected from the group consisting of IGFBP-1,
IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5, and IGFBP-6.
29. The pharmaceutical composition of claim 25 wherein the third
polypeptide comprises full-length IGF.
30. The pharmaceutical composition of claim 25 wherein the third
polypeptide comprises a biologically active fragment of IGF.
31. The pharmaceutical composition of claim 27 wherein the fourth
polypeptide comprises a full-length IGFBP.
32. The pharmaceutical composition of claim 27 wherein the fourth
polypeptide comprises a biologically active fragment of an
IGFBP.
33. The pharmaceutical composition of claim 25 wherein the third
polypeptide is produced by expression of a DNA molecule that
encodes IGF in a host cell, wherein the host cell comprises one
selected from the group consisting of a bacterial cell, a yeast
cell, a mammalian cell, and an insect cell.
34. The pharmaceutical composition of claim 27 wherein the fourth
polypeptide is produced by expression of a DNA molecule that
encodes an IGFBP in a host cell, wherein the host cell comprises
one selected from the group consisting of a bacterial cell, a yeast
cell, a mammalian cell, and an insect cell.
35. The pharmaceutical composition of claim 25 further comprising a
pharmaceutically acceptable carrier.
36. The pharmaceutical composition of claim 27 further comprising a
pharmaceutically acceptable carrier.
37. A method of repairing epithelial tissues comprising applying to
the tissue to be repaired the pharmaceutical composition of claim
25.
38. A method of repairing epithelial tissues comprising applying to
the tissue to be repaired the pharmaceutical composition of claim
27.
39. The method of claim 38, wherein the epithelial tissue comprises
one selected from the group consisting of skin, gastric lining, and
intestinal lining.
40. The method of claim 38, wherein the epithelial tissue comprises
one selected from the group consisting of skin, gastric lining, and
intestinal lining.
41. The method of claim 39, wherein the pharmaceutical composition
is applied in a manner selected from the group consisting of
locally, orally, intradermally, subcutaneously, intraluminally,
intragastrically, and intraperitoneally.
42. The method of claim 40, wherein the pharmaceutical composition
is applied in a manner selected from the group consisting of
locally, orally, intradermally, subcutaneously, intraluminally,
intragastrically, and intraperitoneally.
43. A method of repairing or preventing epithelial cell damage
comprising applying to the cells to be protected or repaired the
pharmaceutical composition of claim 13 and further comprising a
composition comprising IGF.
44. A method of repairing or preventing epithelial cell damage
comprising applying to the cells to be protected or repaired the
pharmaceutical composition of claim 43 and further comprising a
composition comprising an IGFBP.
45-48. (canceled)
49. The pharmaceutical composition of claim 1, comprising one
selected from the group consisting of a cream, a foam, an
injectable solution, a spray, a gel matrix, a sponge, drops, and a
wash.
50. The pharmaceutical composition of claim 25, comprising one
selected from the group consisting of a cream, a foam, an
injectable solution, a spray, a gel matrix, a sponge, drops, and a
wash.
51. The pharmaceutical composition of claim 27, comprising one
selected from the group consisting of a cream, a foam, an
injectable solution, a spray, a gel matrix, a sponge, drops, and a
wash.
Description
FIELD OF THE INVENTION
[0001] This invention relates to the use of a polypeptide having
the biological activity of a platelet derived growth factor (PDGF)
and a polypeptide having the biological activity of keratinocyte
growth factor (KGF) for treatment or prevention of epithelial cell
damage. This invention also relates to the use of a polypeptide
having the biological activity of an insulin like growth factor
(IGF-1 or IGF-2) in combination with the PDGF/KGF combination of
the invention; the PDGF/KGF/IGF combination is useful for treatment
or prevention of epithelial cell damage, being an improvement over
previous methods for treating epithelial cell damage. The invention
further relates to the use of a polypeptide having the biological
activity of an insulin like growth factor binding protein (IGFBP)
in combination with an IGF and further in combination with the
PDGF/KGF combination of the invention. This invention further
relates to pharmaceutical compositions including PDGF/KGF
combination, PDGF/KGF/IGF, and PDGF/KGF/IGF/IGFBP combination for
treatment or prevention of epithelial cell damage. This invention
also relates to use of DNA encoding PDGF, DNA encoding KGF, DNA
encoding an IGF, and DNA encoding an IGFBP for such treatment and
prevention, as a PDGF/KGF combination or as a PDGF/KGF/IGF
combination, or as a PDGF/KGF/IGF/IGFBP combination. Moreover, the
present invention relates to kits containing PDGF and KGF, kits
containing PDGF, KGF, and IGF, and kits containing PDGF, KGF, IGF,
and IGFBP, and/or kits containing DNA encoding the three such
combinations.
BACKGROUND OF THE INVENTION
[0002] The patent application EP 0 619 370 discloses the use of KGF
for wound healing purposes. PDGF may also be used for wound healing
purposes as evidenced by its ability to stimulate mesenchymal
derived cells, as disclosed in U.S. Pat. No. 5,187,263.
[0003] Insulin like growth factors, IGF and IGF-2, have been
described and studied in the art. IGF is described in Rinderknecht,
J. Biol. Chem. 253:2769 (1978), and has been found to act as a
mitogen on a number of different cell types as described in EP 0
128 733. Like insulin, the IGFs stimulate phosphorylation on
specific tyrosine residues within the cytoplasmic domain of the
receptors to which the IGF binds, as described in WO 93/98826.
IGF-II is described in Rinderknecht, FEBS Letters, (1978)
89:283.
[0004] Insulin-like growth factors are also known under the class
name somatomedins, and have been identified in various animal
species as polypeptides that act to stimulate growth of cells in a
variety of tissues and cell types, particularly during development.
Growth promoting effects of somatomedins include enhancement of
cell multiplication and stimulation of cartilage proliferation,
stimulation of transport of amino acids, stimulation of synthesis
of RNA, DNA and protein, and stimulation of incorporation of
sulfate into proteoglycan and of proline into collagen. Much
mammalian postnatal growth is due to stimulation of cartilage
growth by somatomedins and growth in utero may also be
somatomedin-dependent.
[0005] Uses of IGF as a known stimulatory and growth promoting
agent includes use for bone repair and replacement therapy, as
described in EP 303 855; as a means to counteract certain harmful
side effects of carcinostatic drugs, as described in JP 63-196524;
and as a way to increase lactation and meat production in cattle
and other farm animals, as described in U.S. Pat. No.
4,783,524.
[0006] IGF-I has also been found useful in the treatment of
osteoporosis in mammals exhibiting decreased cortical bone mineral
density and those exposed to drugs or environmental conditions that
result in bone density reduction and potentially to an osteoporosis
condition, as described in EP 560 723 and EP 436 469.
[0007] IGF-I has been administered with sodium pentosan polysulfate
(PPS) to severely osteoarthritic canines with the effect of
reducing the severity of the disease by lowering the levels of
active neutral metalloproteinase in the cartilage. In the model of
mildly osteoarthritic canines, therapeutic intervention with IGF-I
and PPS together appeared to successfully maintain cartilage
structure and biochemistry, while IGF alone was ineffective, as
described in Rogachefsky, Osteoarthritis and Cartilage, (1993)
1:105-114.
[0008] IGF binding proteins have been studied extensively, and
presently six IGFBPs are known (IGFBP1-6). IGFBPs form complexes
with IGF-I and IGF-II in plasma and are believed to function
typically as binding proteins for protein hormones, that is
regulating the availability, the activity, and extending the
half-life of the protein hormone ligand that they transport. While
IGFBP-3, a 150 kDa complex, is the most abundant IGFBP in plasma
and is believed to function as a carrier and reservoir of IGF-I in
plasma, IGFBP-1 is a small 25 kDa binding protein produced mainly
in the liver and fibroblasts, and can distribute between the
circulation and the tissues, thus potentially regulating the
bioavailability of IGF in both compartments as described in Tsuboi
et al, J. of Inv. Derm. 104: 199-203 (1995). IGF has been described
as useful for wound healing in combination with IGFBP, as described
in Tsuboi et al, J. of Inv. Derm. 104: 199-203 (1995), Kratz et al,
Scand J. Plast Reconstr Hand Surg 28: 107-112 (1994), and Jyung et
al, Surgery 115: 233-239 (1994).
[0009] IGF expression has been associated with wound healing as
described in Gartner et al, J. Surg. Res. 52: 389-394 (1992), and
Steenfos and Jansson, Eur. J. Surg. 158: 327-331 (1992).
Additionally, IGF has been described as useful when administered in
combination with PDGF for wound healing as described in U.S. Pat.
No. 4,861,757.
[0010] Therefore, it would be advantageous if an improved
composition can be found that would have improved properties over
administration of PDGF alone, over administration of KGF alone,
over PDGF with IGF, and over IGF alone, and over IGF with
IGFBP.
SUMMARY OF THE INVENTION
[0011] It is, thus, an object of the present invention to provide
an improved composition for treatment or prevention of epithelial
cell damage. It is further an object of the present invention to
provide an improved method for such treatment or prevention.
[0012] In accordance one of the objects of the present invention,
there is provided herein a pharmaceutical composition for treatment
or prevention of epithelial cell damage, the composition containing
a first polypeptide having the biological activity of a platelet
derived growth factor (PDGF) and a second polypeptide having the
biological activity of keratinocyte growth factor (KGF).
[0013] In accordance one of the objects of the present invention,
there is provided herein a pharmaceutical composition for treatment
or prevention of epithelial cell damage, the composition containing
a first polypeptide having the biological activity of a platelet
derived growth factor (PDGF), a second polypeptide having the
biological activity of keratinocyte growth factor (KGF), and a
third polypeptide having the biological activity of insulin-like
growth factor-1 (IGF-1).
[0014] Also in accordance with another object of the invention
there is provided kits for treatment and prevention of epithelial
cell damage comprising the pharmaceutical compositions of the
invention as described herein.
