U.S. patent application number 09/977864 was filed with the patent office on 2004-04-01 for hedgehog antagonists, methods and uses related thereto.
Invention is credited to Dudek, Henryk, Karavanov, Irina, Pepicelli, Carmen.
Application Number | 20040060568 09/977864 |
Document ID | / |
Family ID | 22907059 |
Filed Date | 2004-04-01 |
United States Patent
Application |
20040060568 |
Kind Code |
A1 |
Dudek, Henryk ; et
al. |
April 1, 2004 |
Hedgehog antagonists, methods and uses related thereto
Abstract
The present application is directed to compositions and methods
for inhibiting angiogenesis and treating or preventing unwanted
cell proliferation, including tumors, by inhibiting the hedgehog
pathway, e.g., with an antagonist of the hedgehog pathway such as
those disclosed herein.
Inventors: |
Dudek, Henryk; (Wellesley,
MA) ; Karavanov, Irina; (Bethesda, MD) ;
Pepicelli, Carmen; (Cambridge, MA) |
Correspondence
Address: |
ROPES & GRAY LLP
ONE INTERNATIONAL PLACE
BOSTON
MA
02110-2624
US
|
Family ID: |
22907059 |
Appl. No.: |
09/977864 |
Filed: |
October 15, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60240564 |
Oct 13, 2000 |
|
|
|
Current U.S.
Class: |
128/898 |
Current CPC
Class: |
A61P 17/08 20180101;
C07K 16/18 20130101; C07K 16/30 20130101; C07K 2317/73 20130101;
A61P 1/00 20180101; C07J 71/0005 20130101; A61P 17/02 20180101;
A61K 31/4025 20130101; A61P 17/06 20180101; A61P 13/08 20180101;
C07K 16/3038 20130101; A61P 13/10 20180101; C07J 69/00 20130101;
A61P 37/08 20180101; A61P 17/00 20180101; C07J 71/0068 20130101;
G01N 33/574 20130101; A61K 2039/505 20130101; A61P 43/00 20180101;
A61P 35/00 20180101 |
Class at
Publication: |
128/898 |
International
Class: |
A61B 019/00 |
Claims
We claim:
1. A method of inhibiting unwanted cell proliferation comprising,
determining whether cells overexpress a gli gene, and contacting
cells that overexpress a gli gene with an effective amount of a
hedgehog antagonist; whereby said antagonist causes decreased cell
proliferation.
2. A method of claim 1, wherein said gli gene is gli-1.
3. A method of claim 1, wherein said unwanted cell proliferation is
cancer.
4. A method of claim 3, wherein said cancer is urogenital
cancer.
5. A method of claim 3, wherein said cancer is associated with one
or more of lung, prostate, breast, bladder, and colon tissues.
6. A method of claim 5, wherein said form of cancer associated with
breast tissue is selected from inferior ductal carcinoma, inferior
lobular carcinoma, intraductal carcinoma, medullary carcinoma and
tubular carcinoma.
7. A method of claim 5, wherein said cancer associated with lung
tissue is selected from adenocarcinoma, broncho-alveolar
adenocarcinoma and small cell carcinoma.
8. A method of claim 5, wherein said cancer associated with the
prostate is adenocarcinoma.
9. A method of claim 1, wherein said unwanted cell proliferation is
benign prostatic hyperplasia.
10. A method for determining a treatment protocol comprising,
obtaining a tissue sample from a patient, and determining levels of
gli gene expression in said sample, wherein overexpression of a gli
gene indicates that treatment with a hedgehog antagonist is
appropriate.
11. A method of claim 10, wherein said gli gene is gli-1.
12. A method of claim 11, wherein gli-1 expression levels are
determined by measuring gli-1 transcript levels.
13. A method of claim 11, wherein said gli-1 levels are determined
by measuring gli- 1 protein levels.
14. A method of stimulating surfactant production in a lung cell
comprising contacting said cell with an amount of hedgehog
antagonist effective to stimulate surfactant production.
15. A method of stimulating lamellated body formation in a lung
cell comprising contacting said cell with an amount of hedgehog
antagonist effective to stimulate lamellated body formation.
16. A method of claim 14 or 15, wherein said lung cell is present
in the lung tissue of a premature infant.
17. A method of any one of claims 1-15, wherein said hedgehog
antagonist is selected from a small molecule having a molecular
weight less than 2000 daltons, a hedgehog antibody, a patched
antibody, a smoothened antibody, a mutant hedgehog protein, an
antisense nucleic acid, and a ribozyme.
18. A method of claim 17, wherein said small molecule is selected
from cyclopamine, compound A, tomatidine, jervine, AY.sub.9944,
triparanol, compound B and functionally effective derivatives
thereof.
19. A method of determining the likelihood that a cancer will
develop in a tissue, comprising obtaining a tissue sample, and
determining levels of gli gene expression in said sample, wherein
overexpression of a gli gene indicates an increased likelihood that
cancer will develop.
20. A method of claim 19, wherein said gli gene is gli-1.
21. A method for treating a tumor in a patient, comprising
administering to said patient an amount of a hedgehog antagonist
sufficient to decrease the grow and/or proliferation of the tumor,
wherein the tumor is associated with at least one of urogenital,
lung, breast, prostate, bladder, or colon cancer.
22. The method of claim 21, wherein hedgehog antagonist is
administered as part of cancer treatment regimen.
Description
[0001] This application is based on U.S. provisional application
60/240,564, filed Oct. 13, 2000, hereby incorporated by
reference.
BACKGROUND OF THE INVENTION
[0002] Pattern formation is the activity by which embryonic cells
form ordered spatial arrangements of differentiated tissues. The
physical complexity of higher organisms arises during embryogenesis
through the interplay of cell-intrinsic lineage and cell-extrinsic
signaling. Inductive interactions are essential to embryonic
patterning in vertebrate development from the earliest
establishment of the body plan, to the patterning of the organ
systems, to the generation of diverse cell types during tissue
differentiation (Davidson, E., (1990) Development 108: 365-389;
Gurdon, J. B., (1992) Cell 68: 185-199; Jessell, T. M. et al.,
(1992) Cell 68: 257-270). The effects of developmental cell
interactions are varied. Typically, responding cells are diverted
from one route of cell differentiation to another by inducing cells
that differ from both the uninduced and induced states of the
responding cells (inductions). Sometimes cells induce their
neighbors to differentiate like themselves (homeogenetic
induction); in other cases a cell inhibits its neighbors from
differentiating like itself. Cell interactions in early development
may be sequential, such that an initial induction between two cell
types leads to a progressive amplification of diversity. Moreover,
inductive interactions occur not only in embryos, but in adult
cells as well, and can act to establish and maintain morphogenetic
patterns as well as induce differentiation (J. B. Gurdon (1992)
Cell 68:185-199).
[0003] Members of the Hedgehog family of signaling molecules
mediate many important short- and long-range patterning processes
during invertebrate and vertebrate development. In the fly, a
single hedgehog gene regulates segmental and imaginal disc
patterning. In contrast, in vertebrates, a hedgehog gene family is
involved in the control of left-right asymmetry, polarity in the
CNS, somites and limb, organogenesis, chondrogenesis and
spermatogenesis.
[0004] The first hedgehog gene was identified by a genetic screen
in the fruit fly Drosophila melanogaster (Nusslein-Volhard, C. and
Wieschaus, E. (1980) Nature 287, 795-801). This screen identified a
number of mutations affecting embryonic and larval development. In
1992 and 1993, the molecular nature of the Drosophila hedgehog (hh)
gene was reported (C. F., Lee et al. (1992) Cell 71, 33-50), and
since then, several hedgehog homologues have been isolated from
various vertebrate species. While only one hedgehog gene has been
found in Drosophila and other invertebrates, multiple Hedgehog
genes are present in vertebrates.
[0005] The vertebrate family of hedgehog genes includes at least
four members, e.g., paralogs of the single drosophila hedgehog
gene. Exemplary hedgehog genes and proteins are described in PCT
publications WO 95/18856 and WO 96/17924. Three of these members,
herein referred to as Desert hedgehog (Dhh), Sonic hedgehog (Shh)
and India hedgehog (Ihh), apparently exist in all vertebrates,
including fish, birds, and mammals. A fourth member, herein
referred to as tiggie-winkle hedgehog (Thh), appears specific to
fish. Desert hedgehog (Dhh) is expressed principally in the testes,
both in mouse embryonic development and in the adult rodent and
human; India hedgehog (Ihh) is involved in bone development during
embryogenesis and in bone formation in the adult; and Shh, which,
as described above, is primarily involved in morphogenic and
neuroinductive activities. Given the critical inductive roles of
hedgehog polypeptides in the development and maintenance of
vertebrate organs, the identification of hedgehog interacting
proteins is of paramount significance in both clinical and research
contexts.
[0006] The various Hedgehog proteins consist of a signal peptide, a
highly conserved N-terminal region, and a more divergent C-terminal
domain. In addition to signal sequence cleavage in the secretory
pathway (Lee, J. J. et al. (1992) Cell 71:33-50; Tabata, T. et al.
(1992) Genes Dev. 2635-2645; Chang, D. E. et al. (1994) Development
120:3339-3353), Hedgehog precursor proteins undergo an internal
autoproteolytic cleavage which depends on conserved sequences in
the C-terminal portion (Lee et al. (1994) Science 266:1528-1537;
Porter et al. (1995) Nature 374:363-366). This autocleavage leads
to a 19 kD N-terminal peptide and a C-terminal peptide of 26-28 kD
(Lee et al. (1992) supra; Tabata et al. (1992) supra; Chang et al.
(1994) supra; Lee et al. (1994) supra; Bumcrot, D. A., et al.
(1995) Mol. Cell. Biol. 15:2294-2303; Porter et al. (1995) supra;
Ekker, S. C. et al. (1995) Curr. Biol. 5:944-955; Lai, C. J. et al.
(1995) Development 121:2349-2360). The N-terminal peptide stays
tightly associated with the surface of cells in which it was
synthesized, while the C-terminal peptide is freely diffusible both
in vitro and in vivo (Porter et al. (1995) Nature 374:363; Lee et
al. (1994) supra; Bumcrot et al. (1995) supra; Marti, E. et al.
(1995) Development 121:2537-2547; Roelink, H. et al. (1995) Cell
81:445-455). Interestingly, cell surface retention of the
N-terminal peptide is dependent on autocleavage, as a truncated
form of HH encoded by an RNA which terminates precisely at the
normal position of internal cleavage is diffusible in vitro (Porter
et al. (1995) supra) and in vivo (Porter, J. A. et al. (1996) Cell
86, 21-34). Biochemical studies have shown that the autoproteolytic
cleavage of the HH precursor protein proceeds through an internal
thioester intermediate that subsequently is cleaved in a
nucleophilic substitution. It is likely that the nucleophile is a
small lipophilic molecule that becomes covalently bound to the
C-terminal end of the N-peptide (Porter et al. (1996) supra),
tethering it to the cell surface. The biological implications are
profound. As a result of the tethering, a high local concentration
of N-terminal Hedgehog peptide is generated on the surface of the
Hedgehog producing cells. It is this N-terminal peptide which is
both necessary and sufficient for short- and long-range Hedgehog
signaling activities in Drosophila and vertebrates (Porter et al.
(1995) supra; Ekker et al. (1995) supra: Lai et al. (1995) supra;
Roelink, H. et al. (1995) Cell 81:445-455; Porter et al. (1996)
supra: Fietz, M. J. et al. (1995) Curr. Biol. 5:643-651; Fan, C.-M.
et al. (1995) Cell 81:457-465; Marti, E., et al. (1995) Nature
375:322-325; Lopez-Martinez et al. (1995) Curr. Biol 5:791-795;
Ekker, S. C. et al. (1995) Development 121:2337-2347; Forbes, A. J.
et al. (1996) Development 122:1125-1135).
[0007] HH has been implicated in short- and long-range patterning
processes at various sites during Drosophila development. In the
establishment of segment polarity in early embryos, it has
short-range effects that appear to be directly mediated, while in
the patterning of the imaginal discs, it induces long-range effects
via the induction of secondary signals.
[0008] In vertebrates, several hedgehog genes have been cloned in
the past few years. Of these genes, Shh has received most of the
experimental attention, as it is expressed in different organizing
centers, which are the sources of signals that pattern neighboring
tissues. Recent evidence indicates that Shh is involved in these
interactions.
[0009] The expression of Shh starts shortly after the onset of
gastrulation in the presumptive midline mesoderm, the node in the
mouse (Chang et al. (1994) supra; Echelard, Y. et al. (1993) Cell
75:1417-1430), the rat (Roelink, H. et al. (1994) Cell 76:761-775)
and the chick (Riddle, R. D. et al. (1993) Cell 75:1401-1416), and
the shield in the zebrafish (Ekker et al. (1995) supra; Krauss, S.
et al. (1993) Cell 75:1431-1444). In chick embryos, the Shh
expression pattern in the node develops a left-right asymmetry,
which appears to be responsible for the left-right situs of the
heart (Levin, M. et al. (1995) Cell 82:803-814).
[0010] In the CNS, Shh from the notochord and the floorplate
appears to induce ventral cell fates. When ectopically expressed,
Shh leads to a ventralization of large regions of the mid- and
hindbrain in mouse (Echelard et al. (1993) supra; Goodrich, L. V.
et al. (1996) Genes Dev. 10:301-312), Xenopus (Roelink, H. et al.
(1994) supra; Ruiz i Altaba, A. et al. (1995) Mol. Cell. Neurosci.
6:106-121), and zebrafish (Ekker et al. (1995) supra; Krauss et al.
(1993) supra; Hammerschmidt, M., et al. (1996) Genes Dev.
10:647-658). In explants of intermediate neuroectoderm at spinal
cord levels, Shh protein induces floorplate and motor neuron
development with distinct concentration thresholds, floor plate at
high and motor neurons at lower concentrations (Roelink et al.
(1995) supra; Marti et al. (1995) supra; Tanabe, Y. et al. (1995)
Curr. Biol. 5:651-658). Moreover, antibody blocking suggests that
Shh produced by the notochord is required for notochord-mediated
induction of motor neuron fates (Marti et al. (1995) supra). Thus,
high concentrations of Shh on the surface of Shh-producing midline
cells appears to account for the contact-mediated induction of
floorplate observed in vitro (Placzek, M. et al. (1993) Development
117:205-218), and the midline positioning of the floorplate
immediately above the notochord in vivo. Lower concentrations of
Shh released from the notochord and the Doorplate presumably induce
motor neurons at more distant ventrolateral regions in a process
that has been shown to be contact-independent in vitro (Yamada, T.
et al. (1993) Cell 73:673-686). In explants taken at midbrain and
forebrain levels, Shh also induces the appropriate ventrolateral
neuronal cell types, dopaminergic (Heynes, M. et al. (1995) Neuron
15:35-44; Wang, M. Z. et al. (1995) Nature Med. 1:1184-1188) and
cholinergic (Ericson, J. et al. (1995) Cell 81:747-756) precursors,
respectively, indicating that Shh is a common inducer of ventral
specification over the entire length of the CNS. These observations
raise a question as to how the differential response to Shh is
regulated at particular anteroposterior positions.
