U.S. patent application number 11/425470 was filed with the patent office on 2007-01-11 for method for modulating gene expression in epithelial cells.
Invention is credited to David E. Weinstein.
Application Number | 20070010547 11/425470 |
Document ID | / |
Family ID | 32229071 |
Filed Date | 2007-01-11 |
United States Patent
Application |
20070010547 |
Kind Code |
A1 |
Weinstein; David E. |
January 11, 2007 |
METHOD FOR MODULATING GENE EXPRESSION IN EPITHELIAL CELLS
Abstract
The present invention is directed to methods for modulating gene
expression in an epithelial cell.
Inventors: |
Weinstein; David E.; (Dobbs
Ferry, NY) |
Correspondence
Address: |
SANDER RABIN MD JD;CONVERGENT TECHNOLOGY PATENT LAW GROUP
WHITEMAN OSTERMAN & HANNA LLP
ONE COMMERCE PLAZA
ALBANY
NY
12260
US
|
Family ID: |
32229071 |
Appl. No.: |
11/425470 |
Filed: |
June 21, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10290657 |
Nov 8, 2002 |
|
|
|
11425470 |
Jun 21, 2006 |
|
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Current U.S.
Class: |
514/291 |
Current CPC
Class: |
A61K 38/13 20130101 |
Class at
Publication: |
514/291 |
International
Class: |
A61K 31/4745 20060101
A61K031/4745; A61K 31/44 20060101 A61K031/44 |
Claims
1. A method for modulating gene expression in an epithelial cell,
comprising contacting the epithelial cell with an amount of GM-284
effective to modulate gene expression in the epithelial cell.
2. The method of claim 1, wherein the gene is a neuregulin.
3. The method of claim 2, wherein the neuregulin is
neuregulin-.alpha..
4. The method of claim 3, wherein expression of neuregulin-.alpha.
is downregulated.
5. The method of claim 1, wherein the epithelial cell is selected
from the group consisting of a glandular cell, a Langerhans cell, a
neural cell, and a skin cell.
6. The method of claim 5, wherein the skin cell is a
keratinocyte.
7. The method of claim 1, wherein the contacting is effected in
vitro.
8. The method of claim 1, wherein the contacting is effected in
vivo in a subject.
9. The method of claim 8, wherein the contacting is effected in
vivo in a subject by administering GM-284 to the subject.
10. The method of claim 9, wherein GM-284 is administered to the
subject by oral administration, parenteral administration,
sublingual administration, topical administration, transdermal
administration, or osmotic pump.
11. The method of claim 8, wherein the subject is a human.
12. The method of claim 11, wherein the human has a
keratinocyte-associated disorder.
13. The method of claim 12, wherein the keratinocyte-associated
disorder is characterized by a dysregulation of keratinocyte
proliferation.
14. The method of claim 12, wherein the keratinocyte-associated
disorder is selected from the group consisting of leprosy,
periodontal disease, a peripheral neuropathy, a pressure ulcer,
psoriasis, a skin ulceration, a venous stasis ulcer, and a
wound.
15. The method of claim 9, wherein the amount of GM-284 is between
about 0.1 pM and about 5 mM.
16. The method of claim 15, wherein the amount of GM-284 is between
about 5 pM and about 1.5 mM.
Description
[0001] This application is a divisional of U.S. Ser. No. 10/290,657
which was filed on Nov. 8, 2002.
BACKGROUND OF THE INVENTION
[0002] Healing of wounds in skin and other epithelia involves a
complex set of interactions between numerous components, including
epithelial cells, peripheral nerves, and immune cells, as well as
soluble and matrix molecules contributed by the various cell types
(reviewed in Hom et al., Growth factor therapy to improve soft
tissue healing. Facial Plast. Surg., 18:41-52, 2002). Dysfunctions
associated with one or more of these components can lead to
anatomical changes of intact skin and/or alterations in the ability
of wounded epithelium to regain its normal histology and function.
For example, sensory denervation of skin results in alterations in
skin anatomy, including thinning of the epidermis, decreases in
epidermal proliferation, and changes in the gene expression of
Langerhans cells (Huang et al., Influence of cutaneous nerves on
keratinocyte proliferation and epidermal thickness in mice.
Neuroscience, 94:965-73, 1999; Hsieh et al., Epidermal denervation
and its effects on keratinocytes and Langerhans cells. J.
Neurocytol., 25:513-24, 1996; Li et al., Sensory and motor
denervation influence epidermal thickness in rat foot glabrous
skin. Exp. Neurol., 147:452-62, 1997; Laplante et al., Mechanisms
of wound reepithelialization: hints from a tissue-engineered
reconstructed skin to long-standing questions. FASEB J.,
15:2377-89, 2001). Therefore, sensory denervation of skin can
produce an underlying environment that is predisposed to the
establishment of wounds that are refractory to healing.
[0003] There are several clinical examples, including diabetic and
pressure ulcers, in which a similar sequence of events appears to
play a significant role. The importance of dermal innervation was
actually understood in ancient times, and described in detail in
the Bible: leprosy, a disease of the peripheral nerves that leads
to erosive, chronic skin lesions (Weinstein et al., Molecular
mechanism of nerve infection in leprosy. Trends Microbiol.,
7:185-86, 1999), is the first lesion for which this pattern was
described.
[0004] Normal keratinocytes have a relatively high, age-dependent
proliferative index. However, epidermal thickness is maintained at
a relatively constant level throughout most of life, suggesting
that an equilibrium exists among keratinocyte cell birth, squamae
formation, and sloughing of the stratum corneum (Stanulis-Praeger
and Gilchrest, Growth factor responsiveness declines during
adulthood for human skin-derived cells. Mech. Ageing Dev.,
35:185-98, 1986; Laplante et al., Mechanisms of wound
reepithelialization: hints from a tissue-engineered reconstructed
skin to long-standing questions. FASEB J., 15:2377-89, 2001). This
equilibrium is highly influenced by innervation of the skin. Hsieh
and colleagues have shown that, within seventy-two hours of
denervation, there is a marked and significant thinning of the
epidermis (Hsieh and Lin, Modulation of keratinocyte proliferation
by skin innervation. J. Invest. Dermatol., 113:579-86, 1999),
raising the possibility that denervation negatively affects the
keratinocyte mitotic index. In the same study, Hsieh and colleagues
examined BrdU incorporation in denervated rat skin. They noted that
it was reduced to almost half of the incorporation on the
contralateral side of the same animal, and that epidermal thickness
was reduced by 70% within four days (Hsieh and Lin, Modulation of
keratinocyte proliferation by skin innervation. J. Invest.
Dermatol., 113:579-86, 1999). These data are consistent with a
model in which denervation of the skin leads to a reduction in
keratinocyte growth, resulting in epidermal thinning.
[0005] Importantly, the alterations in keratinocyte and Langerhans
cell anatomy and functionality are reversible. By three months
following mechanical nerve transection, axons have regenerated into
denervated areas, and epidermal thickness has returned to baseline,
as has the keratinocyte proliferative rate (Huang et al., Influence
of cutaneous nerves on keratinocyte proliferation and epidermal
thickness in mice. Neuroscience, 94:965-73, 1999).
[0006] One possible explanation for the above findings is that, in
the absence of sensory innervation, there is a decrease in blood
flow to the target field. Such an alteration may result in either
the accumulation of inhibitory molecules--which would then
negatively affect keratinocyte growth--or a reduction in the
delivery of one or more growth factors to the dermal/epidermal
boundaries--which might also reduce keratinocyte proliferation.
This hypothesis is consistent with the known pathophysiology of
diabetes, in which there is microvascular damage, peripheral
neuropathy, and thinning of the skin, with a propensity toward the
development of ulcers. However, while it is an attractive
hypothesis, it lacks support in the literature.
[0007] For example, Monteiro-Riviere and colleagues have
demonstrated in a number of species that there is no correlation
between blood flow and epidermal thickness (Monteiro-Riviere et
al., Interspecies and interregional analysis of the comparative
histologic thickness and laser Doppler blood flow measurements at
five cutaneous sites in nine species. J. Invest. Dermatol.,
95:582-86, 1990; Monteiro-Riviere et al., Laser Doppler
measurements of cutaneous blood flow in ageing mice and rats.
Toxicol. Lett., 57:329-38, 1991). Thus, the epidermal thinning that
occurs after denervation appears to result from a direct
inter-relationship between sensory fibers and keratinocytes. The
foregoing observations also raise questions as to the perceived
etiology of stasis ulcers. While the dogma holds that decubital
ulcers are the result of poor blood flow and blood pooling in the
skin, this has never been demonstrated or tested rigorously. Given
the above-described data, it is possible that these ulcers develop
as a result of pressure-induced peripheral nerve damage and
subsequent thinning of the skin.