[0015] In accordance with a further object of the present
invention, there is provided methods of treatment or prevention of
epithelial cell damage by applying to such cells the pharmaceutical
compositions as described above.
[0016] In accordance with another object of the present invention,
there is provided pharmaceutical compositions for treatment or
prevention of epithelial cell damage by applying to such cells a
pharmaceutical composition comprising a first DNA molecule
including a first nucleotide sequence and a second DNA molecule
including a second nucleotide sequence so that the first nucleotide
sequence encodes PDGF and the second nucleotide sequence encodes
KGF.
[0017] In accordance with another object of the present invention,
there is provided pharmaceutical compositions for treatment or
prevention of epithelial cell damage by applying to such cells a
pharmaceutical composition comprising a first DNA molecule
including a first nucleotide sequence, a second DNA molecule
including a second nucleotide sequence, and a third DNA molecule
including a third nucleotide sequence, so that the first nucleotide
sequence encodes PDGF, the second nucleotide sequence encodes KGF,
and the third nucleotide sequence encodes IGF.
[0018] The pharmaceutical composition may also include a fourth
polynucleotide having the biological activity of insulin-like
growth factor binding protein (IGFBP).
[0019] Another embodiment of the invention is a method of repairing
epithelial tissues comprising applying to the tissue to be repaired
a pharmaceutical composition including KGF, PDGF, and alternatively
IGF or IGF and IGFBP.
[0020] Yet another object of the invention is met with a method of
repairing or preventing epithelial cell damage including applying
to the cells to be protected or repaired the pharmaceutical
composition that includes KGF and PDGF and further includes a
composition with IGF.
[0021] Another object of the invention is a accomplished by a
method of repairing or preventing epithelial cell damage including
applying to the cells to be protected or repaired a pharmaceutical
composition that includes KGF, PDGF and IGF, and that further
includes a composition comprising an IGFBP.
[0022] A further object of the invention is met by a method of
repairing epithelial cell damage comprising applying to the
epithelial cell a pharmaceutical composition comprising a first DNA
molecule, a second DNA molecule, and a third DNA molecule wherein
the first DNA molecule is a first nucleotide sequence encoding
PDGF, the second DNA molecule is a second nucleotide sequence
encoding KGF, and the third DNA molecule is a third nucleotide
sequence encoding IGF.
[0023] Yet a further object of the invention is met by a method of
repairing epithelial cell damage comprising applying to the
epithelial cell a pharmaceutical composition comprising a first DNA
molecule, a second DNA molecule, a third DNA molecule, and a fourth
DNA molecule wherein the first DNA molecule is a first nucleotide
sequence encoding PDGF, the second DNA molecule is a second
nucleotide sequence encoding KGF, the third DNA molecule is a third
nucleotide sequence encoding IGF, and the fourth DNA molecule
comprises a fourth nucleotide sequence encoding an IGFBP.
[0024] Another object of the invention is accomplished by a kit
that has a first DNA molecule, a second DNA molecule and a third
DNA molecule wherein the first DNA molecule is a first nucleotide
sequence encoding PDGF, the second DNA molecule is a second
nucleotide sequence encoding KGF, and the third DNA molecule is a
third nucleotide sequence encoding IGF.
[0025] A final object of the invention is met by a kit that has a
first DNA molecule, a second DNA molecule, a third DNA molecule,
and a fourth DNA molecule wherein the first DNA molecule is a first
nucleotide sequence encoding PDGF, the second DNA molecule is a
second nucleotide sequence encoding KGF, the third DNA molecule is
a third nucleotide sequence encoding IGF, and the fourth DNA
molecule is a fourth nucleotide sequence encoding an IGFBP.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0026] This disclosure herein draws on previously published works
such as scientific articles, published patents or patent
applications. All such published works are incorporated herein by
reference. The present invention can be better understood in light
of the following definitions.
[0027] Definitions
[0028] Unless otherwise expressly provided herein, the term
"platelet derived growth factor" or "PDGF" includes the PDGF A
chain polypeptide and the PDGF B chain polypeptide and to the AA,
BB, and AB dimers, and biologically active fragments, analogs, and
derivatives thereof as described in U.S. Pat. No. 5,187,263;
Waterfield et al., Nature 304:35-39 (1983); Wang et al., J. Biol.
Chem. 259: 10645-48 (1984), Antoniades et al., Biochem. Pharm. 33:
2833-38 (1984); and Westermark et al., Proc. Natl. Acad. Sci USA
83:7197-7200 (1986); U.S. Pat. No. 5,219,759.
[0029] Also unless otherwise expressly provided herein, the term
"keratinocyte growth factor" or "KGF" refers to any one of a mature
polypeptide and biologically active fragments, analogs, and
derivatives thereof as described in WO 90/08771 and WO
95/01434.
[0030] The term "insulin-like growth factor" as used herein
encompasses IGF-I and IGF-II in their substantially purified,
native, recombinantly produced, or chemically synthesized forms,
and includes biologically active fragments, analogues, muteins,
including C-terminal deletion muteins, and derivatives thereof that
retain IGF activity and/or ability to bind the IGF receptors, as
described in, for example, EP 135 094, WO 85/00831, U.S. Pat. No.
4,738,921, WO 92/04363, U.S. Pat. No. 5,158,875, EP 123 228, and EP
128 733. An analog of IGF or an analog of the fragment includes
native IGF that has been modified by one or more amino acid
insertion, deletion, or substitution that does not substantially
affect its properties. Preferably, the analog has increased
activity compared to native IGF. More preferably, at least 2-fold
increase, most preferably, at least 7-10 fold increase. For
example, the analog can include conservative amino acid
substitutions. An IGF analog also includes peptides having one or
more peptide mimics ("peptoids"), such as those described in WO
91/04282. An IGF mutein is polypeptide variant with one or more
amino acids altered to produce a desired characteristic, such as to
replace a cysteine residue with a non-disulfide bond forming amino
acid. Muteins, analogues and derivatives may be generated using
conventional techniques. For example, PCR mutagenesis can be used.
While the following discussion refers to DNA, it is understood that
the technique also finds application with RNA. An example of a PCR
technique is described in WO 92/22653. Another method for making
analogs, muteins, and derivatives, is cassette mutagenesis based on
the technique described by Wells, Gene, (1985) 34:315.
[0031] The term "insulin like growth factor binding protein
(IGFBP)" refers to a binding protein identified to bind an IGF
binding protein as described and identified in Keifer et al, J.
Biol. Chem. 266: 9043-9 (1991), Camacho-Hubner et al, J. Biol.
Chem. 267:11949-56 (1992), McCusker and Clemens, THE INSULIN LIKE
GROWTH FACTORS: STRUCTURE AND BIOLOGICAL FUNCTIONS, Oxford Univ.
Press, N.Y. pp. 110-150 (1992).
[0032] A polypeptide "having the biological activity of PDGF"
refers to a polypeptide having the same or increased capability of
preferentially stimulating the growth of cells of the dermis layer
of the skin. Such a polypeptide can be a full-length PDGF, a
fragment of PDGF, an analog of PDGF bearing amino acid
substitution, deletion or addition or a derivative of PDGF, such as
that described in U.S. Pat. No. 5,149,792 and EP 458 959 B1; and
U.S. Pat. Nos. 4,769,328; 4,801,542; 4,766,073; 4,849,407;
4,845,075; 4,889,919; 5,045,633; and 5,128,321.
[0033] A polypeptide "having the biological activity of KGF" refers
to a polypeptide having the same or increased capability of
preferentially stimulating the growth of cells of the epidermis
layer of the skin. Such a polypeptide can be a full-length KGF, a
fragment of KGF, an analog of KGF bearing an amino acid
substitution, deletion, or addition; or a derivative of KGF as
described in WO 90/08771, and WO 95/01434.
[0034] A polypeptide "having the biological activity of IGF" refers
to a polypeptide having the same or increased capability of acting
as growth factor capable of insulin-like effects such as, for
example, stimulation of phosphorylation of specific tyrosine
residues within the cytoplasmic domain of the receptor to which it
binds as described in WO 93/98826, or, in some cases, for example,
mitogenic effects on certain cells as described in EP 0 128-733.
Such a polypeptide can be full-length IGF, a fragment of IGF, an
analog of IGF bearing an amino acid substitution, deletion, or
addition, or any derivative of IGF.
[0035] A polypeptide "having the biological activity of IGFBP"
refers to a polypeptide having about the same or an increased
capability of acting as an IGF binding protein by binding and
transporting IGF to tissue and cells where IGF-can have a
biological effect. As there are presently at least six IGFBPs
known, many of which significantly different from the other IGFBPs,
specific qualities regarding the biological activity of a given
IGFBP does not include necessarily the entire group of IGF binding
proteins. Thus, the biological activity of an IGFBP may have some
similarities to other IGFBPs, but may also have distinctions that
identify it as a unique IGFBP.
[0036] "Full-length PDGF" or "mature PDGF" and "full-length KGF" or
"mature KGF" and "full-length IGF" or "mature IGF", and "full
length IGFBP" and "mature IGFBP" refers to the respective native
polypeptide as found in human or other mammalian tissues.
[0037] The terms "analog" herein in reference to PDGF, KGF, IGF,
and IGFBP protein refers to truncations, variants, alleles and
derivatives thereof. Unless specifically mentioned otherwise, these
terms encompass the bioactivities of "mature" KGF or "mature" PDGF
"mature" IGF or "mature" IGFBP. Thus, polypeptides that are
identical or contain at least 60%, preferably 70%, more preferably
80%, and most preferably 90% sequence identity to the mature
protein wherever derived, from human or nonhuman sources are
included within this definition. The analogs herein further include
peptides having one or more peptide mimics, also known as peptoids,
that possess the bioactivity of the protein. Included within the
definition are also polypeptides containing one or more analog
amino acid (including, for example, unnatural amino acids, etc.),
polypeptides with substituted linkages, as well as other
modifications known in the art, both naturally occurring and
nonnaturally occurring. The term polypeptide also does not exclude
post-expression modifications of the polypeptide, for example,
glycosylations, acetylations, phosphorylations and the like.
