U.S. patent application number 09/808751 was filed with the patent office on 2002-05-23 for combined treatment with keratinocyte growth factor and epidermal growth factor inhibitor.
This patent application is currently assigned to PFIZER PRODUCTS INC. & OSI PHARMACEUTICALS, INC.. Invention is credited to Miller, Penelope Elizabeth, Moyer, James Dale.
Application Number | 20020061304 09/808751 |
Document ID | / |
Family ID | 22702391 |
Filed Date | 2002-05-23 |
United States Patent
Application |
20020061304 |
Kind Code |
A1 |
Miller, Penelope Elizabeth ;
et al. |
May 23, 2002 |
Combined treatment with keratinocyte growth factor and epidermal
growth factor inhibitor
Abstract
The present invention relates to compositions and methods for
treating the epithelial toxicity caused by administering to a human
cancer patient an epidermal growth factor receptor (EGFR)
inhibitor. The pharmaceutical composition preferably comprises an
EGFR inhibitor and a keratinocyte growth factor (KGF) in a
pharmaceutically-acceptable carrier. The method of treatment
comprises co-administering to the patient a therapeutically
effective amount of KGF with the EGFR inhibitor.
Inventors: |
Miller, Penelope Elizabeth;
(Mystic, CT) ; Moyer, James Dale; (East Lyme,
CT) |
Correspondence
Address: |
LADAS & PARRY
26 WEST 61ST STREET
NEW YORK
NY
10023
US
|
Assignee: |
PFIZER PRODUCTS INC. & OSI
PHARMACEUTICALS, INC.
|
Family ID: |
22702391 |
Appl. No.: |
09/808751 |
Filed: |
March 15, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60190697 |
Mar 20, 2000 |
|
|
|
Current U.S.
Class: |
424/143.1 ;
514/262.1; 514/264.1; 514/265.1; 514/266.4 |
Current CPC
Class: |
A61P 1/12 20180101; A61P
17/00 20180101; A61P 43/00 20180101; C07K 16/2863 20130101; A61P
17/14 20180101; A61K 39/395 20130101; A61K 38/1825 20130101; A61K
2039/505 20130101; A61K 31/517 20130101; A61K 45/06 20130101; A61P
27/02 20180101; A61P 35/00 20180101; A61K 39/395 20130101; A61K
31/535 20130101; A61K 39/395 20130101; A61K 31/505 20130101; A61K
39/395 20130101; A61K 31/00 20130101; A61K 31/517 20130101; A61K
2300/00 20130101; A61K 38/1825 20130101; A61K 2300/00 20130101;
A61K 39/395 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/143.1 ;
514/266.4; 514/264.1; 514/262.1; 514/265.1 |
International
Class: |
A61K 039/395; A61K
031/517; A61K 031/519 |
Claims
1. A pharmaceutical composition comprising an epidermal growth
factor receptor (EGFR) inhibitor and a keratinocyte growth factor
(KGF) in a pharmaceutically acceptable carrier.
2. The pharmaceutical composition of claim 1, wherein the EGFR
inhibitor is a small organic molecule, an antibody or an antibody
fragment that binds specifically to the EGFR.
3. The pharmaceutical composition of claim 1, wherein the EGFR
inhibitor is selected from the group consisting of quinazoline EGFR
inhibitors, pyrido-pyrimidine EGFR inhibitors, pyrimido-pyrimidine
EGFR inhibitors, pyrrolo-pyrimidine EGFR inhibitors,
pyrazolopyrimidine EGFR inhibitors, phenylamino-pyrimidine EGFR
inhibitors, oxindole EGFR inhibitors, indolocarbazole EGFR
inhibitors, phthalazine EGFR inhibitors, isoflavone EGFR
inhibitors, quinalone EGFR inhibitors, and tyrphostin EGFR
inhibitors.
4. The pharmaceutical composition of claim 1, wherein the EGFR
inhibitor is selected from the group consisting of
[6,7-bis(2-methoxyethoxy)-4-quin-
ozolin-4-yl]-(3-ethynylphenyl)amine, ZD1839 (Iressa) and
PC183805.
5. The pharmaceutical composition of claim 1, wherein the EGFR
inhibitor is a monoclonal antibody or an antibody fragment.
6. The pharmaceutical composition of claim 1, wherein the EGFR
inhibitor is monoclonal antibody Mab E7.6.3, or Mab C225, or an
antibody or antibody fragment having the binding specificity
thereof.
7. The pharmaceutical composition of claim 1, wherein the KGF is
human KGF-1, or an analog thereof having at least partial human
KGF-1 activity.
8. The pharmaceutical composition of claim 1, wherein the KGF is
human KGF-2, or an analog thereof having at least partial human
KGF-2 activity.
9. A method of treating the epithelial toxicity resulting from
administration to a patient of an EGFR inhibitor, comprising
co-administering to the patient a therapeutically effective amount
of KGF with the EGFR inhibitor.
10. The method of claim 9, wherein the patient is a human that is
being treated for cancer.
11. The method of claim 9, wherein the epithelial toxicity is a
skin toxicity.
12. The method of claim 11, wherein the skin toxicity is manifested
as a rash.
13. The method of claim 9, wherein the epithelial toxicity is
manifested as corneal thinning.
14. The method of claim 9, wherein the epithelial toxicity is
manifested as diarrhea.
15. The method of claim 9, wherein the EGFR inhibitor and KGF are
co-administered to the patient in the same formulation.
16. The method of claim 9, wherein the EGFR inhibitor and KGF are
co-administered to the patient in different formulations.
17. The method of claim 9, wherein the EGFR inhibitor and KGF are
co-administered to the patient by the same route.
18. The method of claim 9, wherein the EGFR inhibitor and KGF are
co-administered to the patient by different routes.
19. The method of claim 9, wherein the EGFR inhibitor is
administered to the patient by parenteral or oral
administration.
20. The method of claim 9, wherein KGF is administered to the
patient by parenteral or topical administration.
21. The method of claim 9, wherein the EGFR inhibitor is a small
organic molecule, an antibody or an antibody fragment that binds
specifically to the EGFR.
22. The method of claim 9, wherein the EGFR inhibitor is selected
from the group consisting of quinazoline EGFR inhibitors,
pyrido-pyrimidine EGFR inhibitors, pyrimidopyrimidine EGFR
inhibitors, pyrrolo-pyrimidine EGFR inhibitors, pyrazolo-pyrimidine
EGFR inhibitors, phenylamino-pyrimidine EGFR inhibitors, oxindole
EGFR inhibitors, indolocarbazole EGFR inhibitors, phthalazine EGFR
inhibitors, isoflavone EGFR inhibitors, quinalone EGFR inhibitors,
and tyrphostin EGFR inhibitors.