[0011] Shh from the midline also patterns the paraxial regions of
the vertebrate embryo, the somites in the trunk (Fan et al. (1995)
supra) and the head mesenchyme rostral of the somites
(Hammerschmidt et al. (1996) supra). In chick and mouse paraxial
mesoderm explants, Shh promotes the expression of sclerotome
specific markers like Pax1 and Twist, at the expense of the
dermamyotomal marker Pax3. Moreover, filter barrier experiments
suggest that Shh mediates the induction of the sclerotome directly
rather than by activation of a secondary signaling mechanism (Fan,
C.-M. and Tessier-Lavigne, M. (1994) Cell 79, 1175-1186).
[0012] Shh also induces myotomal gene expression (Hammerschmidt et
al. (1996) supra; Johnson, R. L. et al. (1994) Cell 79:1165-1173;
Munsterberg, A. E. et al. (1995) Genes Dev. 9:2911-2922; Weinberg,
E. S. et al. (1996) Development 122:271-280), although recent
experiments indicate that members of the WNT family, vertebrate
homologues of Drosophila wingless, are required in concert
(Munsterberg et al. (1995) supra). Puzzlingly, myotomal induction
in chicks requires higher Shh concentrations than the induction of
sclerotomal markers (Munsterberg et al. (1995) supra), although the
sclerotome originates from somitic cells positioned much closer to
the notochord. Similar results were obtained in the zebrafish,
where high concentrations of Hedgehog induce myotomal and repress
sclerotomal marker gene expression (Hammerschmidt et al. (1996)
supra). In contrast to amniotes, however, these observations are
consistent with the architecture of the fish embryo, as here, the
myotome is the predominant and more axial component of the somites.
Thus, modulation of Shh signaling and the acquisition of new
signaling factors may have modified the somite structure during
vertebrate evolution.
[0013] In the vertebrate limb buds, a subset of posterior
mesenchymal cells, the "Zone of polarizing activity" (ZPA),
regulates anteroposterior digit identity (reviewed in Honig, L. S.
(1981) Nature 291:72-73). Ectopic expression of Shh or application
of beads soaked in Shh peptide mimics the effect of anterior ZPA
grafts, generating a mirror image duplication of digits (Chang et
al. (1994) supra; Lopez-Martinez et al. (1995) supra; Riddle et al.
(1993) supra) (FIG. 2g). Thus, digit identity appears to depend
primarily on Shh concentration, although it is possible that other
signals may relay this information over the substantial distances
that appear to be required for AP patterning (100-150 .mu.m).
Similar to the interaction of HH and DPP in the Drosophila imaginal
discs, Shh in the vertebrate limb bud activates the expression of
Bmp2 (Francis, P. H. et al. (1994) Development 120:209-218), a dpp
homologue. However, unlike DPP in Drosophila, Bmp2 fails to mimic
the polarizing effect of Shh upon ectopic application in the chick
limb bud (Francis et al. (1994) supra). In addition to
anteroposterior patterning, Shh also appears to be involved in the
regulation of the proximodistal outgrowth of the limbs by inducing
the synthesis of the fibroblast growth factor FGF4 in the posterior
apical ectodermal ridge (Laufer, E. et al. (1994) Cell 79:993-1003;
Niswander, L. et al. (1994) Nature 371:609-612).
[0014] The close relationship between Hedgehog proteins and BMPs is
likely to have been conserved at many, but probably not all sites
of vertebrate Hedgehog expression. For example, in the chick
hindgut, Shh has been shown to induce the expression of Bmp4,
another vertebrate dpp homologue (Roberts, D. J. et al. (1995)
Development 121:3163-3174). Furthermore, Shh and Bmp2, 4, or 6 show
a striking correlation in their expression in epithelial and
mesenchymal cells of the stomach, the urogenital system, the lung,
the tooth buds and the hair follicles (Bitgood, M. J. and McMahon,
A. P. (1995) Dev. Biol. 172:126-138). Further, Ihh, one of the two
other mouse Hedgehog genes, is expressed adjacent to Bmp expressing
cells in the gut and developing cartilage (Bitgood and McMahon
(1995) supra).
[0015] Recent evidence suggests a model in which Ihh plays a
crucial role in the regulation of chondrogenic development (Roberts
et al. (1995) supra). During cartilage formation, chondrocytes
proceed from a proliferating state via an intermediate,
prehypertrophic state to differentiated hypertrophic chondrocytes.
Ihh is expressed in the prehypertrophic chondrocytes and initiates
a signaling cascade that leads to the blockage of chondrocyte
differentiation. Its direct target is the perichondrium around the
Ihh expression domain, which responds by the expression of Gli and
Patched (Ptc), conserved transcriptional targets of Hedgehog
signals (see below). Most likely, this leads to secondary signaling
resulting in the synthesis of parathyroid hormone-related protein
(PTHrP) in the periarticular perichondrium. PTHrP itself signals
back to the prehypertrophic chondrocytes, blocking their further
differentiation. At the same time, PTHrP represses expression of
Ihh, thereby forming a negative feedback loop that modulates the
rate of chondrocyte differentiation.
[0016] Patched was originally identified in Drosophila as a segment
polarity gene, one of a group of developmental genes that affect
cell differentiation within the individual segments that occur in a
homologous series along the anterior-posterior axis of the embryo.
See Hooper, J. E. et al. (1989) Cell 59:751; and Nakano, Y. et al.
(1989) Nature 341:508. Patterns of expression of the vertebrate
homologue of patched suggest its involvement in the development of
neural tube, skeleton, limbs, craniofacial structure, and skin.
[0017] Genetic and functional studies demonstrate that patched is
part of the hedgehog signaling cascade, an evolutionarily conserved
pathway that regulates expression of a number of downstream genes.
See Perrimon, N. (1995) Cell 80:517; and Perrimon, N. (1996) Cell
86:513. Patched participates in the constitutive transcriptional
repression of the target genes; its effect is opposed by a secreted
glycoprotein, encoded by hedgehog, or a vertebrate homologue, which
induces transcriptional activation. Genes under control of this
pathway include members of the Wnt and TGF-beta families.
[0018] Patched proteins possess two large extracellular domains,
twelve transmembrane segments, and several cytoplasmic segments.
See Hooper, supra; Nakano, supra; Johnson, R. L. et al. (1996)
Science 272:1668; and Hahn, H. et al. (1996) Cell 85:841. The
biochemical role of patched in the hedgehog signaling pathway is
unclear. Direct interaction with the hedgehog protein has, however,
been reported (Chen, Y. et al. (1996) Cell 87:553), and patched may
participate in a hedgehog receptor complex along with another
transmembrane protein encoded by the smoothened gene. See Perrimon,
supra; and Chen, supra.
[0019] The human homologue of patched was recently cloned and
mapped to chromosome 9q22.3. See Johnson, supra; and Hahn, supra.
This region has been implicated in basal cell nevus syndrome
(BCNS), which is characterized by developmental abnormalities
including rib and craniofacial alterations, abnormalities of the
hands and feet, and spina bifida.
[0020] Sporadic tumors also demonstrated a loss of both functional
alleles of patched. Of twelve tumors in which patched mutations
were identified with a single strand conformational polymorphism
screening assay, nine had chromosomal deletion of the second allele
and the other three had inactivating mutations in both alleles
(Gailani, supra). The alterations did not occur in the
corresponding germline DNA.
[0021] Most of the identified mutations resulted in premature stop
codons or frame shifts (Lench, N. J., et al., Hum. Genet. 1997
October; 100(5-6): 497-502). Several, however, were point mutations
leading to amino acid substitutions in either extracellular or
cytoplasmic domains. These sites of mutation may indicate
functional importance for interaction with extracellular proteins
or with cytoplasmic members of the downstream signaling
pathway.
[0022] The involvement of patched in the inhibition of gene
expression and the occurrence of frequent allelic deletions of
patched in BCC support a tumor suppressor function for this gene.
Its role in the regulation of gene families known to be involved in
cell signaling and intercellular communication provides a possible
mechanism of tumor suppression.
SUMMARY OF THE INVENTION
[0023] In certain aspects, the present invention makes available
methods and reagents for inhibiting undesirable growth states that
occur in cells with an active hedgehog signaling pathway. In one
embodiment, the subject methods may be used to inhibit unwanted
cell proliferation by determining whether cells overexpress a gli
gene, and contacting cells that overexpress a gli gene with an
effective amount of a hedgehog antagonist. In preferred
embodiments, the unwanted cell proliferation is cancer or benign
prostatic hyperplasia.
[0024] Another aspect of the present invention makes available
methods for determining a treatment protocol comprising obtaining a
tissue sample from a patient, and determining levels of gli gene
expression in said sample, wherein overexpression of a gli gene
indicates that treatment with a hedgehog antagonist is
appropriate.
[0025] A further aspect of the invention provides methods for
stimulating surfactant production in a lung cell comprising
contacting said cell with an amount of hedgehog antagonist
effective to stimulate surfactant production. Another aspect of the
invention provides methods for stimulating lamellated body
formation in a lung cell comprising contacting said cell with an
amount of hedgehog antagonist effective to stimulate lamellated
body formation. In preferred embodiments, the lung cell is present
in the lung tissue of a premature infant.
[0026] In other preferred embodiments, hedgehog antagonists of the
invention are selected from a small molecule of less than 2000
daltons, a hedgehog antibody, a patched antibody, a smoothened
antibody, a mutant hedgehog protein, an antisense nucleic acid, and
a ribozyme. In particularly preferred embodiments, the hedgehog
antagonist is selected from one of formulae I through XXV. In
particularly preferred embodiments the hedgehog antagonist is
selected from cyclopamine, compound A, tomatidine, jervine, AY9944,
triparanol, compound B, and functionally effective derivatives
thereof.
[0027] In another aspect, the invention provides methods of
determining the likelihood that a cancer will develop in a tissue,
comprising obtaining a tissue sample, and determining levels of gli
gene expression in said sample, wherein, overexpression of a gli
gene indicates that cancer is more likely to develop.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1 depicts the chemical structures for AY9944,
triparanol, jervine, cyclopamine, tomatidine and cholesterol.
[0029] FIG. 2A depicts the chemical structure for compound A.
[0030] FIG. 2B shows the chemical structure for compound B.
[0031] FIG. 3 shows the chemical structure for agonist Z.
[0032] FIG. 4 depicts gli-1 gene expression in embryonic and adult
mouse lung.
[0033] FIG. 5 shows the inverse relationship between gli-1
expression and the expression of markers of lung maturation.
Between E13.5 and E16.5, the expression of gli-1 decreases while
the expression of the maturation marker, surfactant type C (Sp-C),
increases.
[0034] FIG. 6 shows the effect of compound B treatment of embryonic
mouse lungs on gli-1 expression.
[0035] FIG. 7 shows compound B treatment increases surfactant type
C production in embryonic mouse lungs.
[0036] FIG. 8 shows that type II pneumocytes in compound B-treated
lungs differentiate prematurely, as evidenced by the presence of
surfactant producing lamellated bodies.
[0037] FIG. 9 shows that treatment of embryonic lung cultures with
compound B decreases expression of gli-1.
[0038] FIG. 10 shows that treatment of embryonic lung cultures with
compound B increases expression of the maturation marker Sp-C. The
induction of Sp-C observed following treatment is comparable to
that observed following treatment with known lung maturation factor
hydrocortisone.
[0039] FIG. 11 shows that treatment of embryonic lung cultures with
hedgehog agonists has the opposite effect. Treatment with either
sonic hedgehog or with agonist Z increases gli-1 expression and
decreases Sp-C expression.
[0040] FIG. 12 illustrates gli-1 expression in breast cancer tissue
as visualized by in situ hybridization.
[0041] FIG. 13 shows gli-1 expression in lung cancer visualized by
in situ hybridization
[0042] FIG. 14 illustrates gli-1 expression in prostate cancer as
visualized by in situ hybridization
[0043] FIG. 15 depicts gli-1 expression in benign prostatic
hyperplasia as visualized by in situ hybridization
[0044] FIG. 16 shows: (A). Ptc-lacZ transgene expression in newborn
mouse ptc-1 (d11) lacZ bladder epithelium. LacZ expression can be
detected in the proliferating urothelial cells and, more weakly, in
adjacent mesenchymal cells. (B). Gli-1 expression in adult mouse
bladder epithelium. Gli-1 expression can be detected in the
proliferating urothelial cells.
[0045] FIG. 17 shows the expression of gli-1 and shh in normal
adult bladder and in a commercially available bladder tumor.
[0046] FIG. 18 shows the expression of shh and gli-1 in eight
commercially available bladder cancer cell lines. All eight cell
lines examined express genes involved in hedgehog signaling.
[0047] FIG. 19 shows the expression of shh, ptc-1, smo, gli-1,
gli-2, and gli-3 in eight commercially available bladder cancer
cell lines, as well as in fetal brain.
[0048] FIG. 20 shows a schematic representation of the gli-Luc
assay.
[0049] FIG. 21 shows the results of the gli-Luc assay on bladder
cancer cell co-cultures. Co-culture of S12 cells with either cell
line 5637 or cell line RT4 results in activation of the reporter
gene indicating that these cell lines can activate hedgehog
signaling.
[0050] FIG. 22 shows that the Shh antibody 5E1 inhibits activation
of the reporter gene in RT-4/S12 co-cultures.
[0051] FIGS. 23 and 24 show that administration of the Shh antibody
5E1 inhibits tumor growth in vivo in a nude mouse bladder cancer
model.
[0052] FIG. 25 shows that administration of the Shh antibody 5E1
decreases expression of gli-1 in vivo in a nude mouse bladder
cancer model.
[0053] FIG. 26 shows that shh is expressed in prostate cancer
samples as visualized by in situ hybridization.
[0054] FIG. 27 shows by Q-RT-PCR the expression of gli-1 in normal
adult prostate and in a prostate adenocarcinoma.
[0055] FIG. 28 shows the expression of shh and gli-1 in three
prostate cancer cell lines in comparison with expression in a
normal prostate cell line.
[0056] FIG. 29 shows that prostate cancer cell lines induce
expression of luciferase when co-cultured with S12 cells in the
gli-Luc in vitro assay.
[0057] FIG. 30 shows that the antagonizing antibody 5E1 inhibits
the induction of luciferase in by prostate cancer cells in the
gli-Luc in vitro assay.
[0058] FIG. 31 shows the expression of shh in prostatic epithelium
and stroma in human BPH samples.
[0059] FIG. 32 shows the expression of gli-1 in the prostatic
stroma of human BPH samples as measured by radioactive in situ
hybridization.
[0060] FIG. 33 shows that shh and patched-1 are expressed in a
proximo-distal pattern in normal prostate tissue with the highest
levels of gene expression occurring in the proximo or central
region.
[0061] FIG. 34 shows the expression of shh and gli-1 in BPH
samples, and compares the levels of gene expression to BCC
samples.
[0062] FIG. 35 shows the expression of shh and gli-1 in BPH cell
lines, and compares the levels of gene expression to that of BCC
samples, normal prostate, and prostate cancer.