[0038] The "variants" and "derivatives" herein contain amino acid
substitutions, deletions, or insertions. The amino acid
substitutions can be conservative amino acid substitutions or
substitutions to eliminate non-essential amino acid residues such
as to alter a glycosylation site, a phosphorylation site, an
acetylation site, or to minimize misfolding by substitution or
deletion of one or more cysteine residues that are not necessary
for function. Conservative amino acid substitutions are those that
preserve the general charge, hydrophobicity/hydrophilicity and/or
steric bulk of the amino acid substituted, for example,
substitutions between the members of the following groups are
conservative substitutions: Gly/Ala, Val/Ile/Leu, Asp/Glu, Lys/Arg,
Asn/Gln, Ser/Cys/Thr and Phe/Trp/Tyr.
[0039] The term "polynucleotide" as used herein refers to a DNA
molecule, a RNA molecule or its complementary strand thereof. A
polynucleotide molecule can be single or double stranded.
[0040] A "therapeutically effective amount" as used herein refers
to that amount of a composition that is effective to attain a
desired result which, in the present instance, is repair of
epithelial tissues. That amount can be in a single dose or as part
of a series of doses. The precise amount will vary, from subject to
subject, depending on the subject's age, size, weight, and health,
the nature and severity of the condition to be treated. It is not
possible to specify an exact amount that is therapeutically
effective in advance. However, the effective amount for a given
situation can be determined by routine experimentation or based
upon the experience of the person administering the composition
based on the information provided herein. It is expected that the
dose may fall within a relatively broad range.
[0041] "A pharmaceutically acceptable carrier" herein refers to any
carrier that does not itself induce the production of antibodies
harmful to the individual receiving the composition. Suitable
carriers are typically large, slowly metabolized macromolecules
such as proteins, polysaccharides, polylactic acids, polyglycolic
acids, polymeric amino acids, amino acid copolymers and an inactive
virus particle. Such carriers are well known to those of ordinary
skill in the art. A thorough discussion of pharmaceutically
acceptable excipients can be found in REMINGTON'S PHARMACEUTICAL
SCIENCES (Merck Pub. Co., N.J. 1991). Exemplary pharmaceutically
acceptable carriers can include salts, for example, mineral acid
salts such as hydrochlorides, hydrobromides, phosphates, sulfates,
and the like; and the salts of organic acids such as acetates,
propionates, malonates, benzoates, and the like.
[0042] The "pharmaceutical compositions" herein may further contain
one or more components such as water, saline, glycerol, or ethanol.
Additionally, auxiliary substances, such as wetting or emulsifying
agents, pH buffering substances, stabilizers, antioxidants and the
like may be present in such compositions. The pharmaceutical
compositions herein may be prepared as a cream to be applied
topically, or as liquid solutions or suspensions, or solid forms
suitable for solution or suspension in liquid vehicles for
injection. The pharmaceutical composition herein may be prepared in
liposomal format such as those encapsulated in liposomes or in
DepoFoam.RTM.; as described in U.S. Pat. No. 5,442,120; WO
95/13796; and WO 91/14445.
[0043] "Co-administration" as used herein means administration of
KGF and PDGF according to the method of the invention in
combination with each other, co-administration of KGF/PDGF and an
IGF, and co-administration of KGF/PDGF/IGF/IGFBP. Co-administration
also means administration of a PDGF/KGF combination also in
combination with IGF or PDGF/KGF/IGF combination also in
combination with IGFBP. Co-administration may be simultaneous, for
example, by administering a mixture of KGF and PDGF, or a mixture
of PDGF, KGF, and IGF, or IGF and IGFBP, or may be accomplished by
administration of the agents separately, such as within a short
time period. Co-administration also includes successive
administration of KGF and PDGF, or successive administration of
KGF, PDGF, and IGF, or successive administration of KGF, PDGF, and
co-administration of IGF and IGFBP. Also, in the case of all these
administrations, for example in the case of administration of KGF
and PDGF, one of the two may be administered preventively while the
other is administered thereafter for treatment, within a reasonable
period of time after the preventive administration. For example, in
the case of administration of KGF, PDGF, and IGF, or IGF and IGFBP,
one or two or three may be administered preventively, and one or
two or three may be administered thereafter for treatment, within a
reasonable period of time after the preventive administration.
Dosage treatment for administration or co-administration may be a
single dose schedule or a multiple dose schedule.
[0044] The term "kit" refers to a package containing the specified
material and includes printed instructions for use of the material.
For example, a kit for the method of the invention may include PDGF
and KGF polypeptides separately or in admixture or DNA encoding
PDGF and KGF separately or in admixture, or as a combination of DNA
and polypeptides, for example, the DNA of PDGF and the polypeptide
of KGF. Also for example, a kit for the method of the invention may
include PDGF, KGF, and IGF polypeptides separately or in admixture
or DNA encoding PDGF, KGF and IGF separately or in admixture, or in
a combination of DNA and polypeptides, for example, the DNA of PDGF
and KGF, but the polypeptide of IGF. Also for example, a kit for
the method of the invention may include PDGF, KGF, and IGF and
IGFBP polypeptides separately or in admixture or DNA encoding PDGF,
KGF and IGF IGFBP separately or in admixture, or in a combination
of DNA and polypeptides, for example, the DNA of PDGF and KGF, but
the polypeptide of IGF and the polypeptide IGFBP. "Printed
instructions" may be written or printed on paper or other media, or
committed to electronic media such as magnetic tape,
computer-readable disks or tape, CD-ROM, and the like. Kits may
also include plates, tubes, dishes, diluents, solvents, wash fluid
or other conventional reagents.
[0045] The inventor has discovered, as disclosed herein, that the
combination of PDGF and KGF or biologically active fragments,
analogs or derivatives thereof, is more effective in treatment or
prevention of epithelial cell damage than either PDGF or KGF alone.
In addition, the inventor herein has discovered that the addition
of IGF to the PDGF/KGF combination further improves the treatment
or prevention of epithelial cell damage greatly beyond what might
be expected by any of the three medicaments alone; and further in
addition, the inventor herein has discovered that the addition of
IGF and IGFBP to the PDGF/KGF combination further improves the
treatment or prevention of epithelial cell damage greatly beyond
what might be expected by any of the four medicaments alone, and
even in addition to the improvements to epithelial cell damage
derived by the administration of the PDGF/KGF combination, or the
IGF/IGFBP combination.
[0046] In one embodiment of the present invention, a pharmaceutical
composition contains a therapeutically effective amount of PDGF and
KGF. Each of the PDGF and KGF can be made by any conventional
techniques or can be purified from its natural sources. In a
preferred embodiment of the present invention, each of PDGF and KGF
is made by expression of a polynucleotide sequence encoding the
respective protein in separate hosts or by coexpression thereof in
a single host. The host cell can be prokaryotic or eukaryotic. For
example PDGF can be made as described in U.S. Pat. No. 5,219,759.
KGF can be made as described in WO 95/01434. Other expression
systems can be used as described in greater detail below.
[0047] The pharmaceutical composition, in one embodiment of the
present invention, can be either a composition containing PDGF
alone and KGF alone, or both PDGF and KGF mixed together in a
single composition, in either a single dose or multiple doses. In
another embodiment of the present invention, for gene therapy
purposes, the pharmaceutical composition can be a composition
containing a polynucleotide encoding PDGF alone, a composition
containing polynucleotide encoding KGF alone, or both
polynucleotides mixed together in a single composition.
[0048] The PDGF and KGF compositions, if separately maintained, can
be administered separately or contemporaneously. Direct delivery of
the compositions will generally be accomplished by injection,
either subcutaneously, intradermally, intraperitoneally,
intraluminally, intragastrically, intraintestinally, intravenously
or intramuscularly. Other modes of administration include oral and
pulmonary administration, suppositories, and transdermal
applications. Dosage treatment may be a single dose schedule or a
multiple dose schedule.
[0049] In another embodiment of the present invention, a
pharmaceutical composition contains a therapeutically effective
amount of PDGF, KGF, and IGF. Each of the PDGF, KGF, and IGF can be
made by any conventional techniques or can be purified from its
natural sources. In a preferred embodiment of the present
invention, each of PDGF, KGF, and IGF is made by expression of a
polynucleotide sequence encoding the respective protein in separate
hosts or by coexpression thereof in a single host. The host cell
can be prokaryotic or eukaryotic. IGF can be made as described in
U.S. Pat. No. 4,738,921. IGF can also be synthesized by the solid
phase method as described in Li, PNAS, (1983) 80:2216-2220. In this
method, the polypeptide sequence for IGF-I can be assembled by
coupling the amino acid residues.
[0050] The PDGF, KGF and IGF compositions, if separately
maintained, can be administered separately or contemporaneously.
Direct delivery of the compositions will generally be accomplished
by injection, either subcutaneously, intradermally,
intraperitoneally, intraluminally, intragastrically,
intraintestinally, intravenously or intramuscularly. Other modes of
administration include oral and pulmonary administration,
suppositories, and transdermal applications. Dosage treatment may
be a single dose schedule or a multiple dose schedule.
[0051] In yet another embodiment, a pharmaceutical composition
contains a therapeutically effective amount of PDGF, KGF, and IGF
with IGFBP. Each of the PDGF, KGF, and IGF and IGFBP can be made by
any conventional techniques or can be purified from its natural
sources. In a preferred embodiment of the present invention, each
of PDGF, KGF, and IGF and IGFBP is made by expression of a
polynucleotide sequence encoding the respective protein in separate
hosts or by coexpression thereof in a single host. The host cell
can be prokaryotic or eukaryotic.