[0008] The findings discussed above do not address whether it is
the axons per se, their associated Schwann cells, or both, that
influence keratinocyte biology. However, consistent with other
examples of nerve regeneration (Gondre et al., Accelerated nerve
regeneration mediated by Schwann cells expressing a mutant form of
the POU protein SCIP. J. Cell Biol., 141:493-501, 1998; Weinstein,
D. E., The role of Schwann cells in neural regeneration. The
Neuroscientist, 5:208-16, 1999; Weinstein et al., Molecular
mechanism of nerve infection in leprosy. Trends Microbiol.,
7:185-86, 1999; Strauch et al., The generation of an artificial
nerve, and its use as a conduit for regeneration. J. Reconstr.
Microsurg., 17:589-98, 2001), regenerating sensory fibers in the
skin grow only in association with Schwann cells (Mihara, M.,
Regenerated cutaneous nerves in human epidermal and subepidermal
regions. An electron microscopy study. Arch. Dermatol. Res.,
276:115-22, 1984), suggesting that the Schwann cells play an
integral role in wound healing. This possibility is further
supported by the observation that glial growth factor (GGF) (also
known as NDF and ARIA), which is secreted by Schwann cells
following injury (Carroll et al., Expression of neuregulins and
their putative receptors, ErbB2 and ErbB3, is induced during
Wallerian degeneration. J. of Neurosci., 17:1642-59, 1997),
stimulates keratinocyte proliferation and increases epidermal
thickness (Danilenko et al., Neu differentiation factor upregulates
epidermal migration and integrin expression in excisional wounds.
J. Clin. Invest., 95:842-51, 1995).
[0009] Refractory skin lesions present a huge therapeutic challenge
in patients with a range of underlying pathologies, including
diabetic ulcers, venous stasis ulcers, pressure ulcers, burns, and
trauma. The possibility of increasing the rate of surgical wound
closure represents another, related, challenge, for such an
increase would provide the benefit of limiting post-operative wound
infection. Finally, great benefit would be derived from a means of
enhancing reinnervation of healing skin, as this would likely limit
paresthesias resulting from failed nerve regeneration. To meet some
of these challenges, investigators have developed therapies that
merely stimulate simple re-epithelialization. This approach, while
offering some benefit, is limited, as epidermis will break down in
the absence of repair of the underlying tissue. Accordingly, given
the huge clinical implications associated with wound healing, there
exists a need to develop a new, satisfactory therapy that will
achieve more than mere stimulation of simple
re-epithelialization.
SUMMARY OF THE INVENTION
[0010] The present invention is based on the discovery that
FK506-related compounds, particularly nonimmunosuppressive
derivatives of FK506 such as GM-284, stimulate growth of
keratinocytes independently, and also stimulate the regeneration of
nerve fibers into previously-damaged skin. This dual ability of the
FK506 derivatives is valuable and unexpected--particularly in view
of the fact that numerous other investigators have shown that, in
the face of denervation, keratinocyte proliferation falters, and
skin breaks down.
[0011] In view of the foregoing, the present invention provides a
method for promoting healing of a wound in a subject, by
administering to the subject an amount of an immunophilin ligand
effective to promote healing of the wound in the subject.
[0012] The present invention further provides a use of an
immunophilin ligand to promote healing of a wound in a subject,
wherein the immunophilin ligand is administered to the subject in
an amount effective to promote healing of the wound in the
subject.
[0013] Additionally, the present invention provides a method for
promoting regeneration of epithelial tissue in a subject, by
administering to the subject an amount of an immunophilin ligand
effective to promote regeneration of epithelial tissue in the
subject.
[0014] Also provided is a use of an immunophilin ligand to promote
regeneration of epithelial tissue in a subject, the immunophilin
ligand is administered to the subject in an amount effective to
promote regeneration of epithelial tissue in the subject.
[0015] The present invention is further directed to a method for
enhancing epithelial cell proliferation, by contacting epithelial
tissue with an amount of an immunophilin ligand effective to
enhance epithelial cell proliferation.
[0016] The present invention also provides a use of an immunophilin
ligand to enhance epithelial cell proliferation, wherein epithelial
tissue is contacted with an amount of the immunophilin ligand
effective to enhance epithelial cell proliferation.
[0017] The present invention is also directed to a method for
modulating gene expression in an epithelial cell, by contacting the
epithelial cell with an amount of an immunophilin ligand effective
to modulate gene expression in the epithelial cell.
[0018] Also provided is a use of an immunophilin ligand to modulate
gene expression in an epithelial cell, wherein the epithelial cell
is contacted with an amount of the immunophilin ligand effective to
modulate gene expression in the epithelial cell.
[0019] Finally, the present invention provides a method for
treating a keratinocyte-associated disorder in a subject in need of
treatment therefor, by administering to the subject an amount of an
immunophilin ligand effective to treat the keratinocyte-associated
disorder in the subject.
[0020] Additional aspects of the present invention will be apparent
in view of the description that follows.
BRIEF DESCRIPTION OF THE FIGURES
[0021] FIG. 1 demonstrates that GM-284 increases the rate at which
wounds close. Animals were treated topically with either the
vehicle (veh) or GM-284, once daily, for the indicated times. As
can be seen, GM-284-treated wounds showed improvement by 3 days
(D3), and were virtually closed by the ninth day (D9).
[0022] FIG. 2 depicts quantification of GM-284-mediated skin
closure. The wounds of every animal in the treatment groups were
photographed at the indicated times, and the area of each open
wound was measured using the image-analysis program, NIH Image. As
can be seen, there was a large difference in the closure rates by
day 5. By day 9, the GM-284-treated skin was approximately as
completely healed as that of the two control groups.
[0023] FIG. 3 shows that GM-284 doubles the proliferative rate of
human keratinocytes. Human keratinocytes were thawed, then either
grown in the presence of increasing concentrations of GM-284, or
grown in 5 .mu.M FeSO.sub.4 as a positive control. No difference
was seen in proliferation before the first passage of cells, which
is usual. However, after the cells were reseeded and then grown in
the indicated concentrations of GM-284, a doubling of the
proliferative index of cells grown in the presence of 1 .mu.M
GM-284 was observed.
[0024] FIG. 4 illustrates that topical treatment with GM-284
increases epidermal thickness in vivo. Eight days after
full-thickness skin biopsies and daily treatment with vehicle,
FK506, or GM-284, the original biopsy sites were rebiopsied and
stained with hemotoxylin and eosin. There was no apparent
difference in the epidermal thickness of the vehicle-treated skin
(panel a) or FK506-treated skin (panel b). In contrast, the
GM-284-treated skin showed a marked increase in epidermal thickness
(panel c).
[0025] FIG. 5A shows that topical treatment with GM-284 increases
the size of adenexal structures, and FIG. 5B illustrates that
topical treatment with GM-284 increases the volume of sebaceous
cells. (A) Eight days following full-thickness skin biopsies and
daily treatment with either vehicle or a 1 .mu.M solution of
GM-284, the original biopsy sites were rebiopsied and stained with
hemotoxylin and eosin. While the inventor observed no net increase
in the number of hair follicles (marked by asterisks) or sebaceous
glands (sg), GM-284 treatment increased the size of both
structures. A low-power magnification of vehicle-treated skin
(panel a) and GM-284-treated skin (panel b) shows the overall
increase in hair-follicle size. Higher-power magnification shows an
GM-284-mediated increase in the size of sebaceous glands (cf.
panels c and d). (B) The plump and full appearance of the sebaceous
cells in the GM-284-treated group suggested an overall increase in
cell volume. Micrographs from the two treatment groups were
scanned, and the volume of sebaceous cells was measured in voxels
(n=>300 cells per treatment group). The resulting data were
binned by 3000-voxel increments. These data show an overall
increase in sebaceous-cell volume, mediated by GM-284. There is an
absence of cells smaller than 3000 voxels in the GM-284-treated
group, and an absence of cells over 9000 voxels in the
vehicle-treated group.
[0026] FIG. 6 illustrates that GM-284 induces rapid reinnervation
of injured skin. (A) The inventor examined innervation of
surgically damaged skin (biopsy) following 8 days of treatment with
either GM-284 or vehicle. The skin sections were stained with an
antibody that recognizes myelin basic protein (MBP). Staining of
normal, naive skin from the dorsal trunk of the mouse showed small,
MBP-positive myelinated fibers that coursed parallel to the
epidermis, and, along with Langerhans cells (LC), aligned in
parallel along the nerve fibers of the skin (left panel). After 8
days of treatment with GM-284 following injury, the skin regained
its pre-injury histology, with new, myelinated fibers running
parallel to the epidermis and the Langerhans cells (middle panel).
In contrast, healing skin which had been treated with vehicle alone
still had no myelinated fibers, nor did the Langerhans cells appear
to have any organization relative to the epidermis (right panel).
(B) Unmyelinated fibers also innervate the skin, where the fibers
extend into the epidermis. Consistent with findings of
GM-284-promoted regeneration of the myelinated fibers, it was
discovered that the compound also promotes regeneration of naked
axons into the epidermis. No such fibers were found in the
vehicle-treated controls.