[0063] FIG. 36 shows that the Shh blocking antibody 5E1 decreases
tumor size when administered to mice injected with the Shh
expressing colon cancer cell line HT-29.
[0064] FIG. 37 shows that administration of the Shh blocking
antibody 5E1 after 10 days of tumor growth decreases the rate of
tumor growth and tumor size in mice injected with the Shh
expressing colon cancer cell line HT-29.
DETAILED DESCRIPTION OF THE INVENTION
[0065] I. Overview
[0066] The present invention relates to the discovery that signal
transduction pathways regulated by hedgehog, patched (ptc), gli
and/or smoothened can be inhibited, at least in part, by hedgehog
antagonists. While not wishing to be bound by any theory, in the
case of small molecule antagonists, the modulation of a receptor
may be the mechanism by which these agents act. For example, the
ability of these agents to inhibit proliferation of patched
loss-of-function (ptc.sup.lof) cells may be due to the ability of
such molecules to interact with hedgehog, patched, or smoothened,
or at least to interfere with the ability of those proteins to
activate a hedgehog, ptc, and/or smoothened-mediated signal
transduction pathway.
[0067] It is, therefore, specifically contemplated that these small
molecules which interfere with aspects of hedgehog, ptc, or
smoothened signal transduction activity will likewise be capable of
changing the role of a cell in tissue development from what would
otherwise occur. In preferred embodiments, the cell has a
substantially wild-type hedgehog signaling pathway. It is also
contemplated that hedgehog antagonists are particularly effective
in treating disorders resulting from hyperactivation of the
hedgehog pathway as a result of mutations. Therefore, it is
desirable to have a method for identifying those cells in which the
hedgehog pathway is hyperactive such that antagonist treatment may
be efficiently targeted.
[0068] In certain embodiments, the subject antagonists are organic
molecules having a molecular weight less than 2500 amu, more
preferably less than 1500 amu, and even more preferably less than
750 amu, and are capable of inhibiting at least some of the
biological activities of hedgehog proteins, preferably specifically
in target cells.
[0069] Thus, the methods of the present invention include the use
of small molecules that agonize ptc inhibition of hedgehog
signaling in the regulation of repair and/or functional performance
of a wide range of cells, tissues and organs having the phenotype
of hedgehog gain-of-function and in tissues with wild-type hedgehog
activity. For instance, the subject method has therapeutic and
cosmetic applications ranging from regulation of neural tissues,
bone and cartilage formation and repair, regulation of
spermatogenesis, regulation of smooth muscle, regulation of lung,
liver and tissue of other organs arising from the primitive gut,
regulation of hematopoietic function, regulation of skin and hair
growth, etc. Moreover, the subject methods can be performed on
cells that are provided in culture (in vitro), or on cells in a
whole animal (in vivo). See, for example, PCT publications WO
95/18856 and WO 96/17924 (the specifications of which are expressly
incorporated by reference herein).
[0070] In another aspect, the present invention provides
pharmaceutical preparations comprising, as an active ingredient, a
hedgehog antagonist or ptc agonist such as described herein,
formulated in an amount sufficient to inhibit, in vivo,
proliferation or other biological consequences of hedgehog
gain-of-function.
[0071] The subject treatments using hedgehog antagonists can be
effective for both human and animal subjects. Animal subjects to
which the invention is applicable extend to both domestic animals
and livestock, raised either as pets or for commercial purposes.
Examples are dogs, cats, cattle, horses, sheep, hogs, and
goats.
[0072] II. Definitions
[0073] For convenience, certain terms employed in the
specification, examples, and appended claims are collected
here.
[0074] The phrase "aberrant modification or mutation" of a gene
refers to such genetic lesions as, for example, deletions,
substitution or addition of nucleotides to a gene, as well as gross
chromosomal rearrangements of the gene and/or abnormal methylation
of the gene. Likewise, misexpression of a gene refers to aberrant
levels of transcription of the gene relative to those levels in a
normal cell under similar conditions, as well as non-wild-type
splicing of mRNA transcribed from the gene.
[0075] The term "adenocarcinoma" as used herein refers to a
malignant tumor originating in glandular epithelium.
[0076] The term "angiogenesis", as used herein, refers to the
formation of blood vessels. Specifically, angiogenesis is a
multistep process in which endothelial cells focally degrade and
invade through their own basement membrane, migrate through
interstitial stroma toward an angiogenic stimulus, proliferate
proximal to the migrating tip, organize into blood vessels, and
reattach to newly synthesized basement membrane (see Folkman et
al., Adv. Cancer Res., Vol. 43, pp. 175-203 (1985)).
[0077] "Basal cell carcinomas" exist in a variety of clinical and
histological forms such as nodular-ulcerative, superficial,
pigmented, morphealike, fibroepithelioma and nevoid syndrome. Basal
cell carcinomas are the most common cutaneous neoplasms found in
humans. The majority of new cases of nonmelanoma skin cancers fall
into this category.
[0078] "Benign prostatic hyperplasia", or BPH, is a benign
enlargement of the prostate gland that begins normally after age 50
years probably secondary to the effects of male hormones. If
significant enlargement occurs, it may pinch off the urethra making
urination difficult or impossible.
[0079] "Burn wounds" refer to cases where large surface areas of
skin have been removed or lost from an individual due to heat
and/or chemical agents.
[0080]
[0081] The term "carcinoma" refers to a malignant new growth made
up of epithelial cells tending to infiltrate surrounding tissues
and to give rise to metastases. Exemplary carcinomas include:
"basal cell carcinoma", which is an epithelial tumor of the skin
that, while seldom metastasizing, has potentialities for local
invasion and destruction; "squamous cell carcinoma", which refers
to carcinomas arising from squamous epithelium and having cuboid
cells; "carcinosarcoma", which include malignant tumors composed of
carcinomatous and sarcomatous tissues; "adenocystic carcinoma",
carcinoma marked by cylinders or bands of hyaline or mucinous
stroma separated or surrounded by nests or cords of small
epithelial cells, occurring in the mammary and salivary glands, and
mucous glands of the respiratory tract; "epidermoid carcinoma",
which refers to cancerous cells which tend to differentiate in the
same way as those of the epidermis; i.e., they tend to form prickle
cells and undergo cornification; "nasopharyngeal carcinoma", which
refers to a malignant tumor arising in the epithelial lining of the
space behind the nose; and "renal cell carcinoma", which pertains
to carcinoma of the renal parenchyma composed of tubular cells in
varying arrangements. Other carcinomatous epithelial growths are
"papillomas", which refers to benign tumors derived from epithelium
and having a papillomavirus as a causative agent; and
"epidermoidomas", which refers to a cerebral or meningeal tumor
formed by inclusion of ectodermal elements at the time of closure
of the neural groove.
[0082] The "corium" or "dermis" refers to the layer of the skin
deep to the epidermis, consisting of a dense bed of vascular
connective tissue, and containing the nerves and terminal organs of
sensation. The hair roots, and sebaceous and sweat glands are
structures of the epidermis which are deeply embedded in the
dermis.
[0083] "Dental tissue" refers to tissue in the mouth that is
similar to epithelial tissue, for example gum tissue. The method of
the present invention is useful for treating periodontal
disease.
[0084] "Dermal skin ulcers" refer to lesions on the skin caused by
superficial loss of tissue, usually with inflammation. Dermal skin
ulcers that can be treated by the method of the present invention
include decubitus ulcers, diabetic ulcers, venous stasis ulcers and
arterial ulcers. Decubitus wounds refer to chronic ulcers that
result from pressure applied to areas of the skin for extended
periods of time. Wounds of this type are often called bedsores or
pressure sores. Venous stasis ulcers result from the stagnation of
blood or other fluids from defective veins. Arterial ulcers refer
to necrotic skin in the area around arteries having poor blood
flow.
[0085] The term "ED.sub.50" means the dose of a drug that produces
50% of its maximum response or effect.
[0086] An "effective amount" of, e.g., a hedgehog antagonist, with
respect to the subject method of treatment, refers to an amount of
the antagonist in a preparation which, when applied as part of a
desired dosage regimen brings about, e.g., a change in the rate of
cell proliferation and/or the state of differentiation of a cell
and/or rate of survival of a cell according to clinically
acceptable standards for the disorder to be treated or for the
cosmetic purpose.
[0087] The terms "epithelia", "epithelial" and "epithelium" refer
to the cellular covering of internal and external body surfaces
(cutaneous, mucous and serous), including the glands and other
structures derived therefrom, e.g., corneal, esophegeal, epidermal,
and hair follicle epithelial cells. Other exemplary epithelial
tissue includes: olfactory epithelium, which is the
pseudostratified epithelium lining the olfactory region of the
nasal cavity, and containing the receptors for the sense of smell;
glandular epithelium, which refers to epithelium composed of
secreting cells; squamous epithelium, which refers to epithelium
composed of flattened plate-like cells. The term epithelium can
also refer to transitional epithelium, like that which is
characteristically found lining hollow organs that are subject to
great mechanical change due to contraction and distention, e.g.,
tissue which represents a transition between stratified squamous
and columnar epithelium.
[0088] The term "epithelialization" refers to healing by the growth
of epithelial tissue over a denuded surface.
[0089] The term "epidermal gland" refers to an aggregation of cells
associated with the epidermis and specialized to secrete or excrete
materials not related to their ordinary metabolic needs. For
example, "sebaceous glands" are holocrine glands in the corium that
secrete an oily substance and sebum. The term "sweat glands" refers
to glands that secrete sweat, situated in the corium or
subcutaneous tissue, opening by a duct on the body surface.
[0090] The term "epidermis" refers to the outermost and nonvascular
layer of the skin, derived from the embryonic ectoderm, varying in
thickness from 0.07-1.4 mm. On the palmar and plantar surfaces it
comprises, from within outward, five layers: basal layer composed
of columnar cells arranged perpendicularly; prickle-cell or spinous
layer composed of flattened polyhedral cells with short processes
or spines; granular layer composed of flattened granular cells;
clear layer composed of several layers of clear, transparent cells
in which the nuclei are indistinct or absent; and horny layer
composed of flattened, cornified non-nucleated cells. In the
epidermis of the general body surface, the clear layer is usually
absent.
[0091] "Excisional wounds" include tears, abrasions, cuts,
punctures or lacerations in the epithelial layer of the skin and
may extend into the dermal layer and even into subcutaneous fat and
beyond. Excisional wounds can result from surgical procedures or
from accidental penetration of the skin.
[0092] The "growth state" of a cell refers to the rate of
proliferation of the cell and/or the state of differentiation of
the cell. An "altered growth state" is a growth state characterized
by an abnormal rate of proliferation, e.g., a cell exhibiting an
increased or decreased rate of proliferation relative to a normal
cell.
[0093] The term "hair" refers to a threadlike structure, especially
the specialized epidermal structure composed of keratin and
developing from a papilla sunk in the corium, produced only by
mammals and characteristic of that group of animals. Also, "hair"
may refer to the aggregate of such hairs. A "hair follicle" refers
to one of the tubular-invaginations of the epidermis enclosing the
hairs, and from which the hairs grow. "Hair follicle epithelial
cells" refers to epithelial cells that surround the dermal papilla
in the hair follicle, e.g., stem cells, outer root sheath cells,
matrix cells, and inner root sheath cells. Such cells may be normal
non-malignant cells, or transformed/immortalized cells.
[0094] The term "hedgehog" is used to refer generically to any
member of the hedgehog family, including sonic, indian, desert and
tiggy winkle. The term may be used to indicate protein or gene.
[0095] The term "hedgehog signaling pathway", "hedgehog pathway"
and "hedgehog signal transduction pathway" are all used to refer to
the chain of events normally mediated by hedgehog, smoothened, ptc,
and gli, among others, and resulting in a changes in gene
expression and other phenotypic changes typical of hedgehog
activity. The hedgehog pathway can be activated even in the absence
of a hedgehog protein by activating a downstream component. For
example, overexpression of smoothened will activate the pathway in
the absence of hedgehog. gli and ptc gene expression are indicators
of an active hedgehog signaling pathway.
[0096] The term "hedgehog antagonist" refers to an agent that
potentiates or recapitulates the bioactivity of patched, such as to
repress transcription of target genes. Preferred hedgehog
antagonists can be used to overcome a ptc loss-of-function and/or a
smoothened gain-of-function, the latter also being referred to as
smoothened antagonists. The term `hedgehog antagonist` as used
herein refers not only to any agent that may act by directly
inhibiting the normal function of the hedgehog protein, but also to
any agent that inhibits the hedgehog signalling pathway, and thus
recapitulates the function of ptc. A hedgehog antagonist may be a
small molecule, an antibody (including but not restricted to: a
diabody, single chain antibody, monoclonal antibody, IgG, Ig, IgA,
IgD, IgE, or an antibody fragment comprising at least one pair of
variable regions), an antisense nucleic acid, PNA or ribozyme, or a
mutant hedgehog protein that can disrupt or inhibit hedgehog
signaling. An antibody may be directed to a protein encoded by any
of the genes in the hedgehog pathway, including sonic, indian or
desert hedgehog, smoothened, ptc-1, ptc-2, gli-1, gli-2, gli-3,
etc. In most cases, the antibody would inhibit the activity of the
target protein, but in the case of patched, such an antibody would
be an activator of patched. An antisense nucleic acid would
likewise decrease production of a protein encoded by any of the
genes in the hedgehog pathway, with the exception of patched or
other genes encoding negative regulators of the hedgehog signaling
pathway.
[0097] The term "hedgehog gain-of-function" refers to an aberrant
modification or mutation of a ptc gene, hedgehog gene, or
smoothened gene, or a decrease (or loss) in the level of expression
of such a gene, which results in a phenotype which resembles
contacting a cell with a hedgehog protein, e.g., aberrant
activation of a hedgehog pathway. The gain-of-function may include
a loss of the ability of the ptc gene product to regulate the level
of expression of Ci genes, e.g., Gli1, Gli2, and Gli3. The term
`hedgehog gain-of-function` is also used herein to refer to any
similar cellular phenotype (e.g., exhibiting excess proliferation)
that occurs due to an alteration anywhere in the hedgehog signal
transduction pathway, including, but not limited to, a modification
or mutation of hedgehog itself. For example, a tumor cell with an
abnormally high proliferation rate due to activation of the
hedgehog signalling pathway would have a `hedgehog
gain-of-function` phenotype, even if hedgehog is not mutated in
that cell.
[0098] As used herein, "immortalized cells" refers to cells that
have been altered via chemical and/or recombinant means such that
the cells have the ability to grow through an indefinite number of
divisions in culture.
[0099] "Internal epithelial tissue" refers to tissue inside the
body that has characteristics similar to the epidermal layer in the
skin. Examples include the lining of the intestine. The method of
the present invention is useful for promoting the healing of
certain internal wounds, for example wounds resulting from
surgery.
[0100] The term "keratosis" refers to proliferative skin disorder
characterized by hyperplasia of the horny layer of the epidermis.
Exemplary keratotic disorders include keratosis follicularis,
keratosis palmaris et plantaris, keratosis pharyngea, keratosis
pilaris, and actinic keratosis.