[0052] The PDGF, KGF, IGF, and IGFBP compositions, if separately
maintained, can be administered separately or contemporaneously.
Direct delivery of the compositions will generally be accomplished
by injection, either subcutaneously, intradermally,
intraperitoneally, intraluminally, intragastrically,
intraintestinally, intravenously or intramuscularly. Other modes of
administration include oral and pulmonary administration,
suppositories, and transdermal applications. Dosage treatment may
be a single dose schedule or a multiple dose schedule.
[0053] IGF can be made by conventional recombinant DNA techniques,
as described in Biochem. and Biophys. Res. Comm., (1990)
169:832-839 (IGF II) and Cell Regulation, (1990) 1:197-213, (IGF
II), and Biotechnology News, (11983) 3: 1-3 (IGF-I and II). For
example, IGF can be produced in E. coli as a fusion protein with
the trpE gene under the control of a modified tryptophan operon, as
described in U.S. Pat. No. 4,738,921. Alternatively, IGF can be
synthesized in E. coli under the control of the Vesicular
Stomatitis Virus (VSV) promoter and protector sequences, as
described in EP 478 333. The E. coli expression systems used for
expression herein can be modified as described in U.S. Pat. No.
5,158,875, to include a modified positively charged leader sequence
to enable proper folding of the IGF protein. Moreover, IGF can be
produced in methylotrophic yeast transformants with the IGF coding
sequence linked to a signal sequence which direct secretion and
proteolytic processing of the protein product. The signal sequence
suitable herein includes the S. cerevisiae alpha mating factor
pre-pro sequence in protease deficient P. pastoris strains, as
described in WO 92/04363. DNA constructs for production of IGF-II
can be made and expressed in E. coli as described in WO 89/03423.
Synthesis of recombinant IGF-II can also be achieved by following
the protocol described in EP 434 605, which relates to the
production of recombinant IGF-II with a covalently attached foreign
moiety and lacking the N-terminal attached methionine. IGF can also
be made in yeast as described in EP 123 228 and U.S. patent
application Ser. No. 06/922,199. Another method of producing IGF
using recombinant DNA techniques that is suitable herein is as
described in Biotechnology News, (1983) 10:1-3. IGF-I or IGF-II
coding sequences can be inserted into viral or circular plasmid DNA
vectors to form hybrid vectors, and the resulting hybrid vectors
can be used to transform host microorganisms such as bacteria or
yeast cells. The transformed microorganisms can be grown under
appropriate nutrient conditions to express IGF, as described in EP
135 094. IGF can also be made as described in EP 434 625.
[0054] An IGFBP can be any known IGFBP, for example, IGFBP-1,
IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5, and IGFBP-6. IGFBP can be made
as described in Brinkman et al, EMBO J. 7: 2417-2423 (1988), Mohan
et al, Proc. Natl. Acad Sci USA 86: 833842 (1989), U.S. Pat. No.
5,407,913, WO 92/12243, 92/03471, 92/03470, WO 92/03469, and Brewer
et al, Biochem. Biophys. Res. Commun. 152: 1289-97 (1988).
[0055] The pharmaceutical composition, in one embodiment of the
present invention, can be either a composition containing PDGF
alone, KGF alone, IGF alone, and IGF with IGFBP, or any two of the
four mixed together, and the third and fourth alone, or any three
of the four mixed together, and the fourth alone, or all four mixed
together in a single composition, in either a single dose or
multiple doses. In another embodiment of the present invention, for
gene therapy purposes, the pharmaceutical composition can be a
composition containing a polynucleotide encoding PDGF alone, a
composition containing polynucleotide encoding KGF alone, a
composition containing polynucleotide encoding IGF alone, a
composition containing IGF and IGFBP only, or all four
polynucleotides mixed together in a single composition.
[0056] The PDGF, KGF, IGF, and IGF with IGFBP compositions, if
separately maintained, can be administered separately or
contemporaneously. Additionally, the concentrations of each may be
different, depending on the potency of the factor, and the needs of
the patient. Direct delivery of the compositions will generally be
accomplished by injection, either subcutaneously, intradermally,
intraperitoneally, intraluminally, intragastrically,
intraintestinally, intravenously or intramuscularly. Other modes of
administration include oral and pulmonary administration,
suppositories, and transdermal applications. Dosage treatment may
be a single dose schedule or a multiple dose schedule.
[0057] Where PDGF, KGF, IGF or IGFBP are administered as
polypeptides, either together in a single composition, or in
multiple compositions, the polypeptides can be expressed by any
expression system appropriate for the polypeptide.
[0058] Exemplary expression systems are listed below for generating
the polypeptides of PDGF, KGF, IGF, and IGFBP or for cloning the
polynucleotides encoding the same for application in a gene therapy
protocol.
[0059] Expression in Bacterial Cells
[0060] Bacterial expression systems can be used with the present
constructs. Control elements for use in bacteria include promoters,
optionally containing operator sequences, and ribosome binding
sites. Useful promoters include sequences derived from sugar
metabolizing enzymes, such as galactose, lactose (lac) and maltose.
Additional examples include promoter sequences derived from
biosynthetic enzymes such as tryptophan (trp), the .beta.-lactamase
(bla) promoter system, bacteriophage .lambda.PL, and T7. In
addition, synthetic promoters can be used, such as the tac
promoter. The .beta.-lactamase and lactose promoter systems are
described in Chang et al., Nature (1978) 275: 615, and Goeddel et
al., Nature (1979) 281: 544; the alkaline phosphatase, tryptophan
(trp) promoter system are described in Goeddel et al., Nucleic
Acids Res. (1980) .delta.: 4057 and EP 36,776 and hybrid promoters
such as the tac promoter is described in U.S. Pat. No. 4,551,433
and deBoer et al., Proc. Natl. Acad. Sci. USA (1983) 80: 21-25.
However, other known bacterial promoters useful for expression of
eukaryotic proteins are also suitable. A person skilled in the art
would be able to operably ligate such promoters to the present PDGF
and KGF coding sequences, for example, as described in Siebenlist
et al., Cell (1980) 20: 269, using linkers or adaptors to supply
any required restriction sites. Promoters for use in bacterial
systems also generally will contain a Shine-Dalgano (SD) sequence
operably linked to the DNA encoding the target polypeptide. For
prokaryotic host cells that do not recognize and process the native
target polypeptide signal sequence, the signal sequence can be
substituted by a prokaryotic signal sequence selected, for example,
from the group of the alkaline phosphatase, penicillinase, Ipp, or
heat stable enterotoxin II leaders. The origin of replication from
the plasmid pBR322 is suitable for most Gram-negative bacteria.
[0061] The foregoing systems are particularly compatible with
Escherichia coli. However, numerous other systems for use in
bacterial hosts including Gram-negative or Gram-positive organisms
such as Bacillus spp., Streptococcus spp., Streptomyces spp.,
Pseudomonas species such as P. aeruginosa, Salmonella typhimurium,
or Serratia marcescans, among others. Methods for introducing
exogenous DNA into these hosts typically include the use of
CaCl.sub.2 or other agents, such as divalent cations and DMSO. DNA
can also be introduced into bacterial cells by electroporation,
nuclear injection, or protoplast fusion as described generally in
Sambrook et al. (1989), cited above These examples are illustrative
rather than limiting. Preferably, the host cell should secrete
minimal amounts of proteolytic enzymes. Alternatively, in vitro
methods of cloning, e.g., PCR or other nucleic acid polymerase
reactions, are suitable.
[0062] Also useful for expression of PDGF for the invention are
vectors described in EP 0 622 456-A1, herein incorporated by
reference, that disclose DNA for selection and autonomous
replication in bacterial cells.
[0063] Prokaryotic cells used to produce the target polypeptide of
this invention are cultured in suitable media, as described
generally in Sambrook et al., cited above.
[0064] Expression in Yeast Cells
[0065] Expression and transformation vectors, either
extrachromosomal replicons or integrating vectors, have been
developed for transformation into many yeasts. For example,
expression vectors have been developed for, among others, the
following yeasts: Saccharomyces cerevisiae, as described in Hinnen
et al., Proc. Natl. Acad. Sci. USA (1978) 75: 1929; Ito et al., J.
Bacteriol. (1983) 153: 163; Candida albicans as described in Kurtz
et al., Mol. Cell. Biol. (1986) .delta.: 142; Candida maltosa, as
described in Kunze et al., J. Basic Microbiol. (1985) 25: 141;
Hansenula polymorpha, as described in Gleeson et al., J. Gen.
Microbiol. (1986) 132: 3459 and Roggenkamp et al., Mol. Gen. Genet.
(1986) 202: 302); Kluyveromyces fragilis, as described in Das et
al., J. Bacteriol. (1984) 158:1165; Kluyveromyces lactis, as
described in De Louvencourt et al., J. Bacteriol. (1983) 154: 737
and Van den Berg et al., Bio/Technology (1990) .delta.: 135; Pichia
guillerimondii, as described in Kunze et al., J. Basic Microbiol.
(1985) 25: 141; Pichia pastoris, as described in Cregg et al., Mol.
Cell. Biol. (1985) 5: 3376 and U.S. Pat. Nos. 4,837,148 and
4,929,555; Schizosaccharomyces pombe, as described in Beach and
Nurse, Nature (1981) 300: 706; and Yarrowia lipolytica, as
described in Davidow et al., Curr. Genet. (1985) 10: 380 and
Gaillardin et al., Curr. Genet. (1985) 10: 49, Aspergillus hosts
such as A. nidulans, as described in Ballance et al., Biochem.
Biophys. Res. Commun. (1983) 112: 284-289; Tilburn et al., Gene
(1983) 26: 205-221 and Yelton et al., Proc. Natl. Acad. Sci. USA
(1984) 81: 1470-1474, and A. niger, as described in Kelly and
Hynes, EMBO J. (1985) 4: 475479; Trichoderma reesia, as described
in EP 0 244 234, and filamentous fungi such as, e.g, Neurospora,
Penicillium, Tolypocladium, as described in WO 91/00357.