[0027] FIG. 7 demonstrates that the effects of GM-284 are
reversible. Either GM-284 or vehicle was applied to the dorsal skin
on both sides of the animals, daily for 2 weeks. At that time, a
5-mm biopsy was taken from only one side, and all treatment was
withheld. After an additional 2-week recovery period, a 5-mm biopsy
was taken from the contralateral, intact side. As expected, 2 weeks
of treatment with GM-284, over intact, shaved skin, resulted in
epidermal hypertrophy, while the vehicle had no effect (top
panels). This effect was completely reversed by an additional 2
weeks, when the epidermis had returned to baseline (lower panels).
20.times. magnification
[0028] FIG. 8 illustrates that GM-284 alters neuregulin gene
expression. Previous work has shown that keratinocytes express
neuregulin-.alpha., and that its expression is downregulated
following epidermal wounding. As expected, unwounded,
vehicle-treated keratinocytes strongly express neuregulin-.alpha.
(top panel). In contrast, 2 weeks of topical treatment with GM-284
completely downregulated neuregulin-.alpha. expression (lower
panel). However, allowing the skin to recover for 2 weeks restored
keratinocyte expression of neuregulin-.alpha. (data not shown).
These data are consistent with the notion that GM-284 treatment
alters skin biology toward a pro-healing state.
[0029] FIG. 9 depicts the structure of GM-284.
DETAILED DESCRIPTION OF THE INVENTION
[0030] The inventor has recently described studies that elucidated
a novel, nonimmunosuppressive immunophilin ligand, termed GM-284--a
derivative of the immunosuppressive drug FK506--that enhances
axonal regeneration and induces hypermyelination following
mechanical transection of peripheral nerves. In particular, the
studies demonstrated that GM-284-enhanced nerve regeneration is a
Schwann-cell-dependent effect. During these studies, the inventor
serendipitously observed that the surgical wounds of animals
treated systemically with GM-284 appeared dramatically different
from those of either vehicle- or FK506-treated mice. More detailed
analysis demonstrated unequivocally that the wounds of mice treated
with GM-284 healed at approximately twice the rate as the controls.
Given the biochemical properties of GM-284, and the clinical uses
of FK506 as a topical medication, the inventor investigated the
ability of GM-284 to act as a healing accelerant when applied
topically to full-thickness skin biopsies in mice. Results of that
investigation revealed that GM-284 has the ability to regenerate
wounded epithelial tissue.
[0031] Accordingly, the present invention provides a method for
promoting healing of a wound in a subject in need of wound healing.
As used herein, the term "promoting healing of a wound" means
augmenting, improving, increasing, or inducing closure, healing, or
repair of a wound. The wound may be the result of any affliction
(e.g., disease, injury, surgery), and may be found in any location
of the subject (e.g., an internal wound or an external wound). The
subject may be any animal, but is preferably a mammal (e.g.,
humans, domestic animals, and commercial animals). More preferably,
the subject is a human.
[0032] As disclosed herein, the method of the present invention
comprises administering to a subject in need of wound healing an
amount of an immunophilin ligand effective to promote healing of
the wound in the subject. Immunophilins are ubiquitously-expressed
proteins with peptidyl-proline cis/trans isomerase activity (Galat
and Metcalfe, Peptidylproline cis/trans isomerases. Prog. Biophys.
Mol. Biol., 63:67-118, 1995; Marks, A. R., Cellular functions of
immunophilins. Physiol. Rev., 76:631-49, 1996). As endogenous
intracellular receptors (Kay, J. E., Structure-function
relationships in the FK506-binding protein (FKBP) family of
peptidylprolyl cis-trans isomerases. Biochem. J., 314:361-85,
1996), immunophilins can be further segregated into two distinct
families: FK506-binding proteins (FKBPs) and cyclophilins.
[0033] Unless otherwise indicated, an "immunophilin ligand" is an
agent that is reactive with an immunophilin. As used herein,
"reactive" means the agent has affinity for, binds to, or is
directed against an immunophilin. As further used herein, an
"agent" shall include a protein, polypeptide, peptide, nucleic acid
(including DNA or RNA), antibody, Fab fragment, F(ab').sub.2
fragment, molecule, antibiotic, drug, compound, and any combination
thereof. A Fab fragment is a univalent antigen-binding fragment of
an antibody, which is produced by papain digestion. A F(ab').sub.2
fragment is a divalent antigen-binding fragment of an antibody,
which is produced by pepsin digestion. Additionally, as used
herein, the term "immunophilin ligand" refers to immunophilin
ligands and any analogues and derivatives thereof, including, for
example, a natural or synthetic functional variant of an
immunophilin ligand. Preferably, the immunophilin ligand of the
present invention is a small molecule that binds an immunophilin
receptor.
[0034] It is recognized that FK506 binds with high affinity to
immunophilins (Kay, J. E., Structure-function relationships in the
FK506-binding protein (FKBP) family of peptidylprolyl cis-trans
isomerases. Biochem. J., 314:361-85, 1996). FK506 (tacrolimus)
(Fujisawa Pharmaceutical Co., Ltd, Osaka, Japan) is an
immunosuppressive drug that promotes nerve regeneration (Gold, B.
G., FK506 and the role of immunophilins in nerve regeneration. Mol.
Neurobiol., 15:285-306, 1997; Jost et al., Acceleration of
peripheral nerve regeneration following FK506 administration.
Restor. Neurol. Neurosci., 17:39-44, 2000). A series of compounds,
known as the nonimmunosuppressive immunophilin ligands, have been
synthesized on the basis of FK506. Among these compounds are the
Vertex drug, V10,367 (Vertex Pharmaceuticals, Cambridge Mass.), the
Guilford compound, GPI-1046 (Guilford Pharmaceuticals, Baltimore,
Md.), and a novel nonimmunosuppressive ligand disclosed herein,
termed GM-284. These FK506 mimetics neither bind to, nor inhibit,
calcineurin; therefore, they lack immunosuppressive activity, but
retain the proneuroregenerative activities of the parent compound
(Steiner et al., Neurotrophic immunophilin ligands stimulate
structural and functional recovery in neurodegenerative animal
models. Proc. Natl. Acad. Sci. USA, 94:2019-24, 1997; Hamilton and
Steiner, Immunophilins: beyond immunosuppression. J. Med. Chem.,
41:5119-43, 1998).
[0035] Accordingly, in one embodiment of the present invention, the
immunophilin ligand is FK506 or an FK506 analogue or derivative. As
used herein, an "FK506 derivative" is a chemical substance derived
from FK506, either directly or by modification, truncation, or
partial substitution. FK506 and its analogues and derivatives may
be produced synthetically. The FK506 derivative for use in the
present invention may be nonimmunosuppressive. In a preferred
embodiment of the present invention, the nonimmunosuppressive FK506
derivative is GM-284. This novel compound is a small molecule that
effects transcriptional change in Schwann cells, and promotes wound
healing and regeneration of epithelial tissue and nervous tissue,
as described below. GM-284 is an immunophilin ligand; its
disassociation constant (k.sub.d), as a measure of binding affinity
for recombinant FKBP52, and as determined by solution-phase
tryptophan fluorescence (QTFS), is 139 nm..+-..16.2. The structure
of GM-284 is depicted in FIG. 9.
[0036] GM-284 may be prepared as follows. Triethlyamine (1.908 g,
2.63 mL, 18.89 mmol) followed by ethyl chloriformate (2.05 g, 1.806
mL, 18.86 mmol) is added to a stirred solution of
(2S)-1-(1,2-dioxo-3,3-dimethylpentyl)-2-pyrrolidinecarboxylic acid
(4,557 g, 18.89 mmol) in tetrahydofuran (130 mL) cooled to about
-15.degree. C. (MeOH/ice bath). After stirring at about -15.degree.
C. to about -10.degree. C. for about 30 min, a solid that is
precipitated is removed by filtration and the filtrate and washings
are brought to about a volume of 170 mL with the addition of
tetrahydrofuran. While stirring the solution of the mixed anhydride
(85 mL, 9.32 mmol) at about 0.degree. C., hydrazine monohydrate
(0.48 mL, 9.79 mmol) is added. The mixture is stirred and allowed
to warm to about RT overnight. After removing the solvent in vacuo,
the residue is purified by column chromatography as an eluent to
obtain a colorless solid (0.64 g, 28.7% yield) which was
recrystallized from ether/pentene mp 177-178.degree. C. CIMS 479
(MH.sup.+), 501 (M+Na.sup.-) .sup.1H NMR (300 MHz, CDCl.sub.3,
mixture of rotamers) .delta. (for the major, transrotamer) 9.06 (br
s. 2H), 4.61 (m, 2H), 3.50-3.46 (m, 4H), 2.40-2.36 (m, 2H),
2.13-1.94 (m, 6H), 1.83-1.64 (m, 4H), 1.25 and 1.21 (each s, each
6H), 0.87 (t, 3H). IR(KBr) cm.sup.-1: 3261, 2970, 1706, 1684, 1636.