23. The method of claim 9, wherein the EGFR inhibitor is selected
from the group consisting of
[6,7-bis(2-methoxyethoxy)-4-quinozolin-4-yl]-(3-ethyn-
ylphenyl)amine, ZD1839 (Iressa) and PC183805.
24. The method of claim 9, wherein the EGFR inhibitor is a
monoclonal antibody or an antibody fragment.
25. The method of claim 9, wherein the KGF is human KGF-1, or an
analog thereof having at least partial human KGF-1 activity.
26. The method of claim 9, wherein the KGF is human KGF-2, or an
analog thereof having at least partial human KGF-2 activity.
27. A method of preparing a pharmaceutical composition useful for
treating the epithelial toxicity resulting from administration to a
patient of an EGFR inhibitor, comprising combining a KGF with the
EGFR inhibitor.
28. The method of claim 27, further comprising combining a
pharmaceutically acceptable carrier with the KGF and EGFR
inhibitor.
29. A kit comprising a container comprising an EGFR inhibitor and
KGF.
30. The kit of claim 29, further comprising a sterile diluent.
31. The kit of claim 29, further comprising a package insert
comprising printed instructions directing the use of a combined
treatment of the KGF and EGFR inhibitor to a patient as a method
for treating the epithelial toxicity otherwise resulting from
administration to the patient of the EGFR inhibitor alone.
Description
FIELD OF INVENTION
[0001] The present invention relates to compositions and methods
for treating the epithelial toxicity resulting from the treatment
of cancer patients with EGFR inhibitors. More particularly, the
present invention relates to pharmaceutical compositions comprising
an epidermal growth factor receptor (EGFR) inhibitor in combination
with a keratinocyte growth factor (KGF), and to methods for
treating the epithelial toxicity of EGFR inhibitors administered to
cancer patients, comprising co-administering KGF to said
patients.
BACKGROUND OF THE INVENTION
[0002] Over-expression of the epidermal growth factor receptor
(EGFR), or its ligand TGF.alpha., is frequently associated with
breast, lung and head and neck cancer, and is believed to
contribute to the malignant growth of these tumors. The development
of compounds that inhibit the kinase activity of the EGFR, as well
as antibodies that block EGFR activation, for use as anti-tumor
agents is an area of intense research effort.
[0003] Epidermal growth factor (EGF), acting through its receptor
EGFR, is a mitogen and survival factor for normal human
keratinocytes as well as other epithelial cells (Rheinwald, J. G.
and Green, H., 1977, Nature 265, 421; Rodeck, U. et al., 1997, J.
Cell Science 110, 113). Thus, there is the potential that use of
EGFR inhibitors in chemotherapy would interfere with the normal
renewal of skin and other epithelial tissues such as the cornea and
the lining of the gastrointestinal tract: Toxicity to proliferating
tissues such as skin and the GI tract is frequently a dose-limiting
side effect of cytotoxic agents. Such toxicity may be manifested,
among other symptoms, as a skin rash, diarrhea, corneal thinning,
hair atrophy or loss, hair follicle dysplasia, degeneration,
necrosis or inflammation, interfollicular epidermal hyperplasia, or
a failure to heal or a delayed healing after injury.
[0004] Treatment of normal keratinocytes and EGFR over-expressing
tumor cells with the EGFR inhibitor,
[6,7-bis(2-methoxy-ethoxy)-quinazolin-4-yl-
]-(3-ethynylphenyl)amine, causes cell cycle arrest, as indicated by
an accumulation of cells in the G1 phase of the cell cycle (Moyer
et al., 1997, Cancer Res. 57:4838-4848). Progression of cells from
the G1 phase into the S phase requires the phosphorylation of the
retinoblastoma protein, pRB, which is mediated by cyclin-dependent
kinases. Consistent with its ability reduce the percentage of cells
in S-phase, we have observed that CP-358,774 causes depletion of
hyper- phosphorylated retinoblastoma protein (ppRB) and
accumulation of the cyclin-dependent kinase inhibitor,
p.sub.27.sup.kiP1/waf1 (Moyer et al., supra).
[0005] The keratinocyte growth factor (KGF) family consists of
KGF-1 and KGF-2, also known as FGF-7 and FGF-10, respectively,
reflecting their homology with proteins, in the fibroblast growth
factor superfamily. Various patent publications describe KGFs and
their uses, including PCT International Publication WO 94/23032,
published Oct. 13, 1994; PCT International Publication WO 98/06844,
published Feb. 19, 1998; PCT International Publication WO 98/16243,
published Apr. 23, 1998; PCT International Publication WO 98/16642,
published Apr. 23, 1998; and PCT International Publication WO
98/24813, published Jun. 11, 1998.
[0006] The KGFs are unique among FGFs in that they act exclusively
on epithelial cells. Both KGFs are expressed by stromal cells and
act as paracrine mediators of epithelial cell proliferation (Finch
et al., 1989, Science 245:752; Igarishi et al., 1998, J. Biol.
Chem. 273:13230). KGF-1 and KGF-2 are 57% homologous, and both bind
to the FGFRliiib receptor with high affinity (Igarishi et al.,
1998, supra; Miceli, R., et al. 1999, J. Pharm. Exp. Ther.
290:464). Since KGFs appear to be paracrine factors in the skin
(Marchese, C., et al., 1990, J. Cell Phys. 144:326; Igarashi, M.,
et al., 1998, supra), we investigated whether the KGF pathway can
serve as an alternate means of mitogenic signaling in this tissue,
thereby potentially alleviating the epithelial toxicity caused by
administration of an EGFR inhibitor.
SUMMARY OF THE INVENTION
[0007] The present invention is directed to a pharmaceutical
composition useful to treat the epithelial toxicity associated with
administration to a patient of an epidermal growth factor receptor
(EGFR) inhibitor, which pharmaceutical composition comprises an
EGFR inhibitor and a keratinocyte growth factor (KGF) in a
pharmaceutically acceptable carrier. In a typical treatment
scenario, the EGFR inhibitor is administered as an anti-cancer
agent to a patient in need thereof. In a preferred embodiment, the
EGFR inhibitor is a low molecular weight inhibition or an antibody
that binds specifically to the EGFR and blocks its activation.
[0008] The present invention is further directed to a method of
treating the epithelial toxicity resulting from administration to a
patient of an EGFR inhibitor, comprising co-administering to the
patient a therapeutically effective amount of KGF with the EGFR
inhibitor. In a preferred embodiment, the patient is a human that
is being treated for cancer. In a further preferred embodiment, the
epithelial toxicity is manifested as a skin toxicity.