[0066] Control sequences for yeast vectors are known and include
promoters regions from genes such as alcohol dehydrogenase (ADH),
as described in EP 284,044, enolase, glucokinase,
glucose-6-phosphate isomerase,
glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH),
hexokinase, phosphofructokinase, 3-phosphoglycerate mutase, and
pyruvate kinase (PyK), as described in EP 329,203. The yeast PHO5
gene, encoding acid phosphatase, also provides useful promoter
sequences, as described in Myanohara et al., Proc. Natl. Acad. Sci.
USA (1983) 80: 1. Other suitable promoter sequences for use with
yeast hosts include the promoters for 3-phosphoglycerate kinase, as
described in Hitzeman et al., J. Biol. Chem. (1980) 255: 2073, or
other glycolytic enzymes, such as pyruvate decarboxylase,
triosephosphate isomerase, and phosphoglucose isomerase, as
described in Hess et al., J. Adv. Enzyme Reg. (1968) 7: 149 and
Holland et al., Biochemistry (1978) 17: 4900. Inducible yeast
promoters having the additional advantage of transcription
controlled by growth conditions, include from the list above and
others the promoter regions for alcohol dehydrogenase 2,
isocytochrome C, acid phosphatase, degradative enzymes associated
with nitrogen metabolism, metallothionein,
glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible
for maltose and galactose utilization. Suitable vectors and
promoters for use in yeast expression are further described in
Hitzeman, EP 073,657. Yeast enhancers also are advantageously used
with yeast promoters. In addition, synthetic promoters which do not
occur in nature also function as yeast promoters. For example,
upstream activating sequences (UAS) of one yeast promoter may be
joined with the transcription activation region of another yeast
promoter, creating a synthetic hybrid promoter. Examples of such
hybrid promoters include the ADH regulatory sequence linked to the
GAP transcription activation region, as described in U.S. Pat. Nos.
4,876,197 and 4,880,734. Other examples of hybrid promoters include
promoters which consist of the regulatory sequences of either the
ADH2, GAL4, GAL10, or PHO5 genes, combined with the transcriptional
activation region of a glycolytic enzyme gene such as GAP or PyK,
as described in EP 164,556. Furthermore, a yeast promoter can
include naturally occurring promoters of non-yeast origin that have
the ability to bind yeast RNA polymerase and initiate
transcription.
[0067] Other control elements which may be included in the yeast
expression vectors are terminators, for example, from GAPDH and
from the enolase gene, as described in Holland et al., J. Biol.
Chem. (1981) 256: 1385, and leader sequences which encode signal
sequences for secretion. DNA encoding suitable signal sequences can
be derived from genes for secreted yeast proteins, such as the
yeast invertase gene as described in EP 012,873 and JP 62,096,086
and the a-factor gene, as described in U.S. Pat. Nos. 4,588,684,
4,546,083 and 4,870,008; EP 324,274; and WO 89/02463.
Alternatively, leaders of non-yeast origin, such as an interferon
leader, also provide for secretion in yeast, as described in EP
060,057.
[0068] Methods of introducing exogenous DNA into yeast hosts are
well known in the art, and typically include either the
transformation of spheroplasts or of intact yeast cells treated
with alkali cations. Transformations into yeast can be carried out
according to the method described in Van Solingen et al., J. Bact.
(1977) 130: 946 and Hsiao et al., Proc. Natl. Acad. Sci USA (1979)
76: 3829: However, other methods for introducing DNA into cells
such as by nuclear injection, electroporation, or protoplast fusion
may also be used as described generally in Sambrook et al., cited
above.
[0069] For yeast secretion the native target polypeptide signal
sequence may be substituted by the yeast invertase, .alpha.-factor,
or acid phosphatase leaders. The origin of replication from the
2.mu. plasmid origin is suitable for yeast. A suitable selection
gene for use in yeast is the trp1 gene present in the yeast plasmid
described in Kingsman et al., Gene (1979) 7: 141 or Tschemper et
al., Gene (1980) 10: 157. The trp1 gene provides a selection marker
for a mutant strain of yeast lacking the ability to grow in
tryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or
38,626) are complemented by known plasmids bearing the Leu2
Gene.
[0070] For intracellular production of the present polypeptides in
yeast, a sequence encoding a yeast protein can be linked to a
coding sequence of the PDGF or the KGF polypeptide to produce a
fusion protein that can be cleaved intracellularly by the yeast
cells upon expression. An example, of such a yeast leader sequence
is the yeast ubiquitin gene.
[0071] Expression in Insect Cells
[0072] Baculovirus expression vectors (BEVs) are recombinant insect
viruses in which the coding sequence for a foreign gene to be
expressed is inserted behind a baculovirus promoter in place of a
viral gene, e.g., polyhedrin, as described in Smith and Summers,
U.S. Pat. No. 4,745,051.
[0073] An expression construct herein includes a DNA vector useful
as an intermediate for the infection or transformation of an insect
cell system, the vector generally containing DNA coding for a
baculovirus transcriptional promoter, optionally but preferably,
followed downstream by an insect signal DNA sequence capable of
directing secretion of a desired protein, and a site for insertion
of the foreign gene encoding the foreign protein, the signal DNA
sequence and the foreign gene being placed under the
transcriptional control of a baculovirus promoter, the foreign gene
herein being the coding sequence of the PDGF or the KGF
polypeptide.
[0074] The promoter for use herein can be a baculovirus
transcriptional promoter region derived from any of the over 500
baculoviruses generally infecting insects, such as, for example,
the Orders Lepidoptera, Diptera, Orthoptera, Coleoptera and
Hymenoptera including, for example, but not limited to the viral
DNAs of Autographo californica MNPV, Bombyx mori NPV, rrichoplusia
ni MNPV, Rachlplusia ou MNPV or Galleria mellonella MNPV. Thus, the
baculovirus transcriptional promoter can be, for example, a
baculovirus immediate-early gene IEI or IEN promoter; an
immediate-early gene in combination with a baculovirus
delayed-early gene promoter region selected from the group
consisting of a 39K and a HindIII fragment containing a
delayed-early gene; or a baculovirus late gene promoter. The
immediate-early or delayed-early promoters can be enhanced with
transcriptional enhancer elements.
[0075] Particularly suitable for use herein is the strong
polyhedrin promoter of the baculovirus, which directs a high level
of expression of a DNA insert, as described in Friesen et al.
(1986) "The Regulation of Baculovirus Gene Expression" in: THE
MOLECULAR BIOLOGY OF BACULOVIRUSES (W. Doerfler, ed.); EP 127,839
and EP 155,476; and the promoter from the gene encoding the p10
protein, as described in Vlak et al., J. Gen. Virol. (1988) 69:
765-776.
[0076] The plasmid for use herein usually also contains the
polyhedrin polyadenylation signal, as described in Miller et al.,
Ann. Rev. Microbiol. (1988) 42: 177 and a procaryotic
ampicillin-resistance (amp) gene and an origin of replication for
selection and propagation in E. coli. DNA encoding suitable signal
sequences can also be included and is generally derived from genes
for secreted insect or baculovirus proteins, such as the
baculovirus polyhedrin gene, as described in Carbonell et al., Gene
(1988) 73: 409, as well as mammalian signal sequences such as those
derived from genes encoding human a-interferon as described in
Maeda et al., Nature (1985) 315: 592-594; human gastrin-releasing
peptide, as described in Lebacq-Verheyden et al., Mol. Cell. Biol.
(1988) 8: 3129; human IL-2, as described in Smith et al., Proc.
Natl. Acad. Sci. USA (1985) 82: 8404; mouse IL-3, as described in
Miyajima et al., Gene (1987) 58: 273, and human glucocerebrosidase,
as described in Martin et al., DNA (1988) 7:99.
[0077] Numerous baculoviral strains and variants and corresponding
permissive insect host cells from hosts such as Spodoptera
frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes
albopictus (mosquito), Drosophila melanogaster (fruitfly), and
Bombyx mori host cells have been identified and can be used herein.
See, for example, the description in Luckow et al., Bio/Technology
(1988) 6: 47-55, Miller et al., in GENETIC ENGINEERING (Setlow, J.
K. et al. eds.), Vol. 8 (Plenum Publishing, 1986), pp. 277-279, and
Maeda et al., Nature, (1985) 315: 592-594. A variety of such viral
strains are publicly available, e.g., the L-1 variant of Autographa
californica NPV and the Bm-5 strain of Bombyx mori NPV. Such
viruses may be used as the virus for transfection of host cells
such as Spodoptera frugiperda cells.
[0078] Other baculovirus genes in addition to the polyhedrin
promoter may be employed to advantage in a baculovirus expression
system. These include immediate-early (alpha), delayed-early
(beta), late (gamma), or very late (delta), according to the phase
of the viral infection during which they are expressed. The
expression of these genes occurs sequentially, probably as the
result of a "cascade" mechanism of transcriptional regulation.
Thus, the immediate-early genes are expressed immediately after
infection, in the absence of other viral functions, and one or more
of the resulting gene products induces transcription of the
delayed-early genes. Some delayed-early gene products, in turn,
induce transcription of late genes, and finally, the very late
genes are expressed under the control of previously expressed gene
products from one or more of the earlier classes. One relatively
well defined component of this regulatory cascade is IEI, a
preferred immediate-early gene of Autographo californica nuclear
polyhedrosis virus (AcMNPV). IEI is pressed in the absence of other
viral functions and encodes a product that stimulates the
transcription of several genes of the delayed-early class,
including the preferred 39K gene, as described in Guarino and
Summers, J. Virol. (1986) 57: 563-571 and J. Virol. (1987) 61:
2091-2099 as well as late genes, as described in Guanno and
Summers, Virol. (1988) 162: 444-451.