Anal. Calcd. for C.sub.24H.sub.38N.sub.4O.sub.6: C, 60.23; H, 8.00;
N, 11.71. Found C, 60.30; N, 11.58.
[0037] Pyridine (0.12 mL, 3.35 mmol) followed by thionyl chloride
(0.120 mL, 1.66 mmol) is added to a vigorously stirred ice cold
slurry of the foregoing colorless solid (0.567 g, 1.185 mmol) in
dry ether (400 mL). After stirring the mixture at about 0.degree.
C. for about 2 h the precipitated solids are removed by filtration,
washed quickly with dry ether and the combined filtrates are
evaporated to dryness in vacuo at <40.degree. C. The residue
(0.6235 g foam) is dissolved in dry toluene (24 mL) and heated to
reflux under nitrogen for about 3 h. The residue obtained by
evaporating toluene in vacuo is then purified by column
chromatography on silica gel/CH.sub.2CL.sub.2. Elution with 1%
methanol/methylene chloride yields GM 284. GM 284 (0.354 g, 63.6%
yield) as a colorless solid, recrystallized from ether/pentene, mp
123-124.degree. C.; [.alpha.].sup.24.sub.D-74.60 (c=0.8,
CHCL.sub.3). CIMS 461 (MH.sup.+), 483 (M+Na.sup.+). .sup.1H NMR
(CDCl.sub.3, mixture of rotamers) .delta. (for the major,
transrotamer) 5.32 (d, d, J=3.0, 7.6), 3.59 (m 4H), 2.33-2.07 (m
8H), 1.80-1.65 (m, 4H), 1.23 and 1.20 (each s, each 6H), 0.86 (t,
6H). IR (KBr) cm.sup.-1: 2972, 1704, 1641. Anal. Calcd. for
C.sub.24H.sub.38N.sub.4O.sub.6: C, 62.59; H, 7.88; N 12.16, 11.71.
Found C, 60.68; H 7.75; N, 12.14.
[0038] GM284 may also be prepared as follows. Hexamethydisilazane
(0.123 mL, 0.585 mM), imidazole (10 mg), and tetrabutyl ammonium
fluoride (10 mg) may be added to a solution of the aforesaid
colorless solid (0.112 g, 0.234 mM) in chlorobenzene (10 mL), and
the mixture heated to reflux under nitrogen for about 72 h.
Chromatographic purification of the crude product yields GM-284.
[PCT/US00/16221 at page 84]
[0039] GM284 may further be prepared as follows. To a solution of
the aforesaid colorless solid (0.2018 g, 0.42 mmol) in
tetrahydrofuran (10 mL) may be added
(methoxycarbonylsulfamoyl)-triethlyamine hydroxide inner salt
(Burgess Reagent total 0.3014 g, 1.265 mmol) in three lots, each
added about every 30 min. The mixture is then stirred at about RT
for about 72 h. After removing the solvent in vacuo, flash
chromatography of the reaction residue yields GM-284
[PCT/US00/16221 at pages 84-85]
[0040] GM-284 has been shown to more rapidly restore whisker
movement compared to control in rats that have undergone unilateral
facial nerve compression with resultant paralysis of the whisker
muscle on the affected side.
[0041] It is believed that GM-284 will be effective as a drug to
treat wounds, as well as many types of disorders associated with
epithelial tissue degeneration, keratinocytes, and nervous tissue
degeneration.
[0042] In the method of the present invention, an immunophilin
ligand is administered to a subject in an amount effective to
promote healing of a wound in the subject. As used herein, the
phrase "effective to promote healing of a wound" means effective to
ameliorate or minimize the clinical impairment or symptoms
associated with the wound. For example, where the wound is a
superficial injury to or cut in the skin of the subject, the
clinical impairment or symptoms associated with the wound may be
ameliorated or minimized by inducing or accelerating closure of the
wound; by promoting regeneration of epithelial tissue at the site
of the wound; by enhancing regeneration of at least one damaged
neurite at the site of the wound; and/or by enhancing remyelination
of at least one damaged neurite at the site of the wound. In one
embodiment of the present invention, the effective amount of the
immunophilin ligand (e.g., GM-284) is between about 1 mg/kg and
about 10 mg/kg. More preferably, the effective amount is about 5
mg/kg. In another embodiment of the present invention, the
effective amount of the immunophilin ligand (e.g., GM-284) is
between about 0.1 pM and about 5 mM. More preferably, the effective
amount is between about 5 pM and about 1.5 mM.
[0043] As used herein, the term "promoting regeneration of
epithelial tissue" means augmenting, improving, increasing, or
inducing partial or full growth or regrowth of epithelial tissue
that has degenerated at the site of a wound. As further used
herein, the term "growth" refers to an increase in mass, volume,
and/or thickness of epithelial tissue, and includes an increase in
keratinocyte proliferation. Regeneration, and enhanced
regeneration, of epithelial tissue may be measured or detected by
known procedures, including assays of blood for markers of wound
healing (including collagen subtypes), clinical examination,
electron microscopy, gene expression studies, immunohistochemistry,
light microscopy, and any of the methods, molecular procedures, and
assays disclosed herein. Regeneration of epithelial tissue at the
site of a wound may be promoted, for example, by enhancing
proliferation of keratinocytes at the site of the wound.
[0044] The term "epithelial tissue", as used herein, refers to
tissue on the exterior of the body of a subject, or layering its
interior surfaces, that is covered by continuous cellular sheets
known as epithelial membranes (or epithelia) and the various glands
(both exocrine and endocrine) that develop therefrom, and includes,
without limitation, any or all of the following: endothelium,
mesothelium, and skin (including epidermis and dermis). Examples of
cells of the epidermis include, without limitation, Langerhans
cells, keratinocytes, and melanocytes. Keratinocytes are committed
cells, arising deep in the epidermis, that undergo gradual
transformation into scales of keratin as they become displaced
toward the surface. Epithelial tissue that produces keratin is
referred to herein as "keratinizing epithelial tissue".
[0045] As further used herein, the term "enhancing regeneration of
a damaged neurite" means augmenting, improving, or increasing
partial or full growth or regrowth of a neurite that has
degenerated. As further used herein, the term "growth" refers to an
increase in diameter, length, mass, and/or thickness of a neurite,
a neuron, or myelin, as the case may be. Causes of neurite
degeneration include damage to nervous tissue, death of neurons,
demyelination, and injury, all associated with a wound in the
subject. Regeneration of the neurite may take place in neurites of
both the central nervous system and the peripheral nervous system.
Regeneration, and enhanced regeneration, of neurites may be
measured or detected by known procedures, including Western
blotting for myelin-specific and axon-specific proteins, light or
electron microscopy in conjunction with morphometry, and any of the
methods, molecular procedures, and assays disclosed herein.
[0046] Additionally, as used herein, the term "enhancing
remyelination of a neurite" means augmenting, improving, or
increasing partial or full growth or regrowth of the myelin of a
neurite that has degenerated. The remyelination of the neurite may
take place in the nerves of both the CNS and the PNS.
Remyelination, and enhanced remyelination, of neurites may be
measured or detected by known procedures, including Western
blotting for myelin-specific and axon-specific proteins, electron
microscopy in conjunction with morphometry, and any of the methods,
molecular procedures, and assays disclosed herein.
[0047] The term "nervous tissue", as used herein, includes the
nervous tissue present in both the central nervous system and the
peripheral nervous system, and comprises any or all of the
following: axons, dendrites, fibrils, fibular processes, ganglion
cells, granule cells, grey matter, myelin, neuroglial cells,
neurolimma, neuronal cells or neurons, Schwann cells, stellate
cells, and white matter. As further used herein, a "neuron" is a
conducting or nerve cell of the nervous system that typically
consists of a cell body (perikaryon) that contains the nucleus and
surrounding cytoplasm; several short, radiating processes
(dendrites); and one long process (the axon), which terminates in
twig-like branches (telodendrons), and which may have branches
(collaterals) projecting along its course. Examples of neurons
include, without limitation, autonomic neurons, neurons of the
dorsal root ganglia (DRG), enteric neurons, interneurons, motor
neurons, peripheral neurons, sensory neurons, and neurons of the
spinal cord. In one embodiment of the present invention, the
damaged nervous tissue comprises damaged peripheral neurons.
[0048] Additionally, as used herein, the term "neurite" refers to
processes of neuronal cells, and includes axons and dendrites. For
example, the neurite of the present invention may be a process
extending from a neuron, such as an autonomic neuron, a neuron of
the dorsal root ganglia (DRG), an enteric neuron, an interneuron, a
motor neuron, a peripheral neuron, a sensory neuron, or a neuron of
the spinal cord. Thus, the neurite may be, for example, an
autonomic neuron neurite, a DRG neurite, an enteric neuron neurite,
an interneuron neurite, a motor neuron neurite, a peripheral neuron
neurite, a sensory neuron neurite, and a neurite of the spinal
cord. In one embodiment of the present invention, the neurite is a
peripheral neuron neurite.