[0009] The present invention is further directed to a method of
preparing a pharmaceutical composition useful for treating the
epithelial toxicity resulting from administration to a patient of
an EGFR inhibitor, comprising combining an EGFR inhibitor with a
keratinocyte growth factor (KGF). In a preferred embodiment, the
method further comprises combining a pharmaceutically acceptable
carrier with the EGFR inhibitor and KGF.
[0010] The present invention further provides a kit comprising a
first container comprising an EGFR inhibitor, and a second
container comprising a KGF. A pharmaceutically acceptable carrier
may also be present in either container. The kit may further
comprise a third container comprising a sterile diluent. The kit
may further comprise a package insert comprising printed
instructions directing the use of the combined treatment as a
method for treating the epithelial toxicity resulting from
administration to a patient of an EGFR inhibitor.
DETAILED DESCRIPTION OF THE INVENTION
[0011] The data presented in the Examples herein below demonstrate
that co-administration of a KGF with an EGFR inhibitor is effective
at protecting human epithelial keratinocytes from the cell cycle
arrest normally induced by the EGFR inhibitor alone. Thus, KGF can
advantageously be used to protect normal epithelial cells from the
toxicity otherwise caused by administering an EGFR inhibitor alone
to a patient. Accordingly, the present invention provides a
pharmaceutical composition comprising an EGFR inhibitor and a KGF
in a pharmaceutically acceptable carrier.
[0012] As used herein, the term "EGFR inhibitor" refers to any EGFR
inhibitor that is currently known in the art or that will be
identified in the future, and includes any chemical entity that,
upon administration to a patient, results in inhibition of a
biological activity associated with activation of the EGFRs in the
patient, including any of the downstream biological effects
otherwise resulting from the binding to an EGFR of its natural
ligand. Such EGFR inhibitors include any agent that can block EGFR
activation or any of the downstream biological effects of EGFR
activation that are relevant to treating cancer in a patient. Such
an inhibitor can act by binding directly to the intracellular
domain of the receptor and inhibiting its kinase activity.
Alternatively, such an inhibitor can act by occupying the ligand
binding site or a portion thereof of the EGFR receptor or a portion
thereof, thereby making the receptor inaccessible to its natural
ligand so that its normal biological activity is prevented or
reduced. EGFR inhibitors include but are not limited to low
molecular weight inhibitors, antibodies or antibody fragments,
antisense constructs and ribozymes. In a preferred embodiment, the
EGFR inhibitor is a small organic molecule or an antibody that
binds specifically to the human EGFR.
[0013] EGFR inhibitors that can be used according to the present
invention include but are not limited to those classified in the
art as quinazoline EGFR inhibitors, pyrido-pyrimidine EGFR
inhibitors, pyrimido-pyrimidine EGFR inhibitors, pyrrolo-pyrimidine
EGF R inhibitors, pyrazolo-pyrimidine EGF R inhibitors,
phenylamino-pyrimidine EGFR inhibitors, oxindole EGFR inhibitors,
indolocarbazole EGFR inhibitors, phthalazine EGFR inhibitors,
isoflavone EGFR inhibitors, quinalone EGFR inhibitors, and
tyrphostin EGFR inhibitors.
[0014] Non-limiting examples of low molecular weight EGFR
inhibitors useful in practicing the present invention include any
of the EGFR inhibitors described in the following patent
publications, and all pharmaceutically acceptable salts and
solvates of said EGFR inhibitors: European Patent Application EP
520722, published Dec. 30, 1992; European Patent Application EP
566226, published Oct. 20, 1993; PCT International Publication WO
96/33980, published Oct. 31, 1996; U.S. Pat. No. 5,747,498, issued
May 5, 1998; PCT International Publication WO 96/30347, published
Oct. 3, 1996; European Patent Application EP 787772, published Aug.
6, 1997; PCT International Publication WO 97/30034, published Aug.
21, 1997; PCT International Publication WO 97/30044, published Aug.
21, 1997; PCT International Publication WO 97/38994, published Oct.
23, 1997; PCT International Publicat on WO 97/49688, published Dec.
31, 1997; European Patent Application EP 837063, published Apr. 22,
1998; PCT International Publication WO 98/02434, published Jan. 22,
1998; PCT International Publication WO 97/38983, published Oct. 23,
19 7; PCT International Publication WO 95/19774, published Jul. 27,
1995; PCT International Publication WO 95/19970, published Jul. 27,
1995; PCT International Publication WO 97/13771, published Apr. 17,
1997; PCT International Publication WO 98/02437, published Jan. 22,
1998; PCT International Publication WO 98/02438, published Jan. 22,
1998; PCT International Publication WO 97/32881, published Sep. 12,
1997; German Application DE 19629652, published Jan. 29, 1998; PCT
International Publication WO 98/33798, published Aug. 6, 1998; PCT
International Publication WO 97/32880, published Sep. 12, 1997; PCT
International Publication WO 97/32880 published Sep. 12, 1997;
European Patent Application EP 682027, published Nov. 15, 1995; PCT
International Publication WO 97/02266, published Jan. 23, 1997; PCT
International Publication WO 97/27199, published Jul. 31, 1997; PCT
International Publication WO 98/07726, published Feb. 26, 1998; PCT
International Publication WO 97/34895, published Sep. 25, 1997; PCT
International Publication WO 96/31510, published Oct. 10, 1996; PCT
International Publication WO 98/14449, published Apr. 9, 1998; PCT
Intern ational Publication WO 98/14450, published Apr. 9, 1998; PCT
International Publication WO 98/14451, published Apr. 9, 1998; PCT
International Publication WO 95/09847, published Apr. 13, 1995; PCT
International Publication WO 97/19065, published May 29, 1997; PCT
International Publication WO 98/17662, published April 30, 1998;
U.S. Pat. No. 5,789,427, issued Aug. 4, 1998; U.S. Pat. No.
5,650,415, issued Jul. 22, 1997; U.S. Pat. No. 5,656,643, issued
Aug. 12, 1997; PCT International Publication WO 99/35146, published
Jul. 15, 1999; PCT International Publication WO 99/35132, published
Jul. 15, 1999; PCT International Publication WO 99/07701, published
Feb. 18, 1999; and PCT International Publication WO 92/20642
published Nov. 26, 1992. Additional non-limiting examples of low
molecular weight EGFR inhibitors include any of the EGFR inhibitors
described in Traxler, P., 1998, Exp. Opin. Ther. Patents
8(12):1599-1625.