[0079] Immediate-early genes as described above can be used in
combination with a baculovirus gene promoter region of the
delayed-early category. Unlike the immediate-early genes, such
delayed-early genes require the presence of other viral genes or
gene products such as those of the immediate-early genes. The
combination of immediate-early genes can be made with any of
several delayed-early gene promoter regions such as 39K or one of
the delayed-early gene promoters found on the HindIII fragment of
the baculovirus genome. In the present instance, the 39 K promoter
region can be linked to the foreign gene to be expressed such that
expression can be further controlled by the presence of IEI, as
described in L. A. Guarino and Summers (1986a), cited above;
[0080] Guarino & Summers (1986b) J. Virol., (1986) 60: 215-223,
and Guarino et al. (1986c), J. Virol. (1986) 60: 224-229.
[0081] Additionally, when a combination of immediate-early genes
with a delayed-early gene promoter region is used, enhancement of
the expression of heterologous genes can be realized by the
presence of an enhancer sequence in direct cis linkage with the
delayed-early gene promoter region. Such enhancer sequences are
characterized by their enhancement of delayed-early gene expression
in situations where the immediate-early gene or its product is
limited. For example, the hr5 enhancer sequence can be linked
directly, in cis, to the delayed-early gene promoter region, 39K,
thereby enhancing the expression of the cloned heterologous DNA as
described in Guarino and Summers (1986a), (1986b), and Guarino et
al. (1986).
[0082] The polyhedrin gene is classified as a very late gene.
Therefore, transcription from the polyhedrin promoter requires the
previous expression of an unknown, but probably large number of
other viral and cellular gene products. Because of this delayed
expression of the polyhedrin promoter, state-of-the-art BEVs, such
as the exemplary BEV system described by Smith and Summers in, for
example, U.S. Pat. No. 4,745,051 will express foreign genes only as
a result of gene expression from the rest of the viral genome, and
only after the viral infection is well underway. This represents a
limitation to the use of existing BEVs. The ability of the host
cell to process newly synthesized proteins decreases as the
baculovirus infection progresses. Thus, gene expression from the
polyhedrin promoter occurs at a time when the host cell's ability
to process newly synthesized proteins is potentially diminished for
certain proteins such as human tissue plasminogen activator. As a
consequence, the expression of secretory glycoproteins in BEV
systems is complicated due to incomplete secretion of the cloned
gene product, thereby trapping the cloned gene product within the
cell in an incompletely processed form.
[0083] While it has been recognized that an insect signal sequence
can be used to express a foreign protein that can be cleaved to
produce a mature protein, the present invention is preferably
practiced with a mammalian signal sequence for example the PDGF or
the KGF or the IGF or the IGFBP signal sequence.
[0084] An exemplary insect signal sequence suitable herein is the
sequence encoding for a Lepidopteran adipokinetic hormone (AKH)
peptide. The AKH family consists of short blocked neuropeptides
that regulate energy substrate mobilization and metabolism in
insects. In a preferred embodiment, a DNA sequence coding for a
Lepidopteran Manduca sexta AKH signal peptide can be used. Other
insect AKH signal peptides, such as those from the Orthoptera
Schistocerca gregaria locus can also be employed to advantage.
Another exemplary insect signal sequence is the sequence coding for
Drosophila cuticle proteins such as CPI, CP2, CP3 or CP4.
[0085] Currently, the most commonly used transfer vector that can
be used herein for introducing foreign genes into AcNPV is pAc373.
Many other vectors, known to those of skill in the art, can also be
used herein. Materials and methods for baculovirus/insect cell
expression systems are commercially available in a kit form from
companies such as Invitrogen (San Diego Calif.) ("MaxBac" kit). The
techniques utilized herein are generally known to those skilled in
the art and are fully described in Summers and Smith, A MANUAL OF
METHODS FOR BACULOVIRUS VECTORS AND INSECT CELL CULTURE PROCEDURES,
Texas Agricultural Experiment Station Bulletin No. 1555, Texas
A&M University (1987); Smith et al., Mol. Cell. Biol. (1983) 3:
2156, and Luckow and Summers (1989). These include, for example,
the use of pVL985 which alters the polyhedrin start codon from ATG
to ATT, and which introduces a BamHI cloning site 32 basepairs
downstream from the ATT, as described in Luckow and Summers,
Virology (1989) 17:31.
[0086] Thus, for example, for insect cell expression of the present
polypeptides, the desired DNA sequence can be inserted into the
transfer vector, using known techniques. An insect cell host can be
cotransformed with the transfer vector containing the inserted
desired DNA together with the genomic DNA of wild type baculovirus,
usually by cotransfection. The vector and viral genome are allowed
to recombine resulting in a recombinant virus that can be easily
identified and purified. The packaged recombinant virus can be used
to infect insect host cells to express the PDGF and KGF
polypeptides.
[0087] Other methods that are applicable herein are the standard
methods of insect cell culture, cotransfection and preparation of
plasmids are set forth in Summers and Smith (1987), cited above.
This reference also pertains to the standard methods of cloning
genes into AcMNPV transfer vectors, plasmid DNA isolation,
transferring genes into the AcmMNPV genome, viral DNA purification,
radiolabeling recombinant proteins and preparation of insect cell
culture media.
[0088] The procedure for the cultivation of viruses and cells are
described in Volkman and Summers, J. Virol. (1975) 19: 820-832 and
Volkman, al., J. Virol. (1976) 19: 820-832.
[0089] Expression in Mammalian Cells
[0090] Typical promoters for mammalian cell expression include the
SV40 early promoter, the CMV promoter, the mouse mammary tumor
virus LTR promoter, the adenovirus major late promoter (Ad MLP),
and the herpes simplex virus promoter, among others. Other
non-viral promoters, such as a promoter derived from the murine
metallothionein gene, will also find use in mammalian constructs.
Mammalian expression may be either constitutive or regulated
(inducible), depending on the promoter. Typically, transcription
termination and polyadenylation sequences will also be present,
located 3' to the translation stop codon. Preferably, a sequence
for optimization of initiation of translation, located 5' to the
PDGF or the KGF polypeptide coding sequence, is also present.
Examples of transcription terminator/polyadenylation signals
include those derived from SV40, as described in Sambrook et al.
(1989), cited previously. Introns, containing splice donor and
acceptor sites, may also be designed into the constructs of the
present invention.
[0091] Enhancer elements can also be used herein to increase
expression levels of the mammalian constructs. Examples include the
SV40 early gene enhancer, as described in Dijkema et al., EMBO J.
(1985) 4: 761 and the enhancer/promoter derived from the long
terminal repeat (LTR) of the Rous Sarcoma Virus, as described in
Gorman et al., Proc. Natl. Acad. Sci. USA (1982b) 79: 6777 and
human cytomegalovirus, as described in Boshart et al., Cell (1985)
41: 521. A leader sequence can also be present which includes a
sequence encoding a signal peptide, to provide for the secretion of
the foreign protein in mammalian cells. Preferably, there are
processing sites encoded between the leader fragment and the gene
of interest such that the leader sequence can be cleaved either in
vivo or in vitro. The adenovirus tripartite leader is an example of
a leader sequence that provides for secretion of a foreign protein
in mammalian cells.
[0092] There exist expression vectors that provide for the
transient expression in mammalian cells of DNA encoding the target
polypeptide. In general, transient expression involves the use of
an expression vector that is able to replicate efficiently in a
host cell, such that the host cell accumulates many copies of the
expression vector and, in turn, synthesizes high levels of a
desired polypeptide encoded by the expression vector. Transient
expression systems, comprising a suitable expression vector and a
host cell, allow for the convenient positive identification of
polypeptides encoded by cloned DNAs, as well as for the rapid
screening of such polypeptides for desired biological or
physiological properties. Thus, transient expression systems are
particularly useful for purposes of identifying analogs and
variants of the target polypeptide that have target
polypeptide-like activity.
[0093] The expression vector as disclosed in EP 0622 456 A1 for
expression of PDGF B chain is also useful for the invention for
expression PDGF by a viral promoter functional in mammalian
cells.
[0094] Once complete, the mammalian expression vectors can be used
to transform any of several mammalian cells. Methods for
introduction of heterologous polynucleotides into mammalian cells
are known in the art and include dextran-mediated transfection,
calcium phosphate precipitation, polybrene mediated transfection,
protoplast fusion, electroporation, encapsulation of the
polynucleotide(s) in liposomes, and direct microinjection of the
DNA into nuclei. General aspects of mammalian cell host system
transformations have been described by Axel in U.S. Pat. No.
4,399,216.
[0095] Mammalian cell lines available as hosts for expression are
also known and include many immortalized cell lines available from
the American Type Culture Collection (ATCC), including but not
limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby
hamster kidney (BHK) cells, monkey kidney cells (COS), human
hepatocellular carcinoma cells (e.g., Hep G2), human embryonic
kidney cells, baby hamster kidney cells, mouse sertoli cells,
canine kidney cells, buffalo rat liver cells, human lung cells,
human liver cells, mouse mammary tumor cells, as well as
others.
[0096] The mammalian host cells used to produce the target
polypeptide of this invention may be cultured in a variety of
media. Commercially available media such as Ham's F10 (Sigma),
Minimal Essential Medium ([MEM], Sigma), RPMI-1640 (Sigma), and
Dulbecco's Modified Eagle's Medium ([DMEMI, Sigma) are suitable for
culturing the host cells. In addition, any of the media described
in Ham and Wallace, Meth. Enz. (1979) 58: 44, Barnes and Sato,
Anal. Biochem. (1980) 102: 255, U.S. Pat. Nos. 4,767,704,
4,657,866,4,927,762, or 4,560,655, WO 90/103430, WO 87/00195, and
U.S. RE 30,985, may be used as culture media for the host cells.