[0049] As demonstrated herein, the immunophilin ligand, GM-284, has
the ability to promote wound healing by promoting regeneration of
epithelial tissue at the site of the wound; and/or by enhancing
regeneration or remyelination of at least one damaged neurite
(e.g., a DRG neurite, an interneuron neurite, a motor neuron
neurite, a peripheral neuron neurite, a sensory neuron neurite, or
a neurite of the spinal cord) at the site of the wound. The amount
of immunophilin ligand effective to promote healing of a wound in a
subject in need of wound healing will vary depending upon the
particular factors of each case, including the type of wound, the
severity of the wound, and the method of administration. This
amount may be readily determined by the skilled artisan, based upon
known procedures, including clinical trials, and methods disclosed
herein.
[0050] According to the method of the present invention, an
immunophilin ligand may be administered to a human or animal
subject by known procedures, including, without limitation, oral
administration, parenteral administration (e.g., epifascial,
intracapsular, intracutaneous, intradermal, intramuscular,
intraorbital, intraperitoneal, intraspinal, intrasternal,
intrathecal, intravascular, intravenous, parenchymatous, or
subcutaneous administration), sublingual administration, topical
administration, transdermal administration, and administration
through an osmotic mini-pump. Preferably, the immunophilin ligand
is administered topically.
[0051] For oral administration, the formulation of the immunophilin
ligand may be presented as capsules, tablets, powders, granules, or
as a suspension. The formulation may have conventional additives,
such as lactose, mannitol, cornstarch, or potato starch. The
formulation also may be presented with binders, such as crystalline
cellulose, cellulose derivatives, acacia, cornstarch, or gelatins.
Additionally, the formulation may be presented with disintegrators,
such as cornstarch, potato starch, or sodium
carboxymethylcellulose. The formulation also may be presented with
dibasic calcium phosphate anhydrous or sodium starch glycolate.
Finally, the formulation may be presented with lubricants, such as
talc or magnesium stearate.
[0052] For parenteral administration (i.e., administration by
injection through a route other than the alimentary canal), the
immunophilin ligand may be combined with a sterile aqueous solution
that is preferably isotonic with the blood of the subject. Such a
formulation may be prepared by dissolving a solid active ingredient
in water containing physiologically-compatible substances, such as
sodium chloride, glycine, and the like, and having a buffered pH
compatible with physiological conditions, so as to produce an
aqueous solution, then rendering said solution sterile. The
formulations may be presented in unit or multi-dose containers,
such as sealed ampoules or vials. The formulation may be delivered
by any mode of injection, including, without limitation,
epifascial, intracapsular, intracranial, intracutaneous,
intramuscular, intraorbital, intraperitoneal, intraspinal,
intrasternal, intrathecal, intravascular, intravenous,
parenchymatous, or subcutaneous.
[0053] For transdermal administration, the immunophilin ligand may
be combined with skin penetration enhancers, such as propylene
glycol, polyethylene glycol, isopropanol, ethanol, oleic acid,
N-methylpyrrolidone, dimethyl sulfoxide, and the like, which
increase the permeability of the skin to the immunophilin ligand,
and permit the immunophilin ligand to penetrate through the skin
and into the bloodstream. The ligand/enhancer compositions also may
be further combined with a polymeric substance, such as
ethylcellulose, hydroxypropyl cellulose, ethylene/vinylacetate,
polyvinyl pyrrolidone, and the like, to provide the composition in
gel form, which may be dissolved in solvent, such as methylene
chloride, evaporated to the desired viscosity, and then applied to
backing material to provide a patch. The immunophilin ligand may be
administered transdermally at the site of the wound in the subject
where neural trauma has occurred, or where the wound is localized.
Alternatively, the immunophilin ligand may be administered
transdermally at a site other than the affected area, in order to
achieve systemic administration.
[0054] For topical administration, the immunophilin ligand may be
combined with additional materials that are known for use in
skin-care products, or which are otherwise suitable for topical
application. Such optional materials include, but are not limited
to, disbursing agents, masking agents, preservatives, processing
agents, and additives having specific physicochemical properties,
such as polymeric film formers and the like.
[0055] The immunophilin ligand of the present invention also may be
released or delivered from an osmotic mini-pump or other
time-release device. The release rate from an elementary osmotic
mini-pump may be modulated with a microporous, fast-response gel
disposed in the release orifice. An osmotic mini-pump would be
useful for controlling release, or targeting delivery, of the
immunophilin ligand.
[0056] It is within the confines of the present invention that a
formulation containing GM-284 may be further associated with a
pharmaceutically acceptable carrier, thereby comprising a
pharmaceutical composition. Accordingly, the present invention
further provides a pharmaceutical composition, comprising GM-284
and a pharmaceutically acceptable carrier. The pharmaceutically
acceptable carrier must be "acceptable" in the sense of being
compatible with the other ingredients of the composition, and not
deleterious to the recipient thereof. Examples of acceptable
pharmaceutical carriers include carboxymethylcellulose, crystalline
cellulose, glycerin, gum arabic, lactose, magnesium stearate,
methyl cellulose, powders, saline, sodium alginate, sucrose,
starch, talc, and water, among others. Formulations of the
pharmaceutical composition may be conveniently presented in unit
dosage.
[0057] The formulations of the present invention may be prepared by
methods well known in the pharmaceutical arts. For example, GM-284
may be brought into association with a carrier or diluent, as a
suspension or solution. Optionally, one or more accessory
ingredients (e.g., buffers, flavoring agents, surface active
agents, and the like) also may be added. The choice of carrier will
depend upon the route of administration. The pharmaceutical
composition would be useful for administering the GM-284 of the
present invention to a subject to promote healing of a wound. The
GM-284 is provided in an amount that is effective to promote wound
healing in a subject to whom the pharmaceutical composition is
administered. That amount may be readily determined by the skilled
artisan, as described above.
[0058] The present invention also provides a method for promoting
regeneration of epithelial tissue in a subject. As described above,
regeneration of epithelial tissue in a subject may be promoted by
enhancing proliferation of keratinocytes in the subject.
Accordingly, in one embodiment of the invention, the epithelial
tissue is keratinizing epithelial tissue. In a further embodiment
of the invention, the regeneration of epithelial tissue is promoted
at the site of a wound in the subject, and, thus, contributes to
the promotion of wound healing in the subject.
[0059] The method of the present invention comprises the step of
administering an immunophilin ligand to a subject. In one
embodiment of the present invention, the immunophilin ligand is
FK506 or an FK506 derivative. The FK506 derivative for use in the
present invention may be nonimmunosuppressive. In a preferred
embodiment of the present invention, the nonimmunosuppressive FK506
derivative is GM-284. The immunophilin ligand is administered to a
subject in an amount effective to promote regeneration of
epithelial tissue in the subject, as defined above. In one
embodiment of the present invention, the effective amount of the
immunophilin ligand (e.g., GM-284) is between about 1 mg/kg and
about 10 mg/kg. More preferably, the effective amount is about 5
mg/kg. In another embodiment of the present invention, the
effective amount of the immunophilin ligand (e.g., GM-284) is
between about 0.1 pM and about 5 mM. More preferably, the effective
amount is between about 5 pM and about 1.5 mM.
[0060] The present invention further provides a method for
enhancing epithelial cell proliferation. As used herein in
reference to epithelial cells, the term "enhancing proliferation"
means augmenting, improving, or increasing cell division, cell
number, and/or growth of epithelial cells, and includes increasing
the proliferative rate of keratinocytes and other epithelial cells,
as disclosed herein. Enhancement of the growth and proliferation of
epithelial cells may be measured or detected by known procedures,
including electron microscopy and any of the methods, molecular
procedures, and assays disclosed herein. As further used herein, an
"epithelial cell" is any cell found in, or derived from, epithelial
tissue. Examples of epithelial cells include, without limitation,
follicular cells, glandular cells (e.g., sebaceous cells),
Langerhans cells, neurons and other neural cells, and skin cells.
In a preferred embodiment of the present invention, the epithelial
cell is a keratinocyte.
[0061] The method of the present invention comprises contacting
epithelial tissue with an immunophilin ligand. The epithelial
tissue may be damaged or healthy/undamaged. However, in one
embodiment of the present invention, the epithelial tissue
comprises a wound. In another embodiment of the present invention,
the immunophilin ligand is FK506 or an FK506 derivative. The FK506
derivative for use in the present invention may be
nonimmunosuppressive. Preferably, the nonimmunosuppressive FK506
derivative is GM-284. The immunophilin ligand is contacted with
epithelial tissue in an amount effective to enhance proliferation
of at least one epithelial cell. This amount may be determined by
the skilled artisan using known procedures (e.g., concentration
curves, ELISA, protein-concentration determination,
radioimmunoassay, titration curves, and methods disclosed
herein.
[0062] The method of the present invention may be used to enhance
proliferation of at least one epithelial cell in vitro, or in vivo
in a subject. For example, an immunophilin ligand may be contacted
in vitro with epithelial tissue (e.g., a biopsy or plug of
epithelial tissue removed from a subject) by introducing the
immunophilin ligand to the tissue using conventional procedures.