[0101] "Lamellated bodies" refers to a subcellular structure found
in lung cells that are producing surfactants. Lamellated bodies are
thought to be the site of lung surfactant biosynthesis. The bodies
have a multilayered membranous appearance in an electron
micrograph.
[0102] The term "LD.sub.50" means the dose of a drug that is lethal
in 50% of test subjects.
[0103] The term "nail" refers to the horny cutaneous plate on the
dorsal surface of the distal end of a finger or toe.
[0104] The term "overexpression" as used in reference to gene
expression levels means any level of gene expression in cells of a
tissue that is higher than the normal level of expression for that
tissue. The normal level of expression for a tissue may be assessed
by measuring gene expression in a healthy portion of that
tissue.
[0105] The term "patched loss-of-function" refers to an aberrant
modification or mutation of a ptc gene, or a decreased level of
expression of the gene, which results in a phenotype that resembles
contacting a cell with a hedgehog protein, e.g., aberrant
activation of a hedgehog pathway. The loss-of-function may include
a loss of the ability of the ptc gene product to regulate the level
of expression of Ci genes, e.g., Gli1, Gli2 and Gli3.
[0106] A "patient" or "subject" to be treated by the subject method
can mean either a human or non-human animal.
[0107] The term "prodrug" is intended to encompass compounds that,
under physiological conditions, are converted into the
therapeutically active agents of the present invention. A common
method for making a prodrug is to include selected moieties that
are hydrolyzed under physiological conditions to reveal the desired
molecule. In other embodiments, the prodrug is converted by an
enzymatic activity of the host animal.
[0108] As used herein, "proliferating" and "proliferation" refer to
cells undergoing mitosis.
[0109] Throughout this application, the term "proliferative skin
disorder" refers to any disease/disorder of the skin marked by
unwanted or aberrant proliferation of cutaneous tissue. These
conditions are typically characterized by epidermal cell
proliferation or incomplete cell differentiation, and include, for
example, X-linked ichthyosis, psoriasis, atopic dermatitis,
allergic contact dermatitis, epidermolytic hyperkeratosis, and
seborrheic dermatitis. For example, epidermodysplasia is a form of
faulty development of the epidermis. Another example is
"epidermolysis", which refers to a loosened state of the epidermis
with formation of blebs and bullae either spontaneously or at the
site of trauma.
[0110] As used herein, the term "psoriasis" refers to a
hyperproliferative skin disorder that alters the skin's regulatory
mechanisms. In particular, lesions are formed which involve primary
and secondary alterations in epidermal proliferation, inflammatory
responses of the skin, and an expression of regulatory molecules
such as lymphokines and inflammatory factors. Psoriatic skin is
morphologically characterized by an increased turnover of epidermal
cells, thickened epidermis, abnormal keratinization, inflammatory
cell infiltrates into the dermis layer and polymorphonuclear
leukocyte infiltration into the epidermis layer resulting in an
increase in the basal cell cycle. Additionally, hyperkeratotic and
parakeratotic cells are present.
[0111] The term "skin" refers to the outer protective covering of
the body, consisting of the corium and the epidermis, and is
understood to include sweat and sebaceous glands, as well as hair
follicle structures. Throughout the present application, the
adjective "cutaneous" may be used, and should be understood to
refer generally to attributes of the skin, as appropriate to the
context in which they are used.
[0112] The term "small cell carcinoma" refers to a type of
malignant neoplasm, commonly of the bronchus. Cells of the tumor
have endocrine like characteristics and may secrete one or more of
a wide range of hormones, especially regulatory peptides like
bombesin.
[0113] The term "smoothened gain-of-function" refers to an aberrant
modification or mutation of a smo gene, or an increased level of
expression of the gene, which results in a phenotype that resembles
contacting a cell with a hedgehog protein, e.g., aberrant
activation of a hedgehog pathway. While not wishing to be bound by
any particular theory, it is noted that ptc may not signal directly
into the cell, but rather interact with smoothened, another
membrane-bound protein located downstream of ptc in hedgehog
signaling (Marigo et al., (1996) Nature 384: 177-179). The gene smo
is a segment-polarity gene required for the correct patterning of
every segment in Drosophila (Alcedo et al., (1996) Cell 86:
221-232). Human homologs of smo have been identified. See, for
example, Stone et al. (1996) Nature 384:129-134, and GenBank
accession U84401. The smoothened gene encodes an integral membrane
protein with characteristics of heterotrimeric G-protein-coupled
receptors; i.e., 7-transmembrane regions. This protein shows
homology to the Drosophila Frizzled (Fz) protein, a member of the
wingless pathway. It was originally thought that smo encodes a
receptor of the Hh signal. However, this suggestion was
subsequently disproved, as evidence for ptc being the Hh receptor
was obtained. Cells that express Smo fail to bind Hh, indicating
that smo does not interact directly with Hh (Nusse, (1996) Nature
384: 119-120). Rather, the binding of Sonic hedgehog (SHH) to its
receptor, PTCH, is thought to prevent normal inhibition by PTCH of
smoothened (SMO), a seven-span transmembrane protein.
[0114] Recently, it has been reported that activating smoothened
mutations occur in sporadic basal cell carcinoma, Xie et al. (1998)
Nature 391: 90-2, and primitive neuroectodermal tumors of the
central nervous system, Reifenberger et al. (1998) Cancer Res 58:
1798-803.
[0115] The term "therapeutic index" refers to the therapeutic index
of a drug defined as LD.sub.50/ED.sub.50.
[0116] As used herein, "transformed cells" refers to cells that
have spontaneously converted to a state of unrestrained growth,
i.e., they have acquired the ability to grow through an indefinite
number of divisions in culture. Transformed cells may be
characterized by such terms as neoplastic, anaplastic and/or
hyperplastic, with respect to their loss of growth control.
[0117] "Urogenital" refers to the organs and tissues of the
urogenital tract, which includes among other tissues, the prostate,
ureter, kidney and bladder. A "urogenital cancer" is a cancer of a
urogenital tissue.
[0118] The term "acylamino" is art-recognized and refers to a
moiety that can be represented by the general formula: 1
[0119] wherein R.sub.9 is as defined above, and R'.sub.11
represents a hydrogen, an alkyl, an alkenyl or
--(CH.sub.2).sub.m--R.sub.8, where m and R.sub.8 are as defined
above.
[0120] Herein, the term "aliphatic group" refers to a
straight-chain, branched-chain, or cyclic aliphatic hydrocarbon
group and includes saturated and unsaturated aliphatic groups, such
as an alkyl group, an alkenyl group, and an alkynyl group.
[0121] The terms "alkenyl" and "alkynyl" refer to unsaturated
aliphatic groups analogous in length and possible substitution to
the alkyls described above, but that contain at least one double or
triple bond respectively.
[0122] The terms "alkoxyl" or "alkoxy" as used herein refers to an
alkyl group, as defined above, having an oxygen radical attached
thereto. Representative alkoxyl groups include methoxy, ethoxy,
propyloxy, tert-butoxy and the like. An "ether" is two hydrocarbons
covalently linked by an oxygen. Accordingly, the substituent of an
alkyl that renders that alkyl an ether is or resembles an alkoxyl,
such as can be represented by one of --O-alkyl, --O-alkenyl,
--O-alkynyl, --O--(CH.sub.2).sub.m--R.sub.8, where m and R.sub.8
are described above.
[0123] The term "alkyl" refers to the radical of saturated
aliphatic groups, including straight-chain alkyl groups,
branched-chain alkyl groups, cycloalkyl (alicyclic) groups,
alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted
alkyl groups. In preferred embodiments, a straight chain or
branched chain alkyl has 30 or fewer carbon atoms in its backbone
(e.g., C.sub.1-C.sub.30 for straight chains, C.sub.3-C.sub.30 for
branched chains), and more preferably 20 or fewer. Likewise,
preferred cycloalkyls have from 3-10 carbon atoms in their ring
structure, and more preferably have 5, 6 or 7 carbons in the ring
structure.
[0124] Moreover, the term "alkyl" (or "lower alkyl") as used
throughout the specification, examples, and claims is intended to
include both "unsubstituted alkyls" and "substituted alkyls", the
latter of which refers to alkyl moieties having substituents
replacing a hydrogen on one or more carbons of the hydrocarbon
backbone. Such substituents can include, for example, a halogen, a
hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a
formyl, or an acyl), a thiocarbonyl (such as a thioester, a
thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a
phosphate, a phosphonate, a phosphinate, an amino, an amido, an
amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an
alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a
sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or
heteroaromatic moiety. It will be understood by those skilled in
the art that the moieties substituted on the hydrocarbon chain can
themselves be substituted, if appropriate. For instance, the
substituents of a substituted alkyl may include substituted and
unsubstituted forms of amino, azido, imino, amido, phosphoryl
(including phosphonate and phosphinate), sulfonyl (including
sulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups,
as well as ethers, alkylthios, carbonyls (including ketones,
aldehydes, carboxylates, and esters), --CF.sub.3, --CN and the
like. Exemplary substituted alkyls are described below. Cycloalkyls
can be further substituted with alkyls, alkenyls, alkoxys,
alkylthios, aminoalkyls, carbonyl-substituted alkyls, --CF.sub.3,
--CN, and the like.
[0125] Unless the number of carbons is otherwise specified, "lower
alkyl" as used herein means an alkyl group, as defined above, but
having from one to ten carbons, more preferably from one to six
carbon atoms in its backbone structure. Likewise, "lower alkenyl"
and "lower alkynyl" have similar chain lengths. Throughout the
application, preferred alkyl groups are lower alkyls. In preferred
embodiments, a substituent designated herein as alkyl is a lower
alkyl.
[0126] The term "alkylthio" refers to an alkyl group, as defined
above, having a sulfur radical attached thereto. In preferred
embodiments, the "alkylthio" moiety is represented by one of
--S-alkyl, --S-alkenyl, --S-alkynyl, and
--S--(CH.sub.2).sub.m--R.sub.8, wherein m and R.sub.8 are defined
above. Representative alkylthio groups include methylthio,
ethylthio, and the like.
[0127] The terms "amine" and "amino" are art-recognized and refer
to both unsubstituted and substituted amines, e.g., a moiety that
can be represented by the general formula: 2
[0128] wherein R.sub.9, R.sub.10 and R'.sub.10 each independently
represent a hydrogen, an alkyl, an alkenyl,
--(CH.sub.2).sub.m--R.sub.8, or R.sub.9 and R.sub.10 taken together
with the N atom to which they are attached complete a heterocycle
having from 4 to 8 atoms in the ring structure; R.sub.8 represents
an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle or a
polycycle; and m is zero or an integer in the range of 1 to 8. In
preferred embodiments, only one of R.sub.9 or R.sub.10 can be a
carbonyl, e.g., R.sub.9, R.sub.10 and the nitrogen together do not
form an imide. In even more preferred embodiments, R.sub.9 and
R.sub.10 (and optionally R'.sub.10) each independently represent a
hydrogen, an alkyl, an alkenyl, or --(CH.sub.2).sub.m--R.sub.8.
Thus, the term "alkylamine" as used herein means an amine group, as
defined above, having a substituted or unsubstituted alkyl attached
thereto, i.e., at least one of R.sub.9 and R.sub.10 is an alkyl
group.
[0129] The term "amido" is art-recognized as an amino-substituted
carbonyl and includes a moiety that can be represented by the
general formula: 3
[0130] wherein R.sub.9, R.sub.10 are as defined above. Preferred
embodiments of the amide will not include imides, which may be
unstable.
[0131] The term "aralkyl", as used herein, refers to an alkyl group
substituted with an aryl group (e.g., an aromatic or heteroaromatic
group).
[0132] The term "aryl" as used herein includes 5-, 6-, and
7-membered single-ring aromatic groups that may include from zero
to four heteroatoms, for example, benzene, pyrrole, furan,
thiophene, imidazole, oxazole, thiazole, triazole, pyrazole,
pyridine, pyrazine, pyridazine and pyrimidine, and the like. Those
aryl groups having heteroatoms in the ring structure may also be
referred to as "aryl heterocycles" or "heteroaromatics." The
aromatic ring can be substituted at one or more ring positions with
such substituents as described above, for example, halogen, azide,
alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl,
amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate,
phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,
sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or
heteroaromatic moieties, --CF.sub.3, --CN, or the like. The term
"aryl" also includes polycyclic ring systems having two or more
cyclic rings in which two or more carbons are common to two
adjoining rings (the rings are "fused rings") wherein at least one
of the rings is aromatic, e.g., the other cyclic rings can be
cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or
heterocyclyls.
[0133] The term "carbocycle", as used herein, refers to an aromatic
or non-aromatic ring in which each atom of the ring is carbon.
[0134] The term "carbonyl" is art-recognized and includes such
moieties as can be represented by the general formula: 4
[0135] wherein X is a bond or represents an oxygen or a sulfur, and
R.sub.11 represents a hydrogen, an alkyl, an alkenyl,
--(CH.sub.2).sub.m--R.sub.8 or a pharmaceutically acceptable salt,
R'.sub.11 represents a hydrogen, an alkyl, an alkenyl or
--(CH.sub.2).sub.m--R.sub.8, where m and R.sub.8 are as defined
above. Where X is an oxygen and R.sub.11 or R'.sub.11 is not
hydrogen, the formula represents an "ester". Where X is an oxygen,
and R.sub.11 is as defined above, the moiety is referred to herein
as a carboxyl group, and particularly when R.sub.11 is a hydrogen,
the formula represents a "carboxylic acid". Where X is an oxygen,
and R'.sub.11 is hydrogen, the formula represents a "formate". In
general, where the oxygen atom of the above formula is replaced by
sulfur, the formula represents a "thiocarbonyl" group. Where X is a
sulfur and R.sub.11 or R'.sub.11 is not hydrogen, the formula
represents a "thioester." Where X is a sulfur and R.sub.11 is
hydrogen, the formula represents a "thiocarboxylic acid." Where X
is a sulfur and R.sub.11' is hydrogen, the formula represents a
"thiolformate." On the other hand, where X is a bond, and R.sub.11
is not hydrogen, the above formula represents a "ketone" group.
Where X is a bond, and R.sub.11 is hydrogen, the above formula
represents an "aldehyde" group.
[0136] The term "heteroatom" as used herein means an atom of any
element other than carbon or hydrogen. Preferred heteroatoms are
boron, nitrogen, oxygen, phosphorus, sulfur and selenium.
[0137] The terms "heterocyclyl" or "heterocyclic group" refer to 3-
to 10-membered ring structures, more preferably 3- to 7-membered
rings, whose ring structures include one to four heteroatoms.