Any of these media may be supplemented as necessary with hormones
and/or other growth factors such as insulin, transferrin, or
epidermal growth factor, salts (such as sodium chloride, calcium,
magnesium, and phosphate), buffers (such as HEPES), nucleosides
(such as adenosine and thymidine), antibiotics (such as
Gentamycin(tm) M drug), trace elements (defined as inorganic
compounds usually present at final concentrations in the micromolar
range), and glucose or an equivalent energy source. Any other
necessary supplements may also be included at appropriate
concentrations that would be known to those skilled in the art. The
culture conditions, such as temperature, pH, and the like, are
those previously used with the host cell selected for expression,
and will be apparent to the ordinarily skilled artisan.
[0097] Gene Therapy Administration of PDGF and KGF or PDGF, KGF and
IGF or PDGF, KGF, IGF and IGFBP
[0098] For gene therapy purposes, the polynucleotides encoding PDGF
and KGF, or PDGF, KGF and IGF, or PDGF, KGF, IGF, and IGFBP can be
administered to the animal for expresssion. Further, the
polynucleotides can be administered either as naked polynucleotide,
or can be linked with other polynucleotides for the purposes of
delivery, such as viral vectors. Such polynucleotides can also be
encapsulated in liposomes or other delivery means as conventional
in the art. Examples of the uses of gene therapy protocols for
administration of KGF, PDGF, IGF, and IGFBP are detailed below.
[0099] Where gene therapy techniques are applied to the invention,
KGF and PDGF can be expressed by different vectors or by the same
vector; also KGF, PDGF and IGF can be expressed by different
vectors or by the same vector, also KGF, PDGF, IGF and IGFBP can be
expressed by different vectors or by the same vector. The
regulatory control of each gene product is distinct and a person
skilled in the art of gene therapy and expression can select the
appropriate regulatory sequences for both KGF and PDGF, or where
IGF is also administered, regulatory sequences appropriate for KGF,
PDGF and IGF, and where IGFBP is also administered, regulatory
sequences also appropriate for IGFBP.
[0100] Alternatively, vectors comprising polynucleotide sequences
encoding the KGF and PDGF polypeptides, and also IGF polypeptide
where IGF is also administered, and also IGFBP where IGFBP is also
administered, can be used directly for gene therapy and
administered using standard gene delivery protocols. In this
regard, the nucleotide sequences encoding the KGF and PDGF
polypeptides, and also IGF where IGF is also administered, and
IGFBP where IGFBP is also administered, can be stably integrated
into the host cell genome or maintained on a stable episomal
element in the host cell. Methods for gene delivery are known in
the art, as described in U.S. Pat. No. 5,399,346.
[0101] Gene therapy strategies for delivery of constructs of the
invention can utilize viral or non-viral vector approaches in in
vivo or ex vivo modality. Expression of such coding sequence can be
induced using endogenous mammalian or heterologous promoters.
Expression of the coding sequence in vivo can be either
constitutive or regulated.
[0102] For delivery using viral vectors, any of a number of viral
vectors can be used, as described in Jolly, Cancer Gene Therapy 1:
51-64 (1994). For example, the coding sequence can be inserted into
plasmids designed for expression in retroviral vectors, as
described in Kimura et al., Human Gene Therapy (1994) 5: 845-852,
adenoviral vectors, as described in Connelly et al., Human Gene
Therapy (1995) .delta.: 185-193, adeno-associated viral vectors, as
described in Kaplitt et al., Nature Genetics (1994) .delta.:
148-153 and sindbis vectors. Promoters that are suitable for use
with these vectors include the Moloney retroviral LTR, CMV promoter
and the mouse albumin promoter. Replication incompetent free virus
can be produced and injected directly into the animal or humans or
by transduction of an autologous cell ex vivo, followed by
injection in vivo as described in Zatloukal et al., Proc. Natl.
Acad. Sci. USA (1994) 91: 5148-5152.
[0103] The altered coding sequence can also be inserted into
plasmid for expression of the polypeptide in vivo or ex vivo. For
in vivo therapy, the coding sequence can be delivered by direct
injection into tissue or by intravenous infusion. Promoters
suitable for use in this manner include endogenous and heterologous
promoters such as CMV. Further, a synthetic T7T7/T7OB promoter can
be constructed in accordance with Chen et al. (1994), Nucleic Acids
Res. 22: 2114-2120, where the T7 polymerase is under the regulatory
control of its own promoter and drives the transcription of the
coding sequence, which is also placed under the control of a T7
promoter. The coding sequence can be injected in a formulation
comprising a buffer that can stablize the coding sequence and
facilitate transduction thereof into cells and/or provide
targeting, as described in Zhu et al., Science (1993) 261:
209-211.
[0104] Expression of the coding sequence in vivo upon delivery for
gene therapy purposes by either viral or non-viral vectors can be
regulated for maximal efficacy and safety by use of regulated gene
expression promoters as described in Gossen et al., Proc. Natl.
Acad. Sci. USA (1992) 89:5547-5551. For example, the coding
sequence can be regulated by tetracycline responsive promoters.
These promoters can be regulated in a positive or negative fashion
by treatment with the regulator molecule.
[0105] For non-viral delivery of the coding sequence, the sequence
can be inserted into conventional vectors that contain conventional
control sequences for high level expression, and then be incubated
with synthetic gene transfer molecules such as polymeric
DNA-binding cations like polylysine, protamine, and albumin, linked
to cell targeting ligands such as asialoorosomucoid, as described
in Wu and Wu, J. Biol. Chem. (1987) 262: 4429-4432; insulin, as
described in Hucked et al., Biochem. Pharmacol. 40: 253-263 (1990);
galactose, as described in Plank et al., Bioconjugate Chem.
3:533-539 (1992); lactose, as described in Midoux et al., Nucleic
Acids Res. 21: 871-878 (1993); or transferrin, as described in
Wagner et al., Proc. Natl. Acad. Sci. USA 87:3410-3414 (1990).
Other delivery systems include the use of liposomes to encapsulate
DNA comprising the gene under the control of a variety of
tissue-specific or ubiquitously-active promoters, as described in
Nabel et al., Proc. Natl. Acad. Sci. USA 90: 11307-11311 (1993),
and Philip et al., Mol. Cell Biol. 14: 2411-2418 (1994). Further
non-viral delivery suitable for use includes mechanical delivery
systems such as the biolistic approach, as described in Woffendin
et al., Proc. Natl. Acad. Sci. USA (1994) 91(24): 11581-11585.
Moreover, the coding sequence and the product of expression of such
can be delivered through deposition of photopolymerized hydrogel
materials. Other conventional methods for gene delivery that can be
used for delivery of the coding sequence include, for example, use
of hand held gene transfer particle gun, as described in U.S. Pat.
No. 5,149,655; use of ionizing radiation for activating transferred
gene, as described in U.S. Pat. No. 5,206,152 and PCT application
WO 92/11033.
[0106] Application of gene therapy technology with regard to the
peptides and polypeptides of the invention and their analogues or
variants can be made where it is beneficial to treat a wound by
gene therapy, for example, with chronic wounds such as ulcers, or
with any wound in which the cells at the site of the wound are
responsive to a gene therapy protocol.
[0107] In general, gene therapy can be applied according to the
invention in all situations where it is beneficial to heal a wound
by administering according to a gene therapy protocol, of a
sufficient amount of a peptide of the invention or its analogue,
variant, or dominant negative, for example, for modulating the
normal activity of binding pair interactions. Subsequent
administration may be required depending on the condition of the
wound and the environment with which it is presented.
[0108] Vectors encoding the KGF, PDGF, IGF, and IGFBP polypeptides
can also be packaged in liposomes prior to delivery to the subject
or to cells derived therefrom. Lipid encapsulation is generally
accomplished using liposomes which are able to stably bind or
entrap and retain nucleic acid. The ratio of condensed DNA to lipid
preparation can vary but will generally be around 1:1 (mg
DNA:micromoles lipid), or more of lipid. For a review of the use of
liposomes as carriers for delivery of nucleic acids, see, Hug and
Sleight, Biochim. Biophys. Acta. 1097:1-17 (1991); Straubinger et
al. Methods of Enzymology, 101: 512-527 (1983).
[0109] Liposomal preparations for use in the instant invention
include cationic (positively charged), anionic (negatively charged)
and neutral preparations, with cationic liposomes particularly
preferred. Cationic liposomes have been shown to mediate
intracellular delivery of plasmid DNA (Felgner et al. Proc. Natl.
Acad. Sci. USA (1987) 84:7413-7416); mRNA (Malone et al. Proc.
Natl. Acad. Sci. USA (1989) 86:6077-6081); and purified
transcription factors (Debs et al. J. Biol. Chem. (1990)
265:10189-10192), in functional form.
[0110] Cationic liposomes are readily available. For example,
N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes
are available under the trademark Lipofectin, from GIBCO BRL, Grand
Island, N.Y. (See, also, Felgner et al. Proc. Natl. Acad. Sci. USA
(1987) 84:7413-7416). Other commercially available liposomes
include transfectace (DDAB/DOPE) and DOTAP/DOPE (Boerhinger). Other
cationic liposomes can be prepared from readily available materials
using techniques well known in the art. See, e.g., Szoka et al.
Proc. Natl. Acad. Sci. USA (1978) 75:4194-4198; PCT Publication No.
WO 90/11092 for a description of the synthesis of DOTAP
(1,2-bis(oleoyloxy)-3-(trimethylamm- onio)propane) liposomes.
[0111] Similarly, anionic and neutral liposomes are readily
available, such as from Avanti Polar Lipids (Birmingham, Ala.), or
can be easily prepared using readily available materials. Such
materials include phosphatidyl choline, cholesterol, phosphatidyl
ethanolamine, dioleoylphosphatidyl choline (DOPC),
dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl
ethanolamine (DOPE), among others. These materials can also be
mixed with the DOTMA and DOTAP starting materials in appropriate
ratios. Methods for making liposomes using these materials are well
known in the art.