Alternatively, an immunophilin ligand may be contacted in vivo with
epithelial tissue in a subject by administering the immunophilin
ligand to the subject. It is also within the confines of the
present invention that an immunophilin ligand may be introduced to
epithelial tissue in vitro, using conventional procedures, to
achieve enhanced proliferation of keratinocytes in vitro.
Thereafter, epithelial tissue containing keratinoctyes may be
introduced into a subject to provide keratinocytes in vivo. In such
an ex vivo approach, the epithelial tissue is preferably removed
from the subject, subjected to introduction of the immunophilin
ligand, and then reintroduced into the subject. In one embodiment
of the invention, the enhanced epithelial cell proliferation
promotes healing of a wound in the subject.
[0063] The ability of immunophilin ligands, particularly FK506
derivatives such as GM-284, to enhance proliferation of
keratinocytes in epithelial tissue, as disclosed herein, renders
immunophilin ligands particularly useful for treating conditions
associated with dysregulation of keratinocyte proliferation. It is
believed that immunophilin ligands, including GM-284, would be
effective either alone or in combination with other therapeutic
agents that are typically used in the treatment of these
conditions.
[0064] Accordingly, the present invention provides a method for
treating a keratinocyte-associated disorder in a subject in need of
treatment, comprising contacting epithelial tissue in the subject
with an immunophilin ligand (e.g., by administering the
immunophilin ligand to the subject), thereby treating the
keratinocyte-associated disorder. Keratinocyte-associated disorders
that may be treated by methods disclosed herein include disorders
characterized by a dysregulation of keratinocyte proliferation
(e.g., disorders associated with abnormal keratinocytes, abnormal
keratin, or too few keratinocytes).
[0065] Examples of keratinocyte-associated disorders include,
without limitation, Darier-White's disease; epidermolytic
hyperkeratosis; erythema ab igne; IKK gamma/NEMO deficiency;
leprosy; a neoplasia (e.g., a carcinoma, such as a basal cell or
squamous cell carcinoma; cervical cancer; and oral cancer);
periodontal disease; a peripheral neuropathy (including peripheral
neuropathies associated with such conditions as acute or chronic
inflammatory polyneuropathy, amyotrophic lateral sclerosis (ALS),
collagen vascular disorder (e.g., polyarteritis nodosa, rheumatoid
arthritis, Sjogren's syndrome, or systemic lupus erythematosus),
diphtheria, Guillain-Barre syndrome, hereditary peripheral
neuropathy (e.g., Charcot-Marie-Tooth disease (including type I,
type II, and all subtypes), hereditary motor and sensory neuropathy
(types I, II, and III, and peroneal muscular atrophy), hereditary
neuropathy with liability to pressure palsy (HNPP), infectious
disease (e.g., acquired immune deficiency syndrome (AIDS)), Lyme
disease (e.g., infection with Borreia burgdorferi), invasion of a
microorganism (e.g., leprosy), leukodystrophy, metabolic disease or
disorder (e.g., amyloidosis, diabetes mellitus, hypothyroidism,
porphyria, sarcoidosis, or uremia), neurofibromatosis, nutritional
deficiencies, peroneal nerve palsy, polio, porphyria, postpolio
syndrome, Proteus syndrome, pressure paralysis (e.g., carpal tunnel
syndrome), progressive bulbar palsy, radial nerve palsy, spinal
muscular atrophy, a toxic agent (e.g., barbital, carbon monoxide,
chlorobutanol, dapsone, emetine, heavy metals, hexobarbital, lead,
nitrofurantoin, orthodinitrophenal, phenytoin, pyridoxine,
sulfonamides, triorthocresyl phosphate, the vinca alkaloids, many
solvents, other industrial poisons, and certain AIDS drugs
(including didanosine and zalcitabine), trauma (including neural
trauma--the leading cause of peripheral neuropathy, worldwide), and
ulnar nerve palsy (Beers and Berkow, eds., The Merck Manual of
Diagnosis and Therapy, 17.sup.th ed. (Whitehouse Station, NJ: Merck
Research Laboratories, 1999) chap. 183); porokeratosis; a pressure
ulcer; psoriasis; striate palmoplantar keratoderma; a skin
ulceration (e.g., a diabetic ulcer); a venous stasis ulcer; and a
wound. In a preferred embodiment, the keratinocyte-associated
disorder is leprosy, periodontal disease, a peripheral neuropathy,
a pressure ulcer, psoriasis, a skin ulceration, a venous stasis
ulcer, or a wound.
[0066] In the method of the present invention, an immunophilin
ligand is contacted with epithelial tissue in a subject (e.g.,
administered to a subject), for the purpose of treating a
keratinocyte-associated disorder, in an amount effective to enhance
proliferation of at least one epithelial cell. In one embodiment of
the present invention, the effective amount of the immunophilin
ligand (e.g., GM-284) is between about 0.1 pM and about 5 mM. More
preferably, the effective amount is between about 5 pM and about
1.5 mM.
[0067] Previous work has shown that keratinocytes express
neuregulin-.alpha., and that its expression is downregulated
following epidermal wounding. In the present study, the inventor
has demonstrated that neuregulin-.alpha. is downregulated by the
immunophilin ligand, GM-284. These findings are consistent with the
notion that GM-284 treatment alters skin biology toward a
pro-healing state. In view of the foregoing, the present invention
further provides a method for modulating gene expression in an
epithelial cell, by contacting the epithelial cell with an
immunophilin ligand.
[0068] As used herein, the term "modulating gene expression"
includes altering gene expression by increasing or upregulating
gene expression, or by decreasing or downregulating gene
expression. By way of example, the expression of an epithelial cell
gene may be modulated by contacting an epithelial cell with an
immunophilin ligand. Examples of epithelial cell genes that may be
modulated by the method of the present invention include, without
limitation, a neuregulin and interleukin-1 homologue 1. In one
embodiment of the present invention, the neuregulin is
neuregulin-.alpha.. In a further embodiment of the invention, the
neuregulin is neuregulin-.alpha. and its expression is
downregulated. Modulation of gene expression in epithelial cells
may be measured or detected by known procedures, including
cDNA-array assays of gene expression, Northern blotting, and any of
the methods, molecular procedures, and assays disclosed herein.
[0069] Examples of epithelial cells in which gene expression may be
modulated include, without limitation, follicular cells, glandular
cells (e.g., sebaceous cells), Langerhans cells, neurons and other
neural cells, and skin cells. In a preferred embodiment of the
present invention, the epithelial cell is a keratinocyte. The
epithelial cell may be, for example, in epithelial tissue. In the
method of the present invention, the epithelial cell is contacted
with an amount of immunophilin ligand effective to modulate gene
expression in the epithelial cell. This amount may be readily
determined by the skilled artisan, based upon known procedures,
including analysis of in vivo dose curves and electron microscopy,
and methods disclosed herein.
[0070] The method of the present invention may be used to modulate
gene expression in an epithelial cell in vitro, ex vivo, or in vivo
in a subject, in accordance with methods described above. In one
embodiment of the present invention, the immunophilin ligand is
FK506 or an FK506 derivative. The FK506 derivative for use in the
present invention may be nonimmunosuppressive. In a preferred
embodiment of the present invention, the nonimmunosuppressive FK506
derivative is GM-284.
[0071] It is believed that, by modulating gene expression in
epithelial cells, immunophilin ligands will be useful for the
treatment of conditions associated with dysregulation of
keratinocyte proliferation. It is further believed that
immunophilin ligands, including GM-284, would be effective either
alone or in combination with other therapeutic agents that are
typically used in the treatment of these conditions.
[0072] Accordingly, the present invention provides a method for
treating a keratinocyte-associated disorder in a subject in need of
treatment, comprising contacting an epithelial cell in the subject
with an immunophilin ligand (e.g., by administering the
immunophilin ligand to the subject), thereby treating the
keratinocyte-associated disorder. Examples of
keratinocyte-associated disorders that may be treated by the method
of the present invention are discussed above. In a preferred
embodiment of the present invention, the keratinocyte-associated
disorder is leprosy, periodontal disease, a peripheral neuropathy,
a pressure ulcer, psoriasis, a skin ulceration, a venous stasis
ulcer, or a wound.
[0073] In the method of the present invention, an immunophilin
ligand is contacted with an epithelial cell in a subject (e.g.,
administered to a subject), for the purpose of treating a
keratinocyte-associated disorder, in an amount effective to
modulate gene expression in the epithelial cell. In one embodiment
of the present invention, the effective amount of the immunophilin
ligand (e.g., GM-284) is between about 0.1 pM and about 5 mM. More
preferably, the effective amount is between about 5 pM and about
1.5 mM.