Heterocycles can also be polycycles. Heterocyclyl groups include,
for example, thiophene, thianthrene, furan, pyran, isobenzofuran,
chromene, xanthene, phenoxathiin, pyrrole, imidazole, pyrazole,
isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine,
indolizine, isoindole, indole, indazole, purine, quinolizine,
isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline,
quinazoline, cinnoline, pteridine, carbazole, carboline,
phenanthridine, acridine, pyrimidine, phenanthroline, phenazine,
phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine,
oxolane, thiolane, oxazole, piperidine, piperazine, morpholine,
lactones, lactams such as azetidinones and pyrrolidinones, sultams,
sultones, and the like. The heterocyclic ring can be substituted at
one or more positions with such substituents as described above, as
for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,
hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphate,
phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether,
alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an
aromatic or heteroaromatic moiety, --CF.sub.3, --CN, or the
like.
[0138] As used herein, the term "nitro" means --NO.sub.2; the term
"halogen" designates --F, --Cl, --Br or --I; the term "sulfhydryl"
means --SH; the term "hydroxyl" means --OH; and the term "sulfonyl"
means --SO.sub.2--.
[0139] The terms "polycyclyl" or "polycyclic group" refer to two or
more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls
and/or heterocyclyls) in which two or more carbons are common to
two adjoining rings, e.g., the rings are "fused rings". Rings that
are joined through non-adjacent atoms are termed "bridged" rings.
Each of the rings of the polycycle can be substituted with such
substituents as described above, as for example, halogen, alkyl,
aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro,
sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate,
carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone,
aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic
moiety, --CF.sub.3, --CN, or the like.
[0140] The phrase "protecting group" as used herein means temporary
substituents that protect a potentially reactive functional group
from undesired chemical transformations. Examples of such
protecting groups include esters of carboxylic acids, silyl ethers
of alcohols, and acetals and ketals of aldehydes and ketones,
respectively. The field of protecting group chemistry has been
reviewed (Greene, T. W.; Wuts, P. G. M. Protective Groups in
Organic Synthesis, 2.sup.nd ed.; Wiley: New York, 1991).
[0141] A "selenoalkyl" refers to an alkyl group having a
substituted seleno group attached thereto. Exemplary "selenoethers"
which may be substituted on the alkyl are selected from one of
--Se-alkyl, --Se-alkenyl, --Se-alkynyl, and
--Se--(CH.sub.2).sub.m--R.sub.8, m and R.sub.8 being defined
above.
[0142] As used herein, the term "substituted" is contemplated to
include all permissible substituents of organic compounds. In a
broad aspect, the permissible substituents include acyclic and
cyclic, branched and unbranched, carbocyclic and heterocyclic,
aromatic and nonaromatic substituents of organic compounds.
Illustrative substituents include, for example, those described
herein above. The permissible substituents can be one or more and
the same or different for appropriate organic compounds. For
purposes of this invention, the heteroatoms such as nitrogen may
have hydrogen substituents and/or any permissible substituents of
organic compounds described herein which satisfy the valences of
the heteroatoms. This invention is not intended to be limited in
any manner by the permissible substituents of organic
compounds.
[0143] It will be understood that "substitution" or "substituted
with" includes the implicit proviso that such substitution is in
accordance with permitted valence of the substituted atom and the
substituent, and that the substitution results in a stable
compound, e.g., which does not spontaneously undergo transformation
such as by rearrangement, cyclization, elimination, etc.
[0144] The term "sulfamoyl" is art-recognized and includes a moiety
that can be represented by the general formula: 5
[0145] in which R.sub.9 and R.sub.10 are as defined above.
[0146] The term "sulfate" is art recognized and includes a moiety
that can be represented by the general formula: 6
[0147] in which R.sub.41 is as defined above.
[0148] The term "sulfonamido" is art recognized and includes a
moiety that can be represented by the general formula: 7
[0149] in which R.sub.9 and R'.sub.11 are as defined above.
[0150] The term "sulfonate" is art-recognized and includes a moiety
that can be represented by the general formula: 8
[0151] in which R.sub.41 is an electron pair, hydrogen, alkyl,
cycloalkyl, or aryl.
[0152] The terms "sulfoxido" or "sulfinyl", as used herein, refers
to a moiety that can be represented by the general formula: 9
[0153] in which R.sub.44 is selected from the group consisting of
hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl,
aralkyl, or aryl.
[0154] Analogous substitutions can be made to alkenyl and alkynyl
groups to produce, for example, aminoalkenyls, aminoalkynyls,
amidoalkenyls, amidoalkynyls, iminoalkenyls, iminoalkynyls,
thioalkenyls, thioalkynyls, carbonyl-substituted alkenyls or
alkynyls.
[0155] As used herein, the definition of each expression, e.g.,
alkyl, m, n, etc., when it occurs more than once in any structure,
is intended to be independent of its definition elsewhere in the
same structure.
[0156] The terms triflyl, tosyl, mesyl, and nonaflyl are
art-recognized and refer to trifluoromethanesulfonyl,
p-toluenesulfonyl, methanesulfonyl, and nonafluorobutanesulfonyl
groups, respectively. The terms triflate, tosylate, mesylate, and
nonaflate are art-recognized and refer to trifluoromethanesulfonate
ester, p-toluenesulfonate ester, methanesulfonate ester, and
nonafluorobutanesulfonate ester functional groups and molecules
that contain said groups, respectively.
[0157] The abbreviations Me, Et, Ph, Tf, Nf, Ts, Ms represent
methyl, ethyl, phenyl, trifluoromethanesulfonyl,
nonafluorobutanesulfonyl, p-toluenesulfonyl and methanesulfonyl,
respectively. A more comprehensive list of the abbreviations
utilized by organic chemists of ordinary skill in the art appears
in the first issue of each volume of the Journal of Organic
Chemistry; this list is typically presented in a table entitled
Standard List of Abbreviations. The abbreviations contained in said
list, and all abbreviations utilized by organic chemists of
ordinary skill in the art are hereby incorporated by reference.
[0158] Certain compounds of the present invention may exist in
particular geometric or stereoisomeric forms. The present invention
contemplates all such compounds, including cis- and trans-isomers,
R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the
racemic mixtures thereof, and other mixtures thereof, as falling
within the scope of the invention. Additional asymmetric carbon
atoms may be present in a substituent such as an alkyl group. All
such isomers, as well as mixtures thereof, are intended to be
included in this invention.
[0159] If, for instance, a particular enantiomer of a compound of
the present invention is desired, it may be prepared by asymmetric
synthesis, or by derivation with a chiral auxiliary, where the
resulting diastereomeric mixture is separated and the auxiliary
group cleaved to provide the pure desired enantiomers.
Alternatively, where the molecule contains a basic functional
group, such as amino, or an acidic functional group, such as
carboxyl, diastereomeric salts may be formed with an appropriate
optically active acid or base, followed by resolution of the
diastereomers thus formed by fractional crystallization or
chromatographic means well known in the art, and subsequent
recovery of the pure enantiomers.
[0160] Contemplated equivalents of the compounds described above
include compounds which otherwise correspond thereto, and which
have the same general properties thereof (e.g., the ability to
inhibit hedgehog signaling), wherein one or more simple variations
of substituents are made which do not adversely affect the efficacy
of the compound. In general, the compounds of the present invention
may be prepared by the methods illustrated in the general reaction
schemes as, for example, described below, or by modifications
thereof, using readily available starting materials, reagents and
conventional synthesis procedures. In these reactions, it is also
possible to make use of variants that are in themselves known, but
are not mentioned here.
[0161] For purposes of this invention, the chemical elements are
identified in accordance with the Periodic Table of the Elements,
CAS version, Handbook of Chemistry and Physics, 67th Ed., 1986-87,
inside cover. Also for purposes of this invention, the term
"hydrocarbon" is contemplated to include all permissible compounds
having at least one hydrogen and one carbon atom. In a broad
aspect, the permissible hydrocarbons include acyclic and cyclic,
branched and unbranched, carbocyclic and heterocyclic, aromatic and
nonaromatic organic compounds which can be substituted or
unsubstituted.
[0162] III. Exemplary Compounds and Synthesis Thereof
[0163] Hedgehog antagonists of the invention may be essentially any
composition that inhibits the activity of the hedgehog signaling
pathway, in other words mimicking the effect of patched activity.
Hedgehog antagonists may be small molecules (organic or inorganic),
antisense nucleotides and PNAs, antibodies and altered hedgehog
proteins.
[0164] Small Molecule Antagonists
[0165] Hedgehog antagonist compounds for certain embodiments of the
invention are described in the formulas below and methods of making
the compositions are described in detail in the following U.S.
patent applications: Ser. Nos. 09/663,835, 09/685,244, 09/724,277,
09/687,800, 09/688,018, 60/308,449 and 09/688,076. The exemplary
compounds are divided into five parts. For each of the parts, the
variable groups and numbers (e.g., R.sub.1, L, Z.sub.2) are
individually and distinctly defined, and are internally consistent
but not necessarily consistent from part to part.
[0166] Exemplary Compounds Part 1
[0167] As described in further detail below, it is contemplated
that the subject methods can be carried out using any of a variety
of different steroidal alkaloids which can be readily identified,
e.g., by such drug screening assays as described herein. Steroidal
alkaloids have a fairly complex nitrogen-containing nucleus. Two
exemplary classes of steroidal alkaloids for use in the subject
methods are the Solanum type and the Veratrum type. The above
notwithstanding, in a preferred embodiment, the methods and
compositions of the present invention make use of compounds having
a steroidal alkaloid ring system of cyclopamine.
[0168] There are more than 50 naturally occurring veratrum
alkaloids including veratramine, cyclopamine, cycloposine, jervine,
and muldamine occurring in plants of the Veratrum spp. The
Zigadenus spp., death camas, also produces several veratrum-type of
steroidal alkaloids including zygacine. In general, many of the
veratrum alkaloids (e.g., jervine, cyclopamine and cycloposine)
consist of a modified steroid skeleton attached spiro to a
furanopiperidine. A typical veratrum-type alkaloid may be
represented by: 10
[0169] An example of the Solanum type is solanidine. This steroidal
alkaloid is the nucleus (i.e., aglycone) for two important
glycoalkaloids, solanine and chaconine, found in potatoes. Other
plants in the Solanum family including various nightshades,
Jerusalem cherries, and tomatoes also contain solanum-type
glycoalkaloids. Glycoalkaloids are glycosides of alkaloids. A
typical solanum-type alkaloid may be represented by: 11
[0170] Based on these structures, and the possibility that certain
unwanted side effects can be reduced by some manipulation of the
structure, a wide range of steroidal alkaloids are contemplated as
potential smoothened antagonists for use in the subject method. For
example, compounds useful in the subject methods include steroidal
alkaloids represented in the general formulas (I), or unsaturated
forms thereof and/or seco-, nor- or homo-derivatives thereof:
12
[0171] wherein, as valence and stability permit,
[0172] R.sub.2, R.sub.3, R.sub.4, and R.sub.5, represent one or
more substitutions to the ring to which each is attached, for each
occurrence, independently represent hydrogen, halogens, alkyls,
alkenyls, alkynyls, aryls, hydroxyl, .dbd.O, .dbd.S, alkoxyl,
silyloxy, amino, nitro, thiol, amines, imines, amides, phosphoryls,
phosphonates, phosphines, carbonyls, carboxyls, carboxamides,
anhydrides, silyls, ethers, thioethers, alkylsulfonyls,
arylsulfonyls, selenoethers, ketones, aldehydes, esters, sugar
(e.g., monosaccharide, disaccharide, polysaccharide, etc.),
carbamate (e.g., attached to the steroid at oxygen), carbonate, or
--(CH.sub.2).sub.m--R.sub.8;
[0173] R.sub.6, R.sub.7, and R'.sub.7, are absent or represent,
independently, halogens, alkyls, alkenyls, alkynyls, aryls,
hydroxyl, .dbd.O, .dbd.S, alkoxyl, silyloxy, amino, nitro, thiol,
amines, imines, amides, phosphoryls, phosphonates, phosphines,
carbonyls, carboxyls, carboxamides, anhydrides, silyls, ethers,
thioethers, alkylsulfonyls, arylsulfonyls, selenoethers, ketones,
aldehydes, esters, or --(CH.sub.2).sub.m--R.sub.8, or
[0174] R.sub.6 and R.sub.7, or R.sub.7 and R'.sub.7, taken together
form a ring or polycyclic ring, e.g., which is substituted or
unsubstituted,
[0175] with the proviso that at least one of R.sub.6, R.sub.7, or
R'.sub.7 is present and includes an amine, e.g., as one of the
atoms which makes up the ring;
[0176] R.sub.8 represents an aryl, a cycloalkyl, a cycloalkenyl, a
heterocycle, or a polycycle; and
[0177] m is an integer in the range 0 to 8 inclusive.
[0178] In certain embodiments, R.sub.2 represents .dbd.O, sugar
(e.g., monosaccharide, disaccharide, polysaccharide, etc.),
carbamate (e.g., attached to the steroid at oxygen), ester (e.g.,
attached to the steroid at oxygen), carbonate, or alkoxy.
Substituents such as carbamate, ester, carbonate, and alkoxy may be
substituted or unsubstituted, e.g., may include additional
functional groups such as aryl, aralkyl, heteroaryl, heteroaralkyl,
amide, acylamino, carbonyl, ester, carbamate, urea, ketone,
sulfonamide, etc.
[0179] In certain embodiments, the amine of R.sub.6, R.sub.7, or
R'.sub.7 is a tertiary amine.
[0180] In particular embodiments, R.sub.3, for each occurrence, is
an --OH, alkyl, --O-alkyl, --C(O)-alkyl, or --C(O)--R.sub.8.
[0181] In particular embodiments, R.sub.4, for each occurrence, is
an absent, or represents --OH, .dbd.O, alkyl, --O-alkyl,
--C(O)-alkyl, or --C(O)--R.sub.8.
[0182] In particular embodiments, two of R.sub.6, R.sub.7, and
R'.sub.7 taken together form a nitrogen-containing ring, such as a
furanopiperidine, such as perhydrofuro[3,2-b]pyridine, a
pyranopiperidine, a quinoline, an indole, a pyranopyrrole, a
naphthyridine, a thiofuranopiperidine, or a
thiopyranopiperidine.
[0183] In certain embodiments, the nitrogen-containing ring
comprises a tertiary amine, e.g., by having an extraannular
substituent on the nitrogen atom, e.g., an alkyl substituted with,
for example, aryl, aralkyl, heteroaryl, heteroaralkyl, amide,
acylamino, carbonyl, ester, carbamate, urea, ketone, sulfonamide,
etc. In certain embodiments, the extraannular substituent of the
tertiary amine is a hydrophobic substituent. In certain
embodiments, the hydrophobic extraannular substituent includes an
aryl, heteroaryl, carbocyclyl, heterocyclyl, or polycyclyl group,
such as biotin, a zwitterionic complex of boron, a steroidal
polycycle, etc. In certain embodiments, the hydrophobic substituent
may consist essentially of a combination of alkyl, amido,
acylamino, ketone, ester, ether, halogen, alkenyl, alkynyl, aryl,
aralkyl, urea, or similar functional groups, including between 5
and 40 non-hydrogen atoms, more preferably between 5 and 20
non-hydrogen atoms.
[0184] In particular embodiments, R.sub.8 represents an aryl, a
cycloalkyl, a cycloalkenyl, a heterocycle, or a polycycle, and
preferably R.sub.8 is a piperidine, pyrrolidine, pyridine,
pyrimidine, morpholine, thiomorpholine, pyridazine, etc.