[0015] Specific preferred examples of low molecular weight EGFR
inhibitors that can be used according to the present invention
include
[6,7-bis(2-methoxyethoxy)-4-quinazolin-4-yl]-(3-ethynylphenyl)
amine (U.S Pat. No. 5,747,498 issued May 5, 1998 and Moyer et al.,
1997, supra); C1-1033 and PD183805 (Sherwood et al., 1999, Proc.
Am. Assoc. Cancer Res. 40:723); and ZD1839 (Woodburn et al., 1997,
Proc. Am. Assoc.. Cancer Res. 38:633).
[0016] Antibody-based EGFR inhibitors include any anti-EGFR
antibody or antibody fragment that can partially or completely
block EGFR activation by its natural ligand. Non-limiting examples
of antibody-based EGFR inhibitors include those described in
Modjtahedi, H., et al., 1993, Br. J. Cancer 67:247-253; Teramoto,
T., et al., 1996, Cancer 77:639-645; Goldstein et al., 1995, Clin.
Cancer Res. 1:1311-1318; Huang, S. M., et al., 1999, Cancer Res.
15:59(8):1935-40; and Yang, X., et al., 1999, Cancer Res.
59:1236-1243. Thus, the EGFR inhibitor can be monoclonal antibody
Mab E7.6.3 (Yang, 1999 supra), or Mab C225 (ATCC Accession No.
HB-8508), or an antibody or antibody fragment having the binding
specificity thereof.
[0017] Additional antibody-based EGFR inhibitors can be raised
according to known methods by administering the appropriate antigen
or epitope to a host animal selected, e.g., from pigs, cows,
horses, rabbits, goats, sheep, and mice, among others. Various
adjuvants known in the art can be used to enhance antibody
production.
[0018] Although antibodies useful in practicing the invention can
be polyclonal, monoclonal antibodies are preferred. Monoclonal
antibodies against EGFR can be prepared and isolated using any
technique that provides for the production of antibody molecules by
continuous cell lines in culture. Techniques for production and
isolation include but are not limited to the hybridoma technique
originally described by Kohler and Milstein (Nature, 1975, 256:
495-497); the human B-cell hybridoma technique (Kosbor et al.,
1983, Immunology Today 4:72; Cote et al., 1983, Proc. Nati. Acad.
Sci. USA 80: 2026-2030); and the EBV-hybridoma technique (Cole et
al, 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,
Inc., pp. 77-96).
[0019] Alternatively, techniques described for the production of
single chain antibodies (see, e.g., U.S. Pat. No. 4,946,778) can be
adapted to produce anti-EGFR single chain antibodies.
Antibody-based EGFR inhibitors useful in practicing the present
invention also include anti-EGFR antibody fragments including but
not limited to F(ab').sub.2 fragments, which can be generated by
pepsin digestion of an intact antibody molecule, and Fab fragments,
which can be generated by reducing the disulfide bridges of the
F(ab').sub.2 fragments. Alternatively, Fab and/or scFv expression
libraries can be constructed (see, e.g., Huse et al., 1989, Science
246: 1275-1281) to allow rapid identification of fragments having
the desired specificity to EGFR.
[0020] Techniques for the production and isolation of monoclonal
antibodies and antibody fragments are well-known in the art, and
are additionally described, among other places, in Harlow and Lane,
1988, Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory, and in J. W. Goding, 1986, Monoclonal Anti-bodies:
Principles and Practice, Academic Press, London. Humanized
anti-EGFR antibodies and antibody fragments can also be prepared
according to known techniques such as those described in Vaughn, T.
J. et al., 1998, Nature Biotech. 16:535-539 and references cited
therein, and such antibodies or fragments thereof are also useful
in practicing the present invention.
[0021] EGFR inhibitors for use in the present invention can
alternatively be based on antisense oligonucleotide constructs.
Anti-sense oligonucleotides, including anti-sense RNA molecules and
anti-sense DNA molecules, would act to directly block the
translation of EGFR mRNA by binding thereto and thus preventing
protein translation or increasing mRNA degradation. For example,
antisense oligonucleotides of at least about 15 bases and
complementary to unique regions of the mRNA transcript sequence
encoding EGFR can be synthesized, e.g., by conventional
phosphodiester techniques and administered by e.g., intravenous
injection or infusion.
[0022] Ribozymes can also function as EGFR inhibitors for use in
the present invention. Ribozymes are enzymatic RNA molecules
capable of catalyzing the specific cleavage of RNA. The mechanism
of ribozyme action involves sequence specific hybridization of the
ribozyme molecule to complementary target RNA, followed by
endonucleolytic cleavage. Engineered hammerhead motif ribozyme
molecules that specifically and efficiently catalyze
endonucleolytic cleavage of EGFR mRNA sequences are thereby useful
within the scope of the present invention. Specific ribozyme
cleavage sites within any potential RNA target are initially
identified by scanning the target molecule for ribozyme cleavage
sites, which typically include the following sequences, GUA, GUU,
and GUC. Once identified, short RNA sequences of between about 15
and 20 ribonucleotides corresponding to the region of the target
gene containing the cleavage site can be evaluated for predicted
structural features, such as secondary structure, that can render
the oligonucleotide sequence unsuitable. The suitability of
candidate targets can also be evaluated by testing their
accessibility to hybridization with complementary oligonucleotides,
using, e.g., ribonuclease protection assays.
[0023] Both anti-sense oligonucleotides and ribozymes useful as
EGFR inhibitors can be prepared by known methods. These include
techniques for chemical synthesis such as, e.g., by solid phase
phosphoramadite chemical synthesis. Alternatively, anti-sense RNA
molecules can be generated by in vitro or in vivo transcription of
DNA sequences encoding the RNA molecule. Such DNA sequences can be
incorporated into a wide variety of vectors that incorporate
suitable RNA polymerase promoters such as the T7 or SP6 polymerase
promoters. Various modifications to the oligonucleotides of the
invention can be introduced as a means of increasing intracellular
stability and half-life. Possible modifications include but are not
limited to the addition of flanking sequences of ribonucleotides or
deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or
the use of phosphorothioate or 2'-O-methyl rather than
phosphodiesterase linkages within the oligonucleotide backbone.
[0024] As used herein, "keratinocyte growth factor", or "KGF"
refers to KGF-1 and KGF-2. The nucleotide and amino acid sequences
of KGF-1 are described in Finch et al., 1989, Science 245:752-755.
The amino acid sequence of KGF-2 is described in Igarishi et al.,
1998, J. Biol. Chem. 273:13230.