[0074] The present invention also provides a method for treating a
keratinocyte-associated disorder in a subject in need of treatment,
by administering an immunophilin ligand to the subject, as
described above. Examples of keratinocyte-associated disorders that
may be treated by the method of the present invention are discussed
above. In a preferred embodiment of the present invention, the
keratinocyte-associated disorder is leprosy, periodontal disease, a
peripheral neuropathy, a pressure ulcer, psoriasis, a skin
ulceration, a venous stasis ulcer, or a wound. In one embodiment of
the present invention, the immunophilin ligand is FK506 or an FK506
derivative. The FK506 derivative for use in the present invention
may be nonimmunosuppressive. In a preferred embodiment of the
invention, the nonimmunosuppressive FK506 derivative is GM-284.
[0075] The immunophilin ligand of the present invention is
administered to a subject in need of treatment for a
keratinocyte-associated disorder in an amount that is effective to
treat the keratinocyte-associated disorder in the subject. As used
herein, the phrase "effective to treat a keratinocyte-associated
disorder" means effective to ameliorate or minimize the clinical
impairment or symptoms of the keratinocyte-associated disorder. For
example, where the keratinocyte-associated disorder is a peripheral
neuropathy, the clinical impairment or symptoms of the peripheral
neuropathy may be ameliorated or minimized by alleviating vasomotor
symptoms, increasing deep tendon reflexes, reducing muscle atrophy,
restoring sensory function, and strengthening muscles. The amount
of immunophilin ligand effective to treat a keratinocyte-associated
disorder in a subject in need of treatment therefor will vary
depending upon the particular factors of each case, including the
type of keratinocyte-associated disorder, the stage of the
keratinocyte-associated disorder, the subject's weight, the
severity of the subject's condition, and the method of
administration. This amount may be readily determined by the
skilled artisan, based upon known procedures, including clinical
trials, and methods disclosed herein.
[0076] The present invention is described in the following
Examples, which are set forth to aid in the understanding of the
invention, and should not be construed to limit in any way the
scope of the invention as defined in the claims which follow
thereafter.
EXAMPLES
Example 1
GM-284 Promotes Wound Healing
[0077] In recent studies, the inventor demonstrated myelin and
axonal hypertrophy following mechanical transection of the sciatic
nerve. These studies required surgical exposure of the right lower
quadrant of all study animals. Following exposure of the sciatic
nerve from the sciatic notch to the knee, and manipulation of the
nerve, the muscle and overlaying skin were closed with suture
material. Each animal was subsequently treated with a daily gavage
of either GM-284 (5 mg/kg), FK506 (5 mg/kg), or methylcellulose
vehicle alone. During the post-operative course, it was noted by an
investigator who was blinded to the treatment groups that the
surgical wounds of one group of animals appeared to be
qualitatively improved over those of the other two groups, with
less erythemia and more rapid closure. Following unblinding of the
investigator, it was learned that the group demonstrating improved
healing had received treatment with GM-284.
[0078] To determine whether GM-284 had a direct effect on the skin,
or whether the enhanced wound closure was an indirect effect of
GM-284 that was influenced by healing of the underlying muscle, the
experiments were repeated, with one exception: only the skin was
entered, and the muscle and overlying fascia were left intact.
Under these conditions, the skin of GM-284-treated animals showed
the same improved healing characteristics as were demonstrated when
the muscle was also involved, suggesting that GM-284 acts directly
on the skin proper.
[0079] The foregoing results also raised the possibility that
GM-284 might show similar effects if administered topically,
directly to the surgical site. To investigate this possibility, 10
retired breeders--5 male mice and 5 female mice--were prepared for
surgery, and were subjected to 5-mm skin biopsies using a
disposable biopsy punch. After achieving hemostasis, the wounds
were treated immediately with topical application of either GM-284
or vehicle, or received no treatment at all. The treatment was
repeated daily. As shown in FIG. 1, the GM-284-treated wounds, by 3
days, were significantly more closed than the control groups. By 9
days, the GM-284-treated wounds were virtually closed. Furthermore,
the edges of the GM-284-treated wounds appeared to be better
granulated than the controls (cf. the GM-284-treated skin and the
control skin on D3 and D9 in FIG. 1).
[0080] During the experimental period, the wounds were photographed
daily, and the photographs were scanned for volume analysis. The
open area of each wound was quantified using an image-analysis
program, NIH Image. By the third treatment day, the GM-284-treated
wounds had closed approximately twice as fast as the control
groups. The increased rate of GM-284-mediated wound closure was
maintained at 9 days, when the wounds were approximately one-third
the size of those of the control groups (FIG. 2).
Example 2
GM-284 Doubles the Keratinocyte Proliferative Index
[0081] As detailed above, keratinocyte proliferation is influenced
by a number of factors, including innervation (Hsieh and Lin,
Modulation of keratinocyte proliferation by skin innervation. J.
Invest. Dermatol., 113:579-86, 1999) and growth-factor treatment
(Castagnino et al., Neu differentiation factor/heregulin induction
by hepatocyte and keratinocyte growth factors. Oncogene, 19:640-48,
2000; and reviewed by Werner and Smola, Paracrine regulation of
keratinocyte proliferation and differentiation. Trends Cell Biol.,
11:143-46, 2001). Thus, by culturing human-foreskin keratinocytes
with increasing concentrations of GM-284, the inventor investigated
the possibility that GM-284 was able to influence keratinocyte
growth. As shown in FIG. 3, treatment with 1 .mu.M of GM-284
increased the rate of keratinocyte proliferation by more than 150%
after 14 days of treatment. Not surprisingly, a comparison of the
growth rates of keratinocytes grown in 1 .mu.M or 10 .mu.M GM-284
showed that the increased drug concentration completely ablated the
pro-proliferative activity observed at 1 .mu.M. This type of
drug-dependent inhibition is consistent with the inventor's
previous observation that a higher concentration of GM-284 blocks
gene expression that is induced at a lower drug concentration (data
not shown). Importantly, these data demonstrate that GM-284 has a
direct effect on keratinocytes, and also has an effect on the
underlying nerves (see below).
Example 3
Topical Treatment with GM-284 Increases Epidermal Thickness In
Vivo
[0082] The increased rate of wound closure in the presence of
GM-284, taken with the accelerated keratinocyte proliferation,
suggested to the inventor the possibility that GM-284 treatment
might alter epidermal thickness in vivo. To examine this
possibility, 5-mm, full-skin thickness biopsies were taken from the
hindquarters of both male and female ICR retired breeders. The
wounds were treated daily, for 8 days, with topical administration
of either the DMSO-based vehicle, or FK506 or GM-284 in the
vehicle. Thereafter, the original biopsy site was rebiopsied.
Comparison of epidermal thickness of the FK506- and vehicle-treated
animals showed no observable differences (cf. FIG. 4, panels a and
b). In contrast, the epidermis of the GM-284-treated animals was
much thicker, and appeared to be much more cellular, than the
epidermis of animals under the other two conditions (FIG. 4, panel
c). Without being bound by theory, it is believed that, given the
GM-284-mediated increase in keratinocyte proliferation, the in vivo
epidermal hypertrophy demonstrated herein was a result of
GM-284-induced increases in keratinocyte proliferation.
Example 4
Topical Treatment of Wounds with GM-284 Induces Hypertrophy of
Adenexal Structures
[0083] In addition to the increased epidermal thickness
demonstrated above, treatment with GM-284 also affects intradermal
structures. Using the topical-application-following-skin-biopsy
paradigm detailed above, the lateral aspects of the original
lesions were rebiopsied 8 days after the original lesions. The
tissues were immersion-fixed, dehydrated through step gradients of
alcohols, stained with hemotoxylin and eosin, and mounted for
microscopic analysis, all following standard protocols. It is known
that skin wounds in rodents heal inwards, from the perimeter,
converging at a central point (Cohen, Stress and wound healing.
Acta Anat. (Basel), 103:134-41, 1979). Therefore, the lateral edges
of the original lesions were selected for analysis because these
areas of tissue had the greatest opportunity to regenerate under
the experimental conditions.
[0084] While there was no apparent difference in the regularity of
the adenexal structures in the GM-284-treated skin, as compared
with that of the vehicle control, there was a large increase in the
width and cellularity of the basal aspects of the hair follicles
(FIG. 5A, panels a and b). In addition, the sebaceous glands of the
GM-284-treated skin appeared to be much rounder, larger, and fuller
than those of the controls. Higher-power magnification of these
structures shows both an increase in sebaceous gland cell number
and an apparent increase in the cytoplasmic content of the cells
(FIG. 5A, panels c and d).
[0085] The volumes of the cells of the sebaceous glands were
determined by scanning micrographs taken from animals treated with
either GM-284 or vehicle for 8 days, and measuring cell size with
the NIH Image program. As shown in FIG. 5B, when the cell volumes
were binned according to size, there was a marked shift to the
right as a result of GM-284 treatment. There was an absence of
smaller sebaceous cells in the GM-284 treatment group, and an
absence of larger cells in the vehicle-treated group. The increases
in the cellularity of the adenexal structures of the GM-284-treated
skin was not surprising, as recent reports have demonstrated a
common stem cell which gives rise to keratinocytes, follicular
cells, and sebaceous cells, and which resides in the "bulge" at the
lateral aspect of the hair follicle (Taylor et al., Involvement of
follicular stem cells in forming not only the follicle but also the
epidermis. Cell, 102:451-61, 2000; Merrill et al., Tcf3 and Lef1
regulate lineage differentiation of multipotent stem cells in skin.