[0112] The liposomes can comprise multilammelar vesicles (MLVs),
small unilamellar vesicles (SUVs), or large unilamellar vesicles
(LUVs). The various liposome-nucleic acid complexes are prepared
using methods known in the art. See, e.g., Straubinger et al. in
Methods of immunology (1983), Vol. 101, pp. 512-527; Szoka et al.
Proc. Natl. Acad. Sci. USA (1978) 75:4194-4198; Papahadjopoulos et
al. Biochim. Biophys. Acta (1975) 394:483; Wilson et al. Cell
(1979) 17:77); Deamer and Bangham Biochim. Biophys. Acta (1976)
443:629; Ostro et al. Biochem. Biophys. Res. Commun. (1977) 76:836;
Fraley et al. Proc. Natl. Acad. Sci. USA (1979) 76:3348); Enoch and
Strittmatter Proc. Natl. Acad. Sci. USA (1979) 76:145); Fraley et
al. J. Biol. Chem. (1980) 255:10431; Szoka and Papahadjopoulos
Proc. Natl. Acad. Sci. USA (1978) 75:145; and Schaefer-Ridder et
al. Science (1982) 215:166.
[0113] Liposomes are included within the definition of a
pharmaceutically acceptable carrier. The term "liposomes" refers
to, for example, the liposome compositions described in U.S. Pat.
No. 5,422,120, WO 95/13796, WO 94/23697, WO 91/14445 and EP 524,968
B1. Liposomes may be pharmaceutical carriers for the
polynucleotides or polypeptides of the invention, or for
combination of the two. The therapeutic agent may be conjugated to
a liposome, or may be conjugated to a hydrogel polymer, and the
hydrogel polymer (or a component of a hydrogel polymer) conjugated
or encapsulated by a liposome.
[0114] The recombinant vectors (whether or not encapsulated in
liposomes), may be administered in pharmaceutical compositions as
described above. The pharmaceutical compositions will comprise
sufficient genetic material to produce a therapeutically effective
amount of the analog or analogs, as described above. For purposes
of the present invention, an effective dose will be from about 0.05
mg/kg to about 50 mg/kg of the DNA constructs in the individual to
which it is administered.
[0115] Once formulated, the compositions of the invention can be
administered directly to the subject or, alternatively, in the case
of the vectors described above, delivered ex vivo, to cells derived
from the subject. Methods for the ex vivo delivery and
reimplantation of transformed cells into a subject are known in the
art and described in, for example, WO 93/14778. Generally, such
methods will include dextran-mediated transfection, calcium
phosphate precipitation, polybrene mediated transfection,
protoplast fusion, electroporation, encapsulation of the
polynucleotide(s) in liposomes, and direct microinjection of the
DNA into nuclei, all well known in the art.
[0116] Administration of the therapeutic combinations of the
invention can be accomplished by, for example, topical cream, foam,
injection, aerosol spray, in a gel matrix, a sponge, drops, and a
wash. Administration can be by, for example, local, oral,
intradermal, subcutaneous, intraluminal, intragastric, and
intraperitoneal administration with an appropriate formulation of
the selected composition made up of a combination of the
therapeutics appropriate for a particular treatment.
[0117] The therapeutics of the invention can be administered in a
therapeutically effective dosage and amount, in the process of a
therapeutically effective protocol for treatment of the patient.
The initial and any subsequent dosages administered will depend
upon the patient's age, weight, condition, and the disease, wound,
disorder or biological condition being treated. Depending on the
therapeutic, the dosage and protocol for administration will vary,
and the dosage will also depend on the method of administration
selected, for example, local or systemic administration.
[0118] The wound to which the therapeutic combinations are applied
can be internal or external, and may be directed towards any tissue
exhibiting a wound, for example epithelial tissue. For topical
administration of IGF, a zinc oxide formulation can be applied,
which induces the local production of IGF, as described in Tarnow
et al, Scand J. Plast Reconstr Hand Surg. 28: 255-259 (1994).
Administration of the therapeutic combinations of the invention can
be accomplished with any combination of the therapeutics, for
example, by administering PDGF and KGF followed by IGF with IGFBP,
or by administering PDGF, KGF, IGF, and IGFBP at the same time or
in close proximity in time. The dosages of each therapeutic for a
given wound and a particular patient, are designed to achieve a
maximum effective dose for the therapeutic. The dosages appropriate
for a given treatment may depend on the particular combination of
therapeutics selected for treatment. For example, IGF alone has
been shown to be less potent than IGF administered in conjunction
with IGFBP.
[0119] The doses for a particular wound will be determined on a
patient by patient basis, and depend on the size of the wound, the
type of injury, and the composition that is applied. Doses for the
individual therapeutics have been determined within ranges. For
example, an effective dose of PDGF has been determined to be 5
ng/mm.sup.2 or higher when applied topically as described in U.S.
Pat. No. 4,861,757, and at least 1 ng/ml local concentration of an
isoform of PDGF (for example, PDGF-AA, PDGF-BB, or PDGF-AB)., up to
about 30 ng/ml local concentration applied to a population of
fibrobtasts as described in Lepisto et al, Biochem Biophys Res.
Comm. 209: 393-399 (1995). PDGF can be administered in a
carboxymethylcellulose gel formulation at concentrations of about
10 .mu.g/gm to about 500 .mu.g/gm of gel, about 20 .mu.g/gm to
about 200 .mu.g/gm, and about 30 .mu.g/gm to about 100 .mu.g/gm of
gel, optimally about 100 .mu.g/gm of gel. Efficacy of PDGF has been
achieved within the range of about 3 .mu.g/ml solution to about 300
.mu.g/ml of solution administered.
[0120] About 50 ul of KGF of a concentration of about 5 ug/ml is
effective for wound healing by topical application to epithelial
tissue as described in Sotozono et al, Invest. Opthal. Vis. Science
36: 1524-29 (1995). As described in U.S. Pat. No. 4,861,757, an
effective amount of IGF when co-administered with PDGF is in the
range of at least 2.5 ng/mm.sup.2 to about 5 ng/mm.sup.2, with a
ratio of PDGF to IGF in the range of about 1:10 to about 25:1
weight to weight, with the most effective ratios being PDGF to IGF
of about 1:1 to about 2:1 weight to weight. IGFBP administered in
combination with IGF has been shown to increase wound healing at
dose levels of about 5 ug of IGF with about 1.5 ug of
phosphorylated IGFBP in a molar ration of about 11:1 IGF:IGFBP, as
described in Jyung et al, Surgery 115:233-239 (1994).
[0121] For administration of polypeptide therapeutics, for example,
a PDGF, KGF, IGF and IGFBP polypeptides, the dosage can be in the
range of about 5 .mu.g to about 50 .mu.g/kg of tissue to which the
application is directed, also about 50 .mu.g to about 5 mg/kg, also
about 100 .mu.g to about 500 .mu.g/kg of tissue, and about 200 to
about 250 ug/kg. For polynucleotide therapeutics, for example in a
gene therapy administration protocol, depending on the expression
strength the polynucleotide in the patient, for tissue targeted
administration, vectors containing expressible constructs including
PDGF, KGF, IGF, and IGFBP coding sequences can be administered in a
range of about 100 ng to about 200 mg of DNA for local
administration in a gene therapy protocol, also about 500 ng to
about 50 mg, also about 1 ug to about 2 mg of DNA, about 5 ug of
DNA to about 500 ug of DNA, and about 20 ug to about 100 ug during
a local administration in a gene therapy protocol, and about 250
ug, per injection or administration. Factors such as method of
action and efficacy of transformation and expression are therefore
considerations that will effect the dosage required for ultimate
efficacy for administration of DNA therapeutics. Where greater
expression is desired, over a larger area of tissue, larger amounts
of DNA or the same amounts readministered in a successive protocol
of administrations, or several administrations to different
adjacent or close tissue portions of for example, a wound site may
be required to effect a positive therapeutic outcome. In all cases,
routine experimentation in clinical trials will determine specific
ranges for optimal therapeutic effect, for each therapeutic, each
administrative protocol, and administration to specific patients
will also be adjusted to within effective and safe ranges depending
on the patient condition and responsiveness to initial
administrations.
[0122] Further objects, features, and advantages of the present
invention will become apparent from the detailed description. It
should be understood, however, that the detailed description, while
indicating preferred embodiments of the invention, is given by way
of illustration only, since various changes and modifications
within the spirit and scope of the invention will become apparent
to those skilled in the art from this detailed description. The
following examples are exemplary only, and are not intended to
limit the invention.
[0123] The present invention will now be illustrated by reference
to the following examples which set forth particularly advantageous
embodiments. However, it should be noted that these embodiments are
illustrative and are not to be construed as restricting the
invention in any way.
EXAMPLE 1
[0124] Second degree burns are treated with a spray formulation
including 10 ug/ml of KGF, 30 ng/ml of a PDGF isoform, 10 ng/ml
IGF-1, and 30 ng/ml of IGFBP-1. The spray is allowed to dry in the
air. Re-application is suggested every couple of hours.
EXAMPLE 2
[0125] A suture wound is closed and a topical salve made up of 10%
KGF and 5% PDGF is applied on the suture before bandaging.
Re-application of the salve is directed 3 times daily.
EXAMPLE 3
[0126] A 20% zinc oxide formulation containing also 5% KGF, 2.5%
PDGF, and 10% IGFBP is applied to minor abrasions, sunburns and
chafing for faster healing of these wounds.
EXAMPLE 4
[0127] A liposomal formulation is used to encapsulate KGF, PDGF,
IGF and IGFBP DNA each in a vector for expression. The ration of
DNA by weight is 10:5:2.5:7.5, respectively. The liposomal
composition is administered in a gel capsule by mouth for treatment
of gastrointestinal ulcers with re-administration daily for a
period of about a month, or until significant improvement dictates
reduced frequency of administration.
* * * * *