[0025] Native KGF may be isolated from native human sources or
produced by recombinant DNA techniques, as known in the art. Human
recombinant KGF-1, as expressed from E. coli, is a 19 kDa protein,
and is commercially available (Sigma Chemical Co., St. Louis, Mo.).
KGF analogs reportedly having increased stability over natural KGF
are described in PCT International Publication WO 96/11951, which
published Apr. 25, 1996, and such KGF analogs can be used as the
KGF component in practicing the present invention. Alternatively,
any fragment of the entire KGF polypeptide or analog thereof which
fragment or analog retains complete or even partial KGF activity
can be as used in practicing the present invention. Alternatively,
KGF-2, as disclosed in PCT International Publication WO 98/06844
which published Feb. 19, 1998, or any analogs or peptide fragments
thereof retaining complete or partial KGF-2 activity, can be used
as the KGF component in practicing the present invention. Unless
otherwise indicated, all alternative forms of KGF and KGF analogs
useful in practicing the present invention are referred to
collectively hereinafter as "KGF".
[0026] The present invention further provides a method for treating
the epithelial toxicity resulting from administration to a patient
of an EGFR inhibitor, comprising co-administering to the patient a
therapeutically effective amount of KGF With the EGFR
inhibitor.
[0027] The term "treating" as used herein, unless otherwise
indicated, means reversing, alleviating, inhibiting the progress
of, or preventing, either partially or completely, the epithelial
toxicity, or one or more conditions or symptoms associated with the
epithelial toxicity, caused by administration to a patient of a
dose (single or divided) or series of doses (e.g., a course of
treatment) of an EGFR inhibitor useful in treating cancer. The term
"treatment" as used herein, unless otherwise indicated, refers to
the act of treating as "treating" is defined immediately above.
[0028] As used herein, the term "epithelial toxicity" refers to an
abnormality or dysfunction of the epithelium, and can be manifested
in a patient being treated for cancer by administration of an EGFR
inhibitor by one or more symptoms or conditions selected from skin
rash, diarrhea, corneal thinning, hair atrophy or loss, hair
follicle dysplasia, degeneration, necrosis or inflammation,
interfollicular epidermal hyperplasia, or a failure to heal or a
delayed healing after injury, among other symptoms.
[0029] In a preferred embodiment, the epithelial toxicity is
manifested as a skin toxicity such as acneform or macro-papular
rash.
[0030] As used herein, the term "patient" preferably refers to a
human in need of treatment with an EGFR inhibitor for any purpose,
and more preferably a human in need of such a treatment to treat
cancer. However, the term "patient" can also refer to non-human
animals, preferably mammals such as dogs, cats, horses, cows, pigs,
sheep and non-human primates, among others, that are in need of
treatment with an EGFR inhibitor.
[0031] In a preferred embodiment, the patient is a human in need of
treatment for cancer. The cancer is preferably any cancer
treatable, either partially or completely, by administration of an
EGFR inhibitor. The cancer may be selected from, but is not limited
to, the group consisting of lung cancer, bone cancer, pancreatic
cancer, skin cancer, cancer of the head or neck, cutaneous or
intraocular melanoma, uterine cancer, ovarian cancer, rectal
cancer, cancer of the anal region, stomach cancer, colon cancer,
breast cancer, uterine cancer, carcinoma of the fallopian tubes,
carcinoma of the endometrium, carcinoma of the cervix, carcinoma of
the vagina, carcinoma of the vulva, cancer of the esophagus, cancer
of the small intestine, cancer of the endocrine system, cancer of
the thyroid gland, cancer of the parathyroid gland, cancer of the
adrenal gland, sarcoma of soft tissue, cancer of the urethra,
cancer of the penis, prostate cancer, cancer of the bladder, cancer
of the kidney or ureter, renal cell carcinoma, carcinoma of the
renal pelvis, neoplasms of the central nervous system (CNS), spinal
axis tumors, brain stem glioma, pituitary adenoma, or a combination
of one or more of the foregoing cancers.
[0032] In another embodiment of said method, the patient is a human
being that is in need of treatment for a benign proliferative
disease, including, but not limited to, psoriasis, benign prostatic
hypertrophy or restenosis.
[0033] For purposes of the present invention, "co-administration
of" and "co-administering" KGF with an EGFR inhibitor (both
components referred to hereinafter as the "two active agents")
refer to any administration of the two active agents, either
separately or together, where the two active agents are
administered as part of an appropriate dose regimen designed to
obtain the protective benefit of the combination therapy. Thus, the
two active agents can be administered either as part of the same
pharmaceutical composition or in separate pharmaceutical
compositions. KGF can be administered prior to, at the same time
as, or subsequent to administration of the EGFR inhibitor, or in
some combination thereof, as long as the patient obtains the
protective effect of KGF against the epithelial toxicity that might
otherwise be caused by administration of the EGFR inhibitor alone.
Where the EGFR inhibitor is administered to the patient at repeated
intervals, e.g., during a standard course of treatment, KGF can be
administered prior to, at the same time as, or subsequent to, each
administration of the EGFR inhibitor, or some combination thereof,
or at different intervals in relation to the EGFR inhibitor
treatment, or in a single dose prior to, at any time during, or
subsequent to the course of treatment with the EGFR inhibitor, so
long as the patient obtains the protective effect of KGF against
the epithelial toxicity that might otherwise be caused by
administration of the EGFR inhibitor alone.
[0034] The EGFR inhibitor will typically be administered to the
patient in a dose regimen that provides for the most effective
treatment of the cancer (from both efficacy and safety
perspectives) for which the patient is being treated, as known in
the art, and as disclosed, e.g., in the above-cited publications.
In conducting the treatment method of the present invention, the
EGFR inhibitor can be administered in any effective manner as known
in the art, such as by oral, topical, intravenous,
intra-peritoneal, intramuscular, intra-articular, subcutaneous,
intranasal, intra-ocular, vaginal, rectal, or intradermal routes,
depending upon the type of cancer being treated, the type of EGFR
inhibitor being used (e.g., small molecule, antibody or antisense
construct), and the medical judgement of the prescribing physician
as based, e.g., on the results of published clinical studies.