[0185] In certain preferred embodiments, the definitions outlined
above apply, and the subject compounds are represented by general
formula Ia or unsaturated forms thereof and/or seco-, nor- or
bomo-derivatives thereof: 13
[0186] In certain embodiments, the steroidal alkaloid is
represented in the general formula (II), or unsaturated forms
thereof and/or seco-, nor- or homo-derivatives thereof: 14
[0187] wherein R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6,
R.sub.7, and R'.sub.7 are as defined above, and X represents O or
S, though preferably O.
[0188] In certain embodiments, R.sub.2 represents .dbd.O, sugar
(e.g., monosaccharide, disaccharide, polysaccharide, etc.),
carbamate (e.g., attached to the steroid at oxygen), ester (e.g.,
attached to the steroid at oxygen), carbonate, or alkoxy.
Substituents such as carbamate, ester, carbonate, and alkoxy may be
substituted or unsubstituted, e.g., may include additional
functional groups such as aryl, aralkyl, heteroaryl, heteroaralkyl,
amide, acylamino, carbonyl, ester, carbamate, urea, ketone,
sulfonamide, etc.
[0189] In certain embodiments, the amine of R.sub.6, R.sub.7, or
R'.sub.7 is a tertiary amine, e.g., substituted with a substituted
or unsubstituted alkyl. In certain embodiments, the amine is part
of a bicyclic ring system formed from R.sub.7 and R'.sub.7, e.g., a
furanopiperidine system, and the third substituent is an alkyl
substituted with, for example, aryl, aralkyl, heteroaryl,
heteroaralkyl, amide, acylamino, carbonyl, ester, carbamate, urea,
ketone, sulfonamide, etc. In certain embodiments, the extraannular
substituent of the tertiary amine is a hydrophobic substituent. In
certain embodiments, the hydrophobic extraannular substituent
includes an aryl, heteroaryl, carbocyclyl, heterocyclyl, or
polycyclyl group, such as biotin, a zwitterionic complex of boron,
a steroidal polycycle, etc. In certain embodiments, the hydrophobic
substituent may consist essentially of a combination of alkyl,
amido, acylamino, ketone, ester, ether, halogen, alkenyl, alkynyl,
aryl, aralkyl, urea, or similar functional groups, including
between 5 and 40 non-hydrogen atoms, more preferably between 5 and
20 non-hydrogen atoms.
[0190] In certain preferred embodiments, the definitions outlined
above apply, and the subject compounds are represented by general
formula IIa or unsaturated forms thereof and/or seco-, nor- or
homo-derivatives thereof: 15
[0191] In certain embodiments, the steroidal alkaloid is
represented in the general formula (III), or unsaturated forms
thereof and/or seco-, nor- or homo-derivatives thereof: 16
[0192] wherein
[0193] R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.8 are as
defined above;
[0194] A and B represent monocyclic or polycyclic groups;
[0195] T represents an alkyl, an aminoalkyl, a carboxyl, an ester,
an amide, ether or amine linkage of 1-10 bond lengths;
[0196] T' is absent, or represents an alkyl, an aminoalkyl, a
carboxyl, an ester, an amide, ether or amine linkage of 1-3 bond
lengths, wherein if T and T' are present together, than T and T'
taken together with the ring A or B form a covalently closed ring
of 5-8 ring atoms;
[0197] R.sub.9 represents one or more substitutions to the ring A
or B, which for each occurrence, independently represent halogens,
alkyls, alkenyls, alkynyls, aryls, hydroxyl, .dbd.O, .dbd.S,
alkoxyl, silyloxy, amino, nitro, thiol, amines, imines, amides,
phosphoryls, phosphonates, phosphines, carbonyls, carboxyls,
carboxamides, anhydrides, silyls, ethers, thioethers,
alkylsulfonyls, arylsulfonyls, selenoethers, ketones, aldehydes,
esters, or --(CH.sub.2).sub.m--R.sub.8; and
[0198] n and m are, independently, zero, 1 or 2;
[0199] with the proviso that A, or T, T', and B, taken together,
include at least one amine.
[0200] In certain embodiments, R.sub.2 represents .dbd.O, sugar
(e.g., monosaccharide, disaccharide, polysaccharide, etc.),
carbamate (e.g., attached to the steroid at oxygen), ester (e.g.,
attached to the steroid at oxygen), carbonate, or alkoxy.
Substituents such as carbamate, ester, carbonate, and alkoxy may be
substituted or unsubstituted, e.g., may include additional
functional groups such as aryl, aralkyl, heteroaryl, heteroaralkyl,
amide, acylamino, carbonyl, ester, carbamate, urea, ketone,
sulfonamide, etc.
[0201] In certain embodiments, the amine of A, or T, T', and B, is
a tertiary amine, e.g., substituted with a substituted or
unsubstituted alkyl, e.g., substituted with aryl, aralkyl,
heteroaryl, heteroaralkyl, amide, acylamino, carbonyl, ester,
carbamate, urea, ketone, sulfonamide, etc. In certain embodiments,
the extraannular substituent of the tertiary amine is a hydrophobic
substituent. In certain embodiments, the hydrophobic extraannular
substituent includes an aryl, heteroaryl, carbocyclyl,
heterocyclyl, or polycyclyl group, such as biotin, a zwitterionic
complex of boron, a steroidal polycycle, etc. In certain
embodiments, the hydrophobic substituent may consist essentially of
a combination of alkyl, amido, acylamino, ketone, ester, ether,
halogen, alkenyl, alkynyl, aryl, aralkyl, urea, or similar
functional groups, including between 5 and 40 non-hydrogen atoms,
more preferably between 5 and 20 non-hydrogen atoms.
[0202] In certain preferred embodiments, the definitions outlined
above apply, and the subject compounds are represented by general
formula IIIa or unsaturated forms thereof and/or seco-, nor- or
homo-derivatives thereof: 17
[0203] For example, the subject methods can utilize smoothened
antagonists based on the veratrum-type steroidal alkaloids jervine,
cyclopamine, cycloposine, mukiamine or veratramine, e.g., which may
be represented in the general formula (IV), or unsaturated forms
thereof and/or seco-, nor- or homo-derivatives thereof: 18
[0204] wherein
[0205] R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6 and R.sub.9 are
as defined above;
[0206] R.sub.22 is absent or represents an alkyl, an alkoxyl or
--OH.
[0207] In certain embodiments, R.sub.2 represents .dbd.O, sugar
(e.g., monosaccharide, disaccharide, polysaccharide, etc.),
carbamate (e.g., attached to the steroid at oxygen), ester (e.g.,
attached to the steroid at oxygen), carbonate, or alkoxy.
Substituents such as carbamate, ester, carbonate, and alkoxy may be
substituted or unsubstituted, e.g., may include additional
functional groups such as aryl, aralkyl, heteroaryl, heteroaralkyl,
amide, acylamino, carbonyl, ester, carbamate, urea, ketone,
sulfonamide, etc.
[0208] In certain embodiments, R.sub.9 includes a substituent on
nitrogen, e.g., a substituted or unsubstituted alkyl, e.g.,
substituted with, for example, aryl, aralkyl, heteroaryl,
heteroaralkyl, amide, acylamino, carbonyl, ester, carbamate, urea,
ketone, sulfonamide, etc. In certain embodiments, the extraannular
substituent (e.g., R.sub.9) of the tertiary amine is a hydrophobic
substituent. In certain embodiments, the hydrophobic extraannular
substituent includes an aryl, heteroaryl, carbocyclyl,
heterocyclyl, or polycyclyl group, such as biotin, a zwitterionic
complex of boron, a steroidal polycycle, etc. In certain
embodiments, the hydrophobic substituent may consist essentially of
a combination of alkyl, amido, acylamino, ketone, ester, ether,
halogen, alkenyl, alkynyl, aryl, aralkyl, urea, or similar
functional groups, including between 5 and 40 non-hydrogen atoms,
more preferably between 5 and 20 non-hydrogen atoms.
[0209] In certain preferred embodiments, the definitions outlined
above apply, and the subject compounds are represented by general
formula IVa or unsaturated forms thereof and/or seco-, nor- or
homo-derivatives thereof: 19
[0210] In certain embodiments, the steroidal alkaloid is
represented in the general formula (V) or unsaturated forms thereof
and/or seco-, nor- or homo-derivatives thereof: 20
[0211] wherein R.sub.2, R.sub.3, R.sub.4, R.sub.6 and R.sub.9 are
as defined above;
[0212] In certain embodiments, R.sub.2 represents .dbd.O, sugar
(e.g., monosaccharide, disaccharide, polysaccharide, etc.),
carbamate (e.g., attached to the steroid at oxygen), ester (e.g.,
attached to the steroid at oxygen), carbonate, or alkoxy.
Substituents such as carbamate, ester, carbonate, and alkoxy may be
substituted or unsubstituted, e.g., may include additional
functional groups such as aryl, aralkyl, heteroaryl, heteroaralkyl,
amide, acylamino, carbonyl, ester, carbamate, urea, ketone,
sulfonamide, etc.
[0213] In certain embodiments, R.sub.9 includes a substituent on
nitrogen, e.g., a substituted or unsubstituted alkyl, e.g.,
substituted with, for example, aryl, aralkyl, heteroaryl,
heteroaralkyl, amide, acylamino, carbonyl, ester, carbamate, urea,
ketone, sulfonamide, etc.
[0214] In certain embodiments, the extraannular substituent of the
tertiary amine (e.g., R.sub.9) is a hydrophobic substituent. In
certain embodiments, the hydrophobic extraannular substituent
includes an aryl, heteroaryl, carbocyclyl, heterocyclyl, or
polycyclyl group, such as biotin, a zwitterionic complex of boron,
a steroidal polycycle, etc. In certain embodiments, the hydrophobic
substituent may consist essentially of a combination of alkyl,
amido, acylamino, ketone, ester, ether, halogen, alkenyl, alkynyl,
aryl, aralkyl, urea, or similar functional groups, including
between 5 and 40 non-hydrogen atoms, more preferably between 5 and
20 non-hydrogen atoms.
[0215] In certain preferred embodiments, the definitions outlined
above apply, and the subject compounds are represented by general
formula Va or unsaturated forms thereof and/or seco-, nor- or
homo-derivatives thereof: 21
[0216] Another class of smoothened antagonists can be based on the
veratrum-type steroidal alkaloids resembling verticine and
zygacine, e.g., general formula (VI), or unsaturated forms thereof
and/or seco-, nor- or homo-derivatives thereof: 22
[0217] In certain embodiments, R.sub.2 represents .dbd.O, sugar
(e.g., monosaccharide, disaccharide, polysaccharide, etc.),
carbamate (e.g., attached to the steroid at oxygen), ester (e.g.,
attached to the steroid at oxygen), carbonate, or alkoxy.
Substituents such as carbamate, ester, carbonate, and alkoxy may be
substituted or unsubstituted, e.g., may include additional
functional groups such as aryl, aralkyl, heteroaryl, heteroaralkyl,
amide, acylamino, carbonyl, ester, carbamate, urea, ketone,
sulfonamide, etc.
[0218] In certain preferred embodiments, the definitions outlined
above apply, and the subject compounds are represented by general
formula VIa or unsaturated forms thereof and/or seco-, nor- or
homo-derivatives thereof: 23
[0219] In certain embodiments, the steroidal alkaloid is
represented in the general formula (VII) or unsaturated forms
thereof and/or seco-, nor- or homo-derivatives thereof: 24
[0220] wherein R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.9 are
as defined above.
[0221] In certain embodiments, R.sub.2 represents .dbd.O, sugar
(e.g., monosaccharide, disaccharide, polysaccharide, etc.),
carbamate (e.g., attached to the steroid at oxygen), ester (e.g.,
attached to the steroid at oxygen), carbonate, or alkoxy.
Substituents such as carbamate, ester, carbonate, and alkoxy may be
substituted or unsubstituted, e.g., may include additional
functional groups such as aryl, aralkyl, heteroaryl, heteroaralkyl,
amide, acylamino, carbonyl, ester, carbamate, urea, ketone,
sulfonamide, etc.
[0222] In certain preferred embodiments, the definitions outlined
above apply, and the subject compounds are represented by general
formula VIIa or unsaturated forms thereof and/or seco-, nor- or
homo-derivatives thereof: 25
[0223] In certain embodiments, the subject antagonists and
activators can be chosen on the basis of their selectively for the
smoothened pathway. This selectivity can be for the smoothened
pathway versus other steroid-mediated pathways (such as
testosterone or estrogen mediated activities), as well as
selectivity for particular hedgehog/ptc/smoothened pathways, e.g.,
which isotype specific for ptc (e.g., ptc-1, ptc-2) or hedgehog
(e.g., Shh, Ihh, Dhh, etc.). For instance, the subject method may
employ steroidal alkaloids which do not substantially interfere
with the biological activity of such steroids as aldosterone,
androstane, androstene, androstenedione, androsterone,
cholecalciferol, cholestane, cholic acid, corticosterone, cortisol,
cortisol acetate, cortisone, cortisone acetate,
deoxycorticosterone, digitoxigenin, ergocalciferol, ergosterol,
estradiol-17-.alpha., estradiol-17-.beta., estriol, estrane,
estrone, hydrocortisone, lanosterol, lithocholic acid, mestranol,
.beta.-methasone, prednisone, pregnane, pregnenolone, progesterone,
spironolactone, testosterone, triamcinolone and their derivatives,
at least so far as those activities are unrelated to ptc related
signaling.
[0224] In one embodiment, the subject steroidal alkaloid for use in
the present method has a k.sub.d for members of the nuclear hormone
receptor superfamily of greater than 1 .mu.M, and more preferably
greater than 1 mM, e.g., it does not bind estrogen, testosterone
receptors or the like. Preferably, the subject smoothened
antagonist has no estrogenic activity at physiological
concentrations (e.g., in the range of 1 ng -1 mg/kg).
[0225] In this manner, untoward side effects which may be
associated certain members of the steroidal alkaloid class can be
reduced. For example, using the drug screening assays described
herein, the application of combinatorial and medicinal chemistry
techniques to the steroidal alkaloids provides a means for reducing
such unwanted negative side effects including personality changes,
shortened life spans, cardiovascular diseases and vascular
occlusion, organ toxicity, hyperglycemia and diabetes, Cushnoid
features, "wasting" syndrome, steroidal glaucoma, hypertension,
peptic ulcers, and increased susceptibility to infections. For
certain embodiments, it will be beneficial to reduce the
teratogenic activity relative to jervine, as for example, in the
use of the subject method to selectively inhibit
spermatogenesis.
[0226] In a preferred embodiment, the subject antagonists are
steroidal alkaloids other than spirosolane, tomatidine, jervine,
etc.