Genes Dev., 15:1688-05, 2001). Thus, given the cellular expansion
in all three of these dermal compartments, it is likely that GM-284
acts at the level of the common stem cell.
Example 5
GM-284 Induces Rapid Reinnervation and Remyelination of Healing
Skin
[0086] In the absence of normalizing dermal histoarchitecture, the
permanent re-epithelialization of skin ulcers is difficult. There
is a huge body of literature on this topic: a search by the
inventor of the words "skin" and "ulcer", in papers published
between 1963 and the present, yielded 8545 articles on the subject,
many of which address the issues that are raised herein. Clearly,
the involvement of the nervous system in chronic skin ulcers has
been understood for at least 40 years; yet, the successful
treatment of these wounds, to date, has been elusive.
[0087] As shown above, GM-284 induces rapid healing of wounds; it
does so, in part, by influencing the proliferation of cells within
the dermis and epidermis. In addition, this compound has profound
effects on the regeneration of peripheral nerves. The inventor has
previously shown that, following crushing-nerve injury, GM-284
enhances nerve regeneration, acting on both the axon and the
Schwann cell (data not shown). To investigate whether GM-284 also
enhances sensory-nerve regeneration into skin following wounding,
the inventor examined innervation of skin biopsies after 8 days of
treatment with either GM-284 or vehicle.
[0088] Skin sections were stained with an antibody that recognizes
either myelin basic protein (MBP) or a neuron-specific isoform of
.beta.-tubulin (Easter et al., Initial tract formation in the mouse
brain. J. of Neurosci., 13:285-99, 1993). Staining of normal skin
from the dorsal trunk of the mouse showed small, MBP-positive
myelinated fibers which course parallel to the epidermis. As
described previously, Langerhans cells--antigen-presenting cells
residing in the skin (Steinman and Inaba, The binding of antigen
presenting cells to T lymphocytes. Adv. Exp. Med. Biol., 237:31-41,
1988)--are aligned in parallel along the nerve fibers of the skin
(Asahina et al., Modulation of Langerhans cell function by
epidermal nerves. J. Allergy Clin. Immunol., 96:1178-82, 1995)
(FIG. 6A, left panel). After 8 days of treatment with GM-284
following injury, skin regained its pre-injury histology, with new,
myelinated fibers running parallel to the epidermis and the
Langerhans cells (FIG. 6A, middle panel). In contrast, healing
skin, which had been treated with vehicle alone, still had no
myelinated fibers, nor did the Langerhans cells appear to have any
apparent organization relative to the epidermis (FIG. 6A, right
panel).
[0089] In addition to the myelinated fibers shown above, there are
also unmyelinated fibers that innervate the skin. These fibers
extend into the epidermis, where they contribute to nociception and
skin cooling (Light and Perl, "Peripheral Sensory Systems". In
Peripheral Neuropathy, Dyck, P. J. and Thomas, P. K., eds.
(Philadelphia: W.B. Saunders Company, 1993). Consistent with
findings of GM-284-promoted regeneration of myelinated fibers, it
was discovered herein that the compound also promotes regeneration
of naked axons into the epidermis (FIG. 6B). No such fibers were
found in the vehicle-treated controls.
[0090] Taken together, the foregoing data show that GM-284 promotes
dermal nerve regeneration, and also acts independently on
keratinocytes. However, it is likely that the GM-284-promoted
reinnervation shown herein also contributes to the epidermal
thickening shown in FIG. 4.
Example 6
Treatment of Intact Skin with GM-284 Induces a Reversible Epidermal
Hypertrophy
[0091] The changes in regenerating skin demonstrated above raise
questions concerning the effects of GM-284 on intact dermal
structures. In order to test these effects, the inventor shaved the
dorsal fur from over the hindquarters, bilaterally, and tattooed
5-mm circles on the skin. Either GM-284 or vehicle was applied
daily, to both sides of the animals, for 2 weeks. After that time,
a 5-mm biopsy was taken from only one side of each animal, and all
treatment was withheld. After an additional 2-week "recovery
period", a 5-mm biopsy was taken from the intact side of each
animal. Two weeks of treatment with GM-284, over intact skin,
resulted in epidermal hypertrophy, as shown in FIG. 7 (top panels).
This effect was completely reversed by the end of 2 more weeks,
when the epidermis had returned to baseline (FIG. 7, lower
panels).
Example 7
GM-284 Alters Gene Expression of Neuregulin In Situ and Induces a
Healing-like State in Keratinocytes in the Absence of Injury
[0092] The neuregulins are a large family of EGF-related growth
factors that, along with their receptors, the erbB tyrosine
kinases, have been implicated in a large number of biologies (Xian
and Zhou, Roles of transforming growth factor-alpha and related
molecules in the nervous system. Mol. Neurobiol., 20:157-83, 1999;
Adlkofer and Lai, Role of neuregulins in glial cell development.
Glia, 29:104-11, 2000; Niemann et al., Hepatocyte growth factor and
neuregulin in mammary gland cell morphogenesis. Adv. Exp. Med.
Biol., 480:9-18, 2000), including mediation of axon/Schwann cell
interactions. The inventor has previously shown that neuregulins
influence Schwann cell gene expression, during both development and
regeneration (Wu et al., The POU gene brn-5 is induced by
neuregulin and is restricted to myelinating Schwann cells. Mol.
Cell Neurosci., 17:683-95, 2001). The inventor has also shown that
GM-284 affects Schwann cell gene expression, and enhances
Schwann-cell-mediated nerve regeneration.
[0093] As demonstrated above, GM-284 induces rapid re-entry of
nerve fibers into skin and accelerates wound healing. Previous work
has shown that keratinocytes express neuregulin-.alpha., and that
its expression is downregulated following epidermal wounding
(Danilenko et al., Neu differentiation factor upregulates epidermal
migration and integrin expression in excisional wounds. J. Clin.
Invest., 95:842-51, 1995). As expected, unwounded, vehicle-treated
keratinocytes strongly expressed neuregulin-.alpha. (FIG. 8, top
panel). In contrast, 2 weeks of topical treatment with GM-284
completely downregulated neuregulin-.alpha. expression (FIG. 8,
lower panel). However, when the skin was allowed to recover for 2
weeks, keratinocyte expression of neuregulin-.alpha. was restored
(data not shown). These data are consistent with the notion that
GM-284 treatment alters skin biology toward a pro-healing
state.
[0094] Homeostasis of skin depends upon a complex biology that
involves an active interplay between keratinocytes and the
peripheral nerves that infiltrate and supply target areas. Loss of
innervation, either following trauma or due to pathology, results
in a decrease in keratinocyte proliferation and a thinning of the
epidermis (Lauria et al., Neuropathological alterations in diabetic
truncal neuropathy: evaluation by skin biopsy. J. Neurol.
Neurosurg. Psychiatry, 65:762-66, 1998; Hsieh and Lin, Modulation
of keratinocyte proliferation by skin innervation. J. Invest.
Dermatol., 113:579-86, 1999). Importantly, these effects are
reversible, with epidermal thickness normalizing concomitantly with
reinnervation (Huang et al., Influence of cutaneous nerves on
keratinocyte proliferation and epidermal thickness in mice.
Neuroscience, 94:965-73, 1999).
[0095] In the above Examples, the inventor has demonstrated that a
novel, nonimmunosuppressive immunophilin ligand, GM-284, affects
both components of the regenerating skin: the keratinocytes and the
peripheral nerves. These effects cooperate to increase the rate at
which wounds heal, by increasing keratinocyte proliferation
directly and increasing dermal nerve regeneration directly. In
particular, the inventor's data show that treatment with GM-284
corrects the underlying defects in wounded skin by acting on the
skin adenexa, as well as on keratinocytes and nerves. These
findings are of great interest as efforts increase to generate
artificial, or "lab grown", skin grafts from autologous donors.
Data from Taylor et al. (Involvement of follicular stem cells in
forming not only the follicle but also the epidermis. Cell,
102:451-61, 2000) and Merrill et al. (Tcf3 and Lef1 regulate
lineage differentiation of multipotent stem cells in skin. Genes
Dev., 15:1688-05, 2001) suggest that all three dermal
components--keratinocytes, sebaceous cells, and hair
follicles--share a common stem cell. Therefore, an agent such as
GM-284 might prove an important adjunct in the generation of
artificial skin containing the appropriate structures.
[0096] While the foregoing invention has been described in some
detail for purposes of clarity and understanding, it will be
appreciated by one skilled in the art, from a reading of the
disclosure, that various changes in form and detail can be made
without departing from the true scope of the invention in the
appended claims.
* * * * *