[0035] The amount of EGFR inhibitor administered and the timing of
EGFR inhibitor administration will depend on the type (species,
gender, age, weight, etc.) and condition of the patient being
treated, the severity of the disease or condition being treated,
and on the route of administration. For example, small molecule
EGFR inhibitors can be administered to a patient in doses ranging
from 0.01 to 10 mg/kg of body weight per day or per week in single
or divided doses, or by continuous infusion. Antibody-based EGFR
inhibitors, or antisense or ribozyme constructs, can be
administered to a patient in doses ranging from 0.1 to 100 mg/kg of
body weight per day or per week in single or divided doses, or by
continuous infusion. In some instances, dosage levels below the
lower limit of the aforesaid range may be more than adequate, while
in other cases still larger doses may be employed without causing
any harmful side effect, provided that such larger doses are first
divided into several small doses for administration throughout the
day.
[0036] As used herein, a "therapeutically effective amount of KGF"
refers to that amount of KGF capable of reversing, alleviating,
inhibiting the progress of, or preventing, either completely or
partially, one or more symptoms or conditions in a patient
resulting from epithelial toxicity caused by administration to the
patient of a standard dose or series of doses (e.g., a standard
course of treatment) of an EGFR inhibitor administered to the
patient for the treatment of cancer or other disease, disorder or
condition. The therapeutically effective amount of KGF can be
administered as a single dose, as several divided doses, or can be
continuously infused.
[0037] The prescribing physician can determine what constitutes a
"therapeutically effective amount of KGF" based initially, e.g., on
the results of published clinical trials, and the recommended dose
described in any package insert as present in a kit comprising the
two active agents. The dose of KGF can be adjusted up or down by
the prescribing physician depending on the degree of responsiveness
to KGF treatment by the particular patient. The prescribing
physician will preferably monitor responses to the
co-administration treatment, particularly as those responses relate
to prevention or amelioration of epithelial toxicity otherwise
associated with administration of the EGFR inhibitor alone. In a
preferred embodiment, the prescribing physician will monitor the
condition of the patient's skin, and particularly the prevention or
improvement in any skin rash caused by or otherwise associated with
administration of the EGFR inhibitor. In a further preferred
embodiment, the prescribing physician, by opthalmalogical exam,
will monitor corneal thinning in the patient caused by or otherwise
associated with administration of the EGFR inhibitor. In a further
preferred embodiment, the prescribing physician will monitor
diarrhea in the patient caused by or otherwise associated with
administration of the EGFR inhibitor.
[0038] The KGF will typically be administered to the patient in a
dose regimen that provides for the most effective and safest
treatment of the epithelial toxicity caused by the EGFR inhibitor,
or of one or more conditions or symptoms associated with the
epithelial toxicity. The KGF can be administered in a convenient
manner as known in the art, such as by topical, intravenous,
intraperitoneal, intramuscular, intraarticular, subcutaneous,
intranasal, intraocular, vaginal, rectal or intradermal routes, as
determined by the prescribing physician.
[0039] The amount of KGF administered and the timing of KGF
administration will depend on the type (species, gender, age,
weight, etc.) and condition of the patient being treated, the
severity or potential severity of the epithelial toxicity caused by
the EGFR inhibitor, the route of administration, and the judgement
of the prescribing physician as based, e.g., on the results of
published clinical studies. For example, KGF can be administered
parenterally to a patient in doses ranging from 0.01 to 10 mg/kg of
body weight per day or somewhat less frequently such as 1-4 times
per week in single or divided doses. Also, for example, KGF can be
administered topically to a patient once or more per day in
formulations comprising from about 0.001% (w/v) to about 1.0%
(w/v). In some instances, dosage and concentration levels below the
lower limit of the aforesaid ranges may be more than adequate,
while in other cases still larger doses or concentrations may be
employed without causing any harmful side effects, provided that
such larger doses are first divided into several small doses for
administration throughout the day. In some circumstances, a single
treatment or series of treatments with KGF will be sufficient to
treat the epithelial toxicity caused by the EGFR inhibitor, while
in other circumstances the treatment will continue until sufficient
improvement in the condition is observed by the attending
physician, as determined by one or more standard indices of
epithelial toxicity such as corneal epithelial thickness as
determined, e.g., by a slit-lamp test.
[0040] The present invention further provides a method of preparing
a pharmaceutical composition useful for treating the epithelial
toxicity resulting from administration to a patient of an EGFR
inhibitor, comprising combining an EGFR inhibitor with KGF. In a
preferred embodiment, the method further comprises combining a
pharmaceutically acceptable carrier with the EGFR inhibitor and
KGF.
[0041] The present invention further provides a use for an EGFR
inhibitor combined with KGF in preparing a medicament for heating
cancer in a patient while also treating the epithelial toxicity
resulting from administration to a patient of the EGFR inhibitor
alone. The present invention further provides a use for
co-administered EGFR inhibitor and KGF in preparing an anti-cancer
treatment having reduced epithelial toxicity.
[0042] The EGFR inhibitors and KGF can be administered either
separately or together by the same or different routes, and in a
wide variety of different dosage forms. For example, the EGFR
inhibitor is preferably administered orally or parenterally,
whereas KGF is preferably administered parenterally or
topically.
[0043] The EGFR inhibitor can be administered with various
pharmaceutically acceptable inert carriers in the form of tablets,
capsules, lozenges, troches, hard candies, powders, sprays, creams,
salves, suppositories, jellies, gels, pastes, lotions, ointments,
elixirs, syrups, and the like. Administration of such dosage forms
can be carried out in single or multiple doses. Carriers include
solid diluents or fillers, sterile aqueous media and various
non-toxic organic solvents, etc. Oral pharmaceutical compositions
can be suitably sweetened and/or flavored.
[0044] The KGF can be administered with various pharmaceutically
acceptable inert carriers in the form of sprays, creams, salves,
suppositories, jellies, gels, pastes, lotions, ointments, and the
like. Administration of such dosage forms can be carried out in
single or multiple doses. Carriers include solid diluents or
fillers, sterile aqueous media and various non-toxic organic
solvents, etc.
[0045] The EGFR inhibitor and KGF can be combined together with
various pharmaceutically acceptable inert carriers in the form of
sprays, creams, salves, suppositories, jellies, gels, pastes,
lotions, ointments, and the like. Administration of such dosage
forms can be carried out in single or multiple doses. Carriers
include solid diluents or fillers, sterile aqueous media and,
various non-toxic organic solvents, etc.
[0046] All formulations comprising KGF should be selected so as to
avoid denaturation and loss of biological activity of the KGF
polypeptide.
[0047] Methods of preparing pharmaceutical compositions comprising
an EGFR inhibitor are known in the art, and are described, e.g., in
several of the above-cited publications. Methods of preparing
pharmaceutical compositions comprising KGF are also known in the
art, and are described, e.g., in several of the above-cited
publications. In view of the teaching of the present invention,
methods of preparing pharmaceutical compositions comprising both an
EGFR inhibitor and KGF will be apparent from the above-cited
publications and from other known references, such as Remington's
Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa.,
18.sup.th edition (1990).