[0227] In particular embodiments, the steroidal alkaloid is chosen
for use because it is more selective for one patched isoform over
the next, e.g., 10-fold, and more preferably at least 100- or even
1000-fold more selective for one patched pathway (ptc-1, ptc-2)
over another. Likewise, the steroidal alkaloid may be chosen for
use because it is more selective for one smoothened isoform over
the next, e.g., 10-fold, and more preferably at least 100- or even
1000-fold more selective for one wild-type smoothened protein
(should various isoforms exist) or for activated smoothened mutants
relative to wild-type smoothened. In certain embodiments, the
subject method can be carried out conjointly with the
administration of growth and/or trophic factors, or compositions
that also act on other parts of the hedgehog/smoothened pathway.
For instance, it is contemplated that the subject methods can
include treatment with an agent that modulates cAMP levels, e.g.,
increasing or decreasing intracellular levels of cAMP.
[0228] In one embodiment, the subject method utilizes a smoothened
antagonist, and the conjoint agent elevates cAMP levels in order to
enhance the efficacy of the smoothened antagonist.
[0229] For example, compounds that may activate adenylate cyclase
include forskolin (FK), cholera toxin (CT), pertussis toxin (PT),
prostaglandins (e.g., PGE-1 and PGE-2), colforsin and
.beta.-adrenergic receptor agonists. .beta.-Adrenergic receptor
agonists (sometimes referred to herein as ".beta.-adrenergic
agonists") include albuterol, bambuterol, bitolterol, carbuterol,
clenbuterol, clorprenaline, denopamine, dioxethedrine, dopexamine,
ephedrine, epinephrine, etafedrine, ethylnorepinephrine, fenoterol,
formoterol, hexoprenaline, ibopamine, isoetharine, isoproterenol,
mabuterol, metaproterenol, methoxyphenamine, norepinephrine,
oxyfedrine, pirbuterol, prenalterol, procaterol, propranolol,
protokylol, quinterenol, reproterol, rimiterol, ritodrine,
salmefamol, soterenol, salmeterol, terbutaline, tretoquinol,
tulobuterol, and xamoterol.
[0230] Compounds which may inhibit a cAMP phosphodiesterase include
anrinone, milrinone, xanthine, methylxanthine, anagrelide,
cilostamide, medorinone, indolidan, rolipram,
3-isobutyl-1-methylxanthine (IBMX), chelerythrine, cilostazol,
glucocorticoids, griseolic acid, etazolate, caffeine, indomethacin,
papverine, MDL 12330A, SQ 22536, GDPssS, clonidine, type III and
type IV phosphodiesterase inhibitors, methylxanthines such as
pentoxifylline, theophylline, theobromine, pyrrolidinones and
phenyl cycloalkane and cycloalkene derivatives (described in PCT
publications Nos. WO 92/19594 and WO 92/10190), lisophylline, and
fenoxamine.
[0231] Analogs of cAMP which may be useful in the present method
include dibutyryl-cAMP (db-cAMP), (8-(4)-chlorophenylthio)-cAMP
(cpt-cAMP), 8-[(4-bromo-2,3-dioxobutyl)thio]-cAMP,
2-[(4-bromo-2,3-dioxobutyl)thio]-c- AMP, 8-bromo-cAMP,
dioctanoyl-cAMP, Sp-adenosine 3':5'-cyclic phosphorothioate,
8-piperidino-cAMP, N.sup.6-phenyl-cAMP, 8-methylamino-cAMP,
8-(6-aminohexyl)amino-cAMP, 2'-deoxy-cAMP,
N.sup.6,2'-O-dibutryl-cAMP, N.sup.6,2'-O-disuccinyl-cAMP,
N.sup.6-monobutyryl-cAMP, 2'-O-monobutyryl-cAMP,
2'-O-monobutryl-8-bromo-- cAMP, N.sup.6-monobutryl-2'-deoxy-cAMP,
and 2'-O-monosuccinyl-cAMP.
[0232] Compounds which may reduce the levels or activity of cAMP
include prostaglandylinositol cyclic phosphate (cyclic PIP),
endothelins (ET)-1 and -3, norepinepurine, K252a, dideoxyadenosine,
dynorphins, melatonin, pertussis toxin, staurosporine, G.sub.i
agonists, MDL 12330A, SQ 22536, GDPssS and clonidine,
beta-blockers, and ligands of G-protein coupled receptors.
Additional compounds are disclosed in U.S. Pat. Nos. 5,891,875,
5,260,210, and 5,795,756.
[0233] Above-listed compounds useful in the subject methods may be
modified to increase the bioavailability, activity, or other
pharmacologically relevant property of the compound. For example,
forskolin has the formula: 26
[0234] Modifications of forskolin that have been found to increase
the hydrophilic character of forskolin without severely attenuating
the desired biological activity include acylation of the hydroxyls
at C6 and/or C7 (after removal of the acetyl group) with
hydrophilic acyl groups. In compounds wherein C6 is acylated with a
hydrophilic acyl group, C7 may optionally be deacetylated. Suitable
hydrophilic acyl groups include groups having the structure
--(CO)(CH.sub.2).sub.nX, wherein X is OH or NR.sub.2; R is
hydrogen, a C.sub.1-C.sub.4 alkyl group, or two Rs taken together
form a ring comprising 3-8 atoms, preferably 5-7 atoms, which may
include heteroatoms (e.g., piperazine or morpholine rings); and n
is an integer from 1-6, preferably from 1-4, even more preferably
from 1-2. Other suitable hydrophilic acyl groups include
hydrophilic amino acids or derivatives thereof, such as aspartic
acid, glutamic acid, asparagine, glutamine, seurine, threonine,
tyrosine, etc., including amino acids having a heterocyclic side
chain. Forskolin, or other compounds listed above, modified by
other possible hydrophilic acyl side chains known to those of skill
in the art may be readily synthesized and tested for activity in
the present method.
[0235] Similarly, variants or derivatives of any of the
above-listed compounds may be effective as CAMP antagonists in the
subject method, e.g., in order to decrease CAMP levels and
potentiate the activity of a smoothened activator. Those skilled in
the art will readily be able to synthesize and test such
derivatives for suitable activity.
[0236] Exemplary Compounds Part II
[0237] Additional steroidal alkaloids are contemplated as potential
hedgehog antagonists for use in the subject method. For example,
compounds useful in the subject methods include steroidal alkaloids
represented in the general formulas (VIII), or unsaturated forms
thereof and/or seco-, nor- or homo-derivatives thereof: 27
[0238] wherein, as valence and stability permit,
[0239] R.sub.2, R.sub.3, R.sub.4, and R.sub.5, represent one or
more substitutions to the ring to which each is attached, for each
occurrence, independently represent hydrogen, halogens, alkyls,
alkenyls, alkynyls, aryls, hydroxyl, .dbd.O, .dbd.S, alkoxyl,
silyloxy, amino, nitro, thiol, amines, imines, amides, phosphoryls,
phosphonates, phosphines, carbonyls, carboxyls, carboxamides,
anhydrides, silyls, ethers, thioethers, alkylsulfonyls,
arylsulfonyls, selenoethers, ketones, aldehydes, esters, sugar
(e.g., monosaccharide, disaccharide, polysaccharide, etc.),
carbamate (e.g., attached to the steroid at oxygen), carbonate, or
--(CH.sub.2).sub.m--R.sub.8;
[0240] R.sub.6, R.sub.7, and R'.sub.7, are absent or represent,
independently, halogens, alkyls, alkenyls, alkynyls, aryls,
hydroxyl, .dbd.O, .dbd.S, alkoxyl, silyloxy, amino, nitro, thiol,
amines, imines, amides, phosphoryls, phosphonates, phosphines,
carbonyls, carboxyls, carboxamides, anhydrides, silyls, ethers,
thioethers, alkylsulfonyls, arylsulfonyls, selenoethers, ketones,
aldehydes, esters, or --(CH.sub.2).sub.m--R.sub.8, or
[0241] R.sub.6 and R.sub.7, or R.sub.7 and R'.sub.7, taken together
form a ring or polycyclic ring, e.g., which is substituted or
unsubstituted,
[0242] with the proviso that at least one of R.sub.6, R.sub.7, or
R'.sub.7 is present and includes a primary or secondary amine;
[0243] R.sub.8 represents an aryl, a cycloalkyl, a cycloalkenyl, a
heterocycle, or a polycycle; and
[0244] m is an integer in the range 0 to 8 inclusive.
[0245] In preferred embodiments,
[0246] R.sub.2 and R.sub.3, for each occurrence, is an --OH, alkyl,
--O-alkyl, --C(O)-alkyl, or --C(O)--R.sub.8;
[0247] R.sub.4, for each occurrence, is an absent, or represents
--OH, .dbd.O, alkyl, --O-alkyl, --C(O)-alkyl, or
--C(O)--R.sub.8;
[0248] R.sub.6, R.sub.7, and R'.sub.7 each independently represent,
hydrogen, alkyls, alkenyls, alkynyls, amines, imines, amides,
carbonyls, carboxyls, carboxamides, ethers, thioethers, esters, or
--(CH.sub.2).sub.m--R.sub.8, or
[0249] R.sub.7, and R'.sub.7 taken together form a
furanopiperidine, such as perhydrofuro[3,2-b]pyridine, a
pyranopiperidine, a quinoline, an indole, a pyranopyrrole, a
naphthyridine, a thiofuranopiperidine, or a
thiopyranopiperidine
[0250] with the proviso that at least one of R.sub.6, R.sub.7, or
R'.sub.7 is present and includes a primary or secondary amine;
[0251] R.sub.8 represents an aryl, a cycloalkyl, a cycloalkenyl, a
heterocycle, or a polycycle, and preferably R.sub.8 is a
piperidine, pyrimidine, morpholine, thiomorpholine, pyridazine,
[0252] In certain preferred embodiments, the definitions outlined
above apply, and the subject compounds are represented by general
formula VIIIa or unsaturated forms thereof and/or seco-, nor- or
homo-derivatives thereof: 28
[0253] In preferred embodiments, the subject hedgehog antagonists
can be represented in one of the following general formulas (IX) or
unsaturated forms thereof and/or seco-, nor- or homo-derivatives
thereof: 29
[0254] wherein R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6,
R.sub.7, and R'.sub.7 are as defined above, and X represents O or
S, though preferably O.
[0255] In certain preferred embodiments, the definitions outlined
above apply, and the subject compounds are represented by general
formula IXa or unsaturated forms thereof and/or seco-, nor- or
homo-derivatives thereof: 30
[0256] In certain embodiments, the subject hedgehog antagonists are
represented by the general formula (X) or unsaturated forms thereof
and/or seco-, nor- or homo-derivatives thereof: 31
[0257] wherein
[0258] R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.8 are as
defined above;
[0259] A and B represent monocyclic or polycyclic groups;
[0260] T represents an alkyl, an aminoalkyl, a carboxyl, an ester,
an amide, ether or amine linkage of 1-10 bond lengths;
[0261] T' is absent, or represents an alkyl, an aminoalkyl, a
carboxyl, an ester, an amide, ether or amine linkage of 1-3 bond
lengths, wherein if T and T' are present together, than T and T'
taken together with the ring A or B form a covalently closed ring
of 5-8 ring atoms;
[0262] R.sub.9 represents one or more substitutions to the ring A
or B, which for each occurrence, independently represent halogens,
alkyls, alkenyls, alkynyls, aryls, hydroxyl, .dbd.O, .dbd.S,
alkoxyl, silyloxy, amino, nitro, thiol, amines, imines, amides,
phosphoryls, phosphonates, phosphines, carbonyls, carboxyls,
carboxamides, anhydrides, silyls, ethers, thioethers,
alkylsulfonyls, arylsulfonyls, selenoethers, ketones, aldehydes,
esters, or --(CH.sub.2).sub.m--R.sub.8; and
[0263] n and m are, independently, zero, 1 or 2;
[0264] with the proviso that A and R.sub.9, or T, T' B and R.sub.9,
taken together include at least one primary or secondary amine.
[0265] In certain preferred embodiments, the definitions outlined
above apply, and the subject compounds are represented by general
formula Xa or unsaturated forms thereof and/or seco-, nor- or
homo-derivatives thereof: 32
[0266] For example, the subject methods can utilize hedgehog
antagonists based on the veratrum-type steroidal alkaloids jervine,
cyclopamine, cycloposine, mukiamine or veratramine, e.g., which may
be represented in the general formula (XI) or unsaturated forms
thereof and/or seco-, nor- or homo-derivatives thereof: 33
[0267] wherein
[0268] R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6 and R.sub.9 are
as defined above;
[0269] R.sub.22 is absent or represents an alkyl, an alkoxyl or
--OH.
[0270] In certain preferred embodiments, the definitions outlined
above apply, and the subject compounds are represented by general
formula XIa or unsaturated forms thereof and/or seco-, nor- or
homo-derivatives thereof: 34
[0271] In even more preferred embodiments, the subject antagonists
are represented in the formulas (XII) or unsaturated forms thereof
and/or seco-, nor- or homo-derivatives thereof: 35
[0272] wherein R.sub.2, R.sub.3, R.sub.4, R.sub.6 and R.sub.9 are
as defined above;
[0273] In certain preferred embodiments, the definitions outlined
above apply, and the subject compounds are represented by general
formula XIIa or unsaturated forms thereof and/or seco-, nor- or
homo-derivatives thereof: 36
[0274] Another class of hedgehog antagonists can be based on the
veratrum-type steroidal alkaloids resembling verticine and
zygacine, e.g., represented in the general formulas (VI) or
unsaturated forms thereof and/or seco-, nor- or homo-derivatives
thereof: 37
[0275] wherein R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.9 are
as defined above.
[0276] In certain preferred embodiments, the definitions outlined
above apply, and the subject compounds are represented by general
formula XIIIa or unsaturated forms thereof and/or seco-, nor- or
homo-derivatives thereof: 38
[0277] Still another class of potential hedgehog antagonists are
based on the solanum-type steroidal alkaloids, e.g., solanidine,
which may be represented in the general formula (XIV) or
unsaturated forms thereof and/or seco-, nor- or homo-derivatives
thereof: 39
[0278] wherein R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.9 are
as defined above.
[0279] In certain preferred embodiments, the definitions outlined
above apply, and the subject compounds are represented by general
formula XIVa or unsaturated forms thereof and/or seco-, nor- or
homo-derivatives thereof: 40
[0280] In certain embodiments, the subject antagonists can be
chosen on the basis of their selectively for the hedgehog pathway.
This selectivity can for the hedgehog pathway versus other
steroid-mediated pathways (such as testosterone or estrogen
mediated activities), as well as selectivity for particular
hedgehog pathways, e.g., which isotype specific for hedgehog (e.g.,
Shh, Ihh, Dhh) or the patched receptor (e.g., ptc-1, ptc-2). For
instance, the subject method may employ steroidal alkaloids which
do not substantially interfere with the biological activity of such
steroids as aldosterone, androstane, androstene, androstenedione,
androsterone, cholecalciferol, cholestane, cholic acid,
corticosterone, cortisol, cortisol acetate, cortisone, cortisone
acetate, deoxycorticosterone, digitoxigenin, ergocalciferol,
ergosterol, estradiol-17-.alpha., estradiol-17-.beta., estriol,
estrane, estrone, hydrocortisone, lanosterol, lithocholic acid,
mestranol, .beta.-methasone, prednisone, pregnane, pregnenolone,
progesterone, spironolactone, testosterone, triamcinolone and their
derivatives, at least so far as those activities are unrelated to
ptc related signaling.