[0048] For oral administration of EGFR inhibitor, tablets
containing one or both of the active agents are combined with any
of various excipients such as micro-crystalline cellulose, sodium
citrate, calcium carbonate, dicalcium phosphate and glycine, along
with various disintegrants such as starch (and preferably corn,
potato or tapioca starch), alginic acid and certain complex
silicates, together with granulation binders like polyvinyl
pyrrolidone, sucrose, gelatin and acacia. Additionally, lubricating
agents such as magnesium stearate, sodium lauryl sulfate and talc
are often very useful for tableting purposes. Solid compositions of
a similar type may also be employed as fillers in gelatin capsules;
preferred materials in this connection also include lactose or milk
sugar as well as high molecular weight polyethylene glycols. When
aqueous suspensions and/or elixirs are desired for oral
administration, the EGFR inhibitor may be combined with various
sweetening or flavoring agents, coloring matter or dyes, and, if so
desired, emulsifying and/or suspending agents as well, together
with such diluents as water, ethanol, propylene glycol, glycerin
and various like combinations thereof.
[0049] For parenteral administration of either or both of the
active agents, solutions in either sesame or peanut oil or in
aqueous propylene glycol may be employed, as well as sterile
aqueous solutions comprising the active agent or a corresponding
water-soluble salt thereof. Such sterile aqueous solutions are
preferably suitably buffered, and are also preferably rendered
isotonic, e.g., with sufficient saline or glucose. These particular
aqueous solutions are especially suitable for intravenous,
intramuscular, subcutaneous and intraperitoneal injection purposes.
The oily solutions are suitable for intra-articular, intramuscular
and subcutaneous injection purposes. The preparation of all these
solutions under sterile conditions is readily accomplished by
standard pharmaceutical techniques well known to those skilled in
the art. Any parenteral formulation selected for administration of
KGF should be selected so as to avoid denaturation and loss of
biological activity of the KGF polypeptide.
[0050] Additionally, it is possible to topically administer either
or both of the active agents, and this may preferably be done by
way of creams, lotions, jellies, gels, pastes, ointments, salves
and the like, in accordance with standard pharmaceutical practice.
For example, a topical formulation comprising either an EGFR
inhibitor or KGF in about 0.1% (w/v) to about 5% (w/v)
concentration can be prepared. In a preferred embodiment, a topical
formulation of KGF can be prepared and will be particularly
effective where the epithelial toxicity caused by the EGFR
inhibitor is manifested as a skin toxicity. Any topical formulation
selected for KGF should be selected so as to avoid denaturation and
loss of biological activity of the KGF polypeptide.
[0051] For veterinary purposes, the active agents can be
administered separately or together to animals using any of the
forms and by any of the routes described above. In a preferred
embodiment, the EGFR inhibitor is administered in the form of a
capsule, bolus, tablet, liquid drench, by injection or as an
implant. As an alternative, the EGFR inhibitor can be administered
with the animal feedstuff, and for this purpose a concentrated feed
additive or premix may be prepared for a normal animal feed. The
KGF is preferably administered in the form of liquid drench, by
injection or as an implant. Such formulations are prepared in a
conventional manner in accordance with standard veterinary
practice.
[0052] The present invention further provides a kit comprising a
single container comprising both an EGFR inhibitor and KGF. The
present invention further provides a kit comprising a first
container comprising an EGFR inhibitor and a second container
comprising KGF. In a preferred embodiment, the kit containers may
further comprise a pharmaceutically acceptable carrier. The kit may
further comprise a sterile diluent, which is preferably stored in a
separate additional container. The kit may further comprise a
package insert comprising printed instructions directing the use of
the combined treatment as a method for treating the epithelial
toxicity resulting from administration to a patient of an EGFR
inhibitor.
[0053] The following examples are illustrative only, and are not
intended to limit the scope of the present invention.
EXAMPLE 1
MEASURING PROTECTION OF KERATINOCYTES BY KGF
[0054] The protective effect of KGF on keratinocytes was measured
in two types of assays, the first to determine its protective
effect on cell cycle phase distribution against a small molecule
EGFR inhibitor, and the second to determine its protective effect
against ppRB depletion and p27.sup.kiP1/waf1 up regulation caused
by the EGFR inhibitor. The effect of KGF on cell cycle phase
distribution was measured by bivariate analysis of
bromodeoxyuridine incorporation and propidium iodide uptake by DiFi
colon carcinoma cells using flow cytometry. The protective effect
of KGF against ppRB depletion and p27.sup.kiP1/waf1 upregulation
was measured by immunoblotting of lysates from treated and control
DiFi cells with antibodies specific for RB and p27.sup.kip1/waf1,
respectively. The methods are described in detail below.
[0055] All experiments were carried out with normal human neonatal
epidermal keratinoctyes, NHEK (Clonetics, San Diego, Calif.). Cells
were maintained in Keratinocyte Growth Medium, KGM-2 (Clonetics).
Human recombinant KGF expressed in E. coli as a 19 kDa protein was
purchased from Sigma Chemical Co. (St. Louis, Mo.).
FLOW CYTOMETRIC MEASUREMENT OF CELL CYCLE ARREST
[0056] NHEK cells were seeded in 60-mm dishes in KGM-2. When the
cells were semiconfluent, the medium was replaced with fresh medium
containing the test agents: vehicle (0.125% DMSO); vehicle +20
ng/ml KGF; vehicle +0.3 .mu.M CP-358, 774-01 +/-20 ng/ml KGF; or
vehicle +1 .mu.M CP-358, 774-01 +/-20 ng/ml KGF. The cells were
incubated at 37.degree. C. in a 5% CO.sub.2, humidified atmosphere.
After 24 hr, bromodeoxyuridine (BrdU) was added to the medium at a
final concentration of 10 .mu.M for 30 min. The cells were then
harvested using 0.25% trypsin/1 mM EDTA and counted using a Coulter
counter. Aliquots of 2.times.10.sup.6 cells were collected by
centrifugation at 220.times. g for 5 min, washed in 2 ml phosphate
buffered saline (PBS) containing 2.5% fetal calf serum, resuspended
in 0.2 ml PBS, and fixed by the addition of 5 ml 70% ethanol at
-20.degree. C. After a 30 min incubation on ice, the cells were
collected by centrifugation at 500.times. g for 5 min and
resuspended in 25 .mu.l PBS. One ml of 0.2 mg/ml pepsin in 2 N HCl
was added and the cells were incubated for 30 min at rm temp. Cells
were collected by centrifugation at 1500.times. g for 5 min and
washed with 1 ml 0.1 M sodium borate, pH 8.5. Cells were pelleted
at 1500.times. g for 5 min, resuspended in 1 ml of fluorescein
isothiocyanate (FITC)-conjugated anti-BrdU (Becton Dickinson, San
Jose, Calif.) diluted fifty-fold in PBS containing 0.5% Tween 20
and 1% bovine serum albumin, and incubated at ambient temperature
in the dark for 30 min. After a final wash with PBS with 0.5% Tween
20 and 1% BSA, the cells were resuspended in PBS containing 10
.mu.g/ml propidium iodide and 10 .mu.g/ml RNase, filtered through a
35 .mu.m mesh and analyzed on a FACSCalibur flow cytometer (Becton
Dickinson) equipped with an argon laser with an emission wavelength
of 488 nm. FITC fluorescence data was acquired at 515-545 nm, and
propidium iodide fluorescence data was acquired at 564-606 nm. A
minimum of 20,000 cells was analyzed per sample. Data was analyzed
using CellOuest software (Becton-Dickinson) and the percent of
cells in each phase (G1, S, G2) of the cell cycle was
calculated.
MEASUREMENT OF PPRB AND P27.sup.KIP1/WAF1 PROTEIN LEVELS
[0057] NHEK cells were seeded in 6-well plates and grown until
semi-confluent in KGM-2. Fresh medium containing vehicle or CP-358,
774-01 +/-20 ng/ml KGF was added, and the cells were incubated for
24 hr in a 5% CO.sub.2, humidified atmosphere. Cells were washed
with 50 mM Tris-HCl pH 7.4, 140 mM sodium chloride, 3.3 mM
potassium chloride and 500 .mu.M sodium orthovanadate, and lysed by
boiling for 10 min in SDS sample buffer (50 mM Tris-HCl, pH 6.8,
100 mM DTT, 2% SDS, 0.1% bromphenol blue, 10% glycerol). Total
protein concentration of the lysates was determined using the BCA
protein assay (Pierce Chemicals, Rockford, Ill.). Ten .mu.g of
protein was resolved on a 7.5% (RB) or 4-20% (p27) polyacrylamide
gel (Owl Separation Systems, Portsmouth, N.H.), and transferred to
an Immobilon-P membrane (Millipore, Bedford, Mass.) for 2 hr at 250
mA. Membranes were blocked overnight in 4% nonfat dry milk in TBST
(50 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1% Tween 20) and probed
with 1 .mu.g/ml monoclonal antibody G3-245 (Pharmingen, San Diego,
Calif.) for RB detection or with 0.1 .mu.g/ml anti-p27kip1 Clone 57
monoclonal antibody (Transduction Labs, Lexington, Ky.) followed by
horseradish peroxidase-conjugated goat anti-mouse IgG (Pharmingen)
diluted 1:1000. The identity of the lower band in RB blots as
hypophosphorylated RB (pRB) was confirmed by use of an antibody,
Clone G99-549 (Pharmingen), specific for this form.
RESULTS
CELL CYCLE PHASE DISTRIBUTION
[0058] Treatment of keratinocytes for 24 hours with
N-(3-ethynylphenylamino)-6,7-bis(2-methoxyethoxy-4-quinazolineamine
hydrochloride salt (Compound A) caused a concentration dependent
accumulation of cells in the G1 phase of the cell cycle and a
corresponding decrease in the S-phase fraction (Table 1).
Co-treatment with KGF reversed this effect, while treatment with
KGF alone had no significant effect on cell cycle phase
distribution.
1TABLE 1 Treatment % G1 % S % G2 Vehicle 52 34 12 20 ng/ml KGF 52
34 13 0.3 .mu.M Compound A 75 13 11 0.3 .mu.M Compound A + 20 ng/ml
KGF 53 34 12 1 .mu.M Compound A 84 6.0 10 1 .mu.M Compound A + 20
ng/ml KGF 50 35 13
[0059] ppRB and p27.sup.kip1/baf1 Levels
[0060] Hyperphosphorylated (ppRB) and hypophosphorylated (pRB)
retino-blastoma protein can be resolved because of their different
rates of migration on polyacrylamide electrophoresis gels. Both
forms of the protein have a MW of .about.105 kDa, but ppRB migrates
slower than pRB, resulting in the appearance of an upper (ppRB)
and/or a lower (pRB) band on blots that have been probed with the
monoclonal antibody, G3-245, which recognizes both forms of the
protein.
[0061] In untreated proliferating keratinocytes, all apparent RB
immuno-reactivity was in the hyperphosphorylated RB (upper band).
After treatment with 0.3 .mu.M Compound A, the ppRB signal was
dramatically reduced and a lower band corresponding to pRB
appeared. However, cells treated simultaneously with both 0.3 .mu.M
Compound A and 20 ng/ml KGF resembled control cells and exhibited
only the upper band. Thus, despite the presence of the EGFR
inhibitor, these cells contained predominantly hyperphosphorylated
RB (i.e., the form of RB that is permissive for cell cycle
progression). This is consistent with the BrdU labeling assay
described above, in which cells treated simultaneously with
Compound A and KGF had an S-phase fraction similar to that of
controls. In keratinocytes treated with 1 .mu.M Compound A, the
ppRB band was completely abolished and, as at the lower
concentration of Compound A, co-treatment with KGF completely
restored ppRB to control levels.
[0062] pRB is phosphorylated by cyclin-dependent kinases (cdk's),
which are positively regulated by cyclins and negatively regulated
by the cdk inhibitors p21.sup.cip1 and p27.sup.kip1/waf1. Lysates
of keratinocytes treated with 0.3 .mu.M Compound A exhibited a
3.9-fold induction of p27.sup.kip1/waf1 protein levels, consistent
with the effect of the compound on pRB phosphorylation. Treatment
with 1 .mu.M Compound A resulted in a 5.2-fold induction of
p27.sup.kip1/waf1. Co-treatment with KGF partially restored
p27.sup.kip1/waf1 to control levels.
[0063] All patents, patent applications, and publications cited
above are incorporated herein by reference in their entirety.
[0064] The present invention is not limited in scope by the
specific embodiments described, which are intended as single
illustrations of individual aspects of the invention. Functionally
equivalent compositions and methods are within the scope of the
invention. Indeed, various modifications of the invention, in
addition to those. shown and described herein, will become apparent
to those skilled in the art from the foregoing description. Such
modifications are intended to fall within the scope of the appended
claims.
* * * * *