[0281] In one embodiment, the subject steroidal alkaloid for use in
the present method has a k.sub.d for members of the nuclear hormone
receptor superfamily of greater than 1 .mu.M, and more preferably
greater than 1 mM, e.g., it does not bind estrogen, testosterone
receptors or the like. Preferably, the subject hedgehog antagonist
has no estrogenic activity at physiological concentrations (e.g.,
in the range of 1 ng -1 mg/kg).
[0282] In this manner, untoward side effects which may be
associated certain members of the steroidal alkaloid class can be
reduced. For example, using the drug screening assays described
herein, the application of combinatorial and medicinal chemistry
techniques to the steroidal alkaloids provides a means for reducing
such unwanted negative side effects including personality changes,
shortened life spans, cardiovascular diseases and vascular
occlusion, organ toxicity, hyperglycemia and diabetes, Cushnoid
features, "wasting" syndrome, steroidal glaucoma, hypertension,
peptic ulcers, and increased susceptibility to infections. For
certain embodiments, it will be beneficial to reduce the
teratogenic activity relative to jervine, as for example, in the
use of the subject method to selectively inhibit
spermatogenesis.
[0283] In a preferred embodiment, the subject antagonists are
steroidal alkaloids other than spirosolane, tomatidine, jervine,
etc.
[0284] In certain preferred embodiments, the subject inhibitors
inhibit a hedgehog signal transduction pathway with an ED.sub.50 of
1 mM or less, more preferably of 1 .mu.M or less, and even more
preferably of 1 nM or less.
[0285] In certain embodiments, the subject inhibitors inhibit a
hedgehog signal transduction pathway with an ED.sub.50 of 1 mM or
less, more preferably 1 .mu.M or less, and even more preferably 1
nM or less.
[0286] In particular embodiments, the steroidal alkaloid is chosen
for use because it is more selective for one patched isoform over
the next, e.g., 10-fold, and more preferably at least 100- or even
1000-fold more selective for one patched pathway (ptc-1, ptc-2)
over another.
[0287] Exemplary Compounds Part III
[0288] As described in further detail below, it is contemplated
that the subject methods can be carried out using a variety of
different small molecules which can be readily identified, for
example, by such drug screening assays as described herein. For
example, compounds useful in the subject methods include compounds
may be represented by general formula (XV): 41
[0289] wherein, as valence and stability permit,
[0290] R.sub.1 and R.sub.2, independently for each occurrence,
represent H, lower alkyl, aryl (e.g., substituted or
unsubstituted), aralkyl (e.g., substituted or unsubstituted, e.g.,
--(CH.sub.2).sub.naryl), or heteroaryl (e.g., substituted or
unsubstituted), or heteroaralkyl (e.g., substituted or
unsubstituted, e.g., --(CH.sub.2).sub.nheteroaralkyl-);
[0291] L, independently for each occurrence, is absent or
represents --(CH.sub.2).sub.n-alkyl, -alkenyl-, -alkynyl-,
--(CH.sub.2).sub.nalkenyl- -, --(CH.sub.2).sub.nalkynyl-,
--(CH.sub.2).sub.nO(CH.sub.2).sub.p--,
--(CH.sub.2).sub.nNR.sub.2(CH.sub.2).sub.p--,
--(CH.sub.2).sub.nS(CH.sub.- 2).sub.p--,
--(CH.sub.2).sub.nalkenyl(CH.sub.2).sub.p--,
--(CH.sub.2).sub.nalkynyl(CH.sub.2).sub.p--, --O(CH.sub.2).sub.n--,
--NR.sub.2(CH.sub.2).sub.n--, or --S(CH.sub.2).sub.n--;
[0292] X.sub.1 and X.sub.2 can be selected, independently, from
--N(R.sub.8)--, --O--, --S--, --Se--, --N.dbd.N--, --ON.dbd.CH--,
--(R.sub.8)N--N(R.sub.8)--, --ON(R.sub.8)--, a heterocycle, or a
direct bond between L and Y.sub.1 or Y.sub.2, respectively;
[0293] Y.sub.1 and Y.sub.2 can be selected, independently, from
--C(.dbd.O)--, --C(.dbd.S)--, --S(O.sub.2)--, --S(O)--,
--C(.dbd.NCN)--, --P(.dbd.O)(OR.sub.2)--, a heteroaromatic group,
or a direct bond between X.sub.1 and Z.sub.1 or X.sub.2 and
Z.sub.2, respectively;
[0294] Z.sub.1 and Z.sub.2 can be selected, independently, from
--N(R.sub.8)--, --O--, --S--, --Se--, --N.dbd.N--,
--ON.dbd.CH----R.sub.8N--NR.sub.8--, --ONR.sub.8--, a heterocycle,
or a direct bond between Y.sub.1 or Y.sub.2, respectively, and
L;
[0295] R.sub.8, independently for each occurrence, represents H,
lower alkyl, --(CH.sub.2).sub.naryl (e.g., substituted or
unsubstituted), --(CH.sub.2).sub.nheteroaryl (e.g., substituted or
unsubstituted), or two R.sub.8 taken together may form a 4- to
8-membered ring, e.g., with X.sub.1 and Z.sub.1 or X.sub.2 and
Z.sub.1, which ring may include one or more carbonyls;
[0296] p represents, independently for each occurrence, an integer
from 0 to 10, preferably from 0 to 3; and
[0297] n, individually for each occurrence, represents an integer
from 0 to 10, preferably from 0 to 5.
[0298] In certain embodiments, R.sub.1 represents a substituted or
unsubstituted heteroaryl group.
[0299] In certain embodiments, X.sub.1 and X.sub.2 can be selected
from --N(R.sub.8)--, --O--, --S--, a direct bond, and a
heterocycle, Y.sub.1 and Y.sub.2 can be selected from
--C(.dbd.O)--, --C(.dbd.S)--, and --S(O.sub.2)--, and Z.sub.1 or
Z.sub.2 can be selected from --N(R.sub.8)--, --O--, --S--, a direct
bond, and a heterocycle.
[0300] In certain related embodiments, X.sub.1-Y.sub.1-Z.sub.1 or
X.sub.2-Y.sub.2-Z.sub.2 taken together represents a urea
(N--C(O)--N) or an amide (N--C(O) or C(O)--N).
[0301] In certain embodiments, X.sub.1 or X.sub.2 represents a
diazacarbocycle, such as a piperazine.
[0302] In certain embodiments, R.sub.1 represents a fused
cycloalkyl-aryl or cycloalkyl-heteroaryl system, for example:
42
[0303] wherein W is a substituted or unsubstituted aryl or
heteroaryl ring fused to the cycloalkyl ring and m is an integer
from 1-4 inclusive, e.g., from 1-3, or from 1-2. The fused system
may be bound to L from any carbon of the fused system, including
the position depicted above. In certain embodiments, R.sub.1 may
represent a tetrahydronaphthyl group, and preferably
Y.sub.1-X.sub.1-L-R.sub.1 taken together represent a
tetrahydronaphthyl amide group, such as: 43
[0304] In embodiments wherein Y.sub.1 and Z.sub.1 are absent and
X.sub.1 comprises a pyrimidone, compounds useful in the present
invention may be represented by general formula (XVI): 44
[0305] wherein, as valence and stability permit,
[0306] R.sub.1 and R.sub.2, independently for each occurrence,
represent H, lower alkyl, --(CH.sub.2).sub.naryl (e.g., substituted
or unsubstituted), or --(CH.sub.2).sub.nheteroaryl (e.g.,
substituted or unsubstituted);
[0307] L, independently for each occurrence, is absent or
represents --(CH.sub.2).sub.n-alkyl, -alkenyl-, -alkynyl-,
--(CH.sub.2).sub.nalkenyl- -, --(CH.sub.2).sub.nalkynyl-,
--(CH.sub.2).sub.nO(CH.sub.2).sub.p--,
--(CH.sub.2).sub.nNR.sub.2(CH.sub.2).sub.p--,
--(CH.sub.2).sub.nS(CH.sub.- 2).sub.p--,
--(CH.sub.2).sub.nalkenyl(CH.sub.2).sub.p--,
--(CH.sub.2).sub.nalkynyl(CH.sub.2).sub.p--, --O(CH.sub.2).sub.n--,
--NR.sub.2(CH.sub.2).sub.n--, or --S(CH.sub.2).sub.n--;
[0308] X can be selected from --N(R.sub.8)--, --O--, --S--, --Se--,
--N.dbd.N--, --ON.dbd.CH--, --(R.sub.8)N--N(R.sub.8)--,
--ON(R.sub.8)--, a heterocycle, or a direct bond between L and
Y;
[0309] Y can be selected from --C(.dbd.O)--, --C(.dbd.S)--,
--S(O.sub.2)--, --S(O)--, --C(.dbd.NCN)--, --P(.dbd.O)(OR.sub.2)--,
a heteroaromatic group, or a direct bond between X and Z;
[0310] Z can be selected from --N(R.sub.8)--, --O--, --S--, --Se--,
--N.dbd.N--, --ON.dbd.CH--, --R.sub.8N--NR.sub.8--, --ONR.sub.8--,
a heterocycle, or a direct bond between Y and L;
[0311] R.sub.8, independently for each occurrence, represents H,
lower alkyl, --(CH.sub.2).sub.naryl (e.g., substituted or
unsubstituted), --(CH.sub.2).sub.nheteroaryl (e.g., substituted or
unsubstituted), or two R.sub.8 taken together may form a 4- to
8-membered ring, e.g., with X and Z, which ring may include one or
more carbonyls;
[0312] W represents a substituted or unsubstituted aryl or
heteroaryl ring fused to the pyrimidone ring;
[0313] p represents, independently for each occurrence, an integer
from 0 to 10, preferably from 0 to 3; and
[0314] n, individually for each occurrence, represents an integer
from 0 to 10, preferably from 0 to 5.
[0315] In embodiments wherein Y.sub.1 and Z.sub.1 are absent and
X.sub.1 comprises a pyrimidone, compounds useful in the present
invention may be represented by general formula (XVII): 45
[0316] wherein, as valence and stability permit,
[0317] R.sub.1 and R.sub.2, independently for each occurrence,
represent H, lower alkyl, aryl (e.g., substituted or
unsubstituted), aralkyl (e.g., substituted or unsubstituted, e.g.,
--(CH.sub.2).sub.naryl), or heteroaryl (e.g., substituted or
unsubstituted), or heteroaralkyl (e.g., substituted or
unsubstituted, e.g., --(CH.sub.2).sub.nheteroaralkyl-);
[0318] L, independently for each occurrence, is absent or
represents --(CH.sub.2).sub.n-alkyl, -alkenyl-, -alkynyl-,
--(CH.sub.2).sub.nalkenyl- -, --(CH.sub.2).sub.nalkynyl-,
--(CH.sub.2).sub.nO(CH.sub.2).sub.p--,
--(CH.sub.2).sub.nNR.sub.2(CH.sub.2).sub.p--,
(CH.sub.2).sub.nS(CH.sub.2)- .sub.p--,
--(CH.sub.2).sub.nalkenyl(CH.sub.2).sub.p--,
--(CH.sub.2).sub.nalkynyl(CH.sub.2).sub.p--, --O(CH.sub.2).sub.n--,
--NR.sub.2(CH.sub.2).sub.n--, or --S(CH.sub.2).sub.n--, which may
optionally be substituted with a group selected from H, substituted
or unsubstituted lower alkyl, alkenyl, or alkynyl, cycloalkylalkyl
(e.g., substituted or unsubstituted, e.g.,
--(CH.sub.2).sub.ncycloalkyl), (e.g., substituted or
unsubstituted), aryl (e.g., substituted or unsubstituted), aralkyl
(e.g., substituted or unsubstituted, e.g., --(CH.sub.2).sub.naryl),
or heteroaryl (e.g., substituted or unsubstituted), or
heteroaralkyl (e.g., substituted or unsubstituted, e.g.,
--(CH.sub.2).sub.nheteroaralkyl-), preferably from H, lower alkyl,
--(CH.sub.2).sub.naryl (e.g., substituted or unsubstituted), or
--(CH.sub.2).sub.nheteroaryl (e.g., substituted or
unsubstituted);
[0319] X can be selected from --N(R.sub.8)--, --O--, --S--, --Se--,
--N.dbd.N--, --ON.dbd.CH--, --(R.sub.8)N--N(R.sub.8)--,
--ON(R.sub.8)--, a heterocycle, or a direct bond between L and
Y;
[0320] Y can be selected from --C(.dbd.O)--, --C(.dbd.S)--,
--S(O.sub.2)--, --S(O)--, --C(.dbd.NCN)--, --P(.dbd.O)(OR.sub.2)--,
a heteroaromatic group, or a direct bond between X and Z;
[0321] Z can be selected from --N(R.sub.8)--, --O--, --S--, --Se--,
--N.dbd.N--, --ON.dbd.CH--, --R.sub.8N--NR.sub.8--, --ONR.sub.8--,
a heterocycle, or a direct bond between Y and L;
[0322] R.sub.8, independently for each occurrence, represents H,
lower alkyl, aryl (e.g., substituted or unsubstituted), aralkyl
(e.g., substituted or unsubstituted, e.g., --(CH.sub.2).sub.naryl),
or heteroaryl (e.g., substituted or unsubstituted), or
heteroaralkyl (e.g., substituted or unsubstituted, e.g.,
--(CH.sub.2).sub.nheteroaralkyl-), or two R.sub.8 taken together
may form a 4- to 8-membered ring, e.g., with X and Z, which ring
may include one or more carbonyls;
[0323] W represents a substituted or unsubstituted aryl or
heteroaryl ring fused to the pyrimidone ring;
[0324] p represents, independently for each occurrence, an integer
from 0 to 10, preferably from 0 to 3; and
[0325] n, individually for each occurrence, represents an integer
from 0 to 10, preferably from 0 to 5.
[0326] In certain embodiments, R.sub.1 represents a substituted or
unsubstituted aryl or heteroaryl group, e.g., a phenyl ring, a
pyridine ring, etc. In certain embodiments wherein -LR.sub.1
represents a substituted aryl or heteroaryl group, R.sub.1 is
preferably not substituted with an isopropoxy (Me.sub.2CHO--)
group. In certain embodiments wherein -LR.sub.1 represents a
substituted aryl or heteroaryl group, R.sub.1 is preferably not
substituted with an ether group. In certain embodiments,
substituents on R.sub.1 (e.g., other than hydrogen) are selected
from halogen, cyano, alkyl, alkenyl, alkynyl, aryl, hydroxyl,
(unbranched alkyl-O--), silyloxy, amino, nitro, thiol, amino,
imino, amido, phosphoryl, phosphonate, phosphine, carbonyl,
carboxyl, carboxamide, anhydride, silyl, thioether, alkylsulfonyl,
arylsulfonyl, sulfoxide, selenoether, ketone, aldehyde, ester, or
--(CH.sub.2).sub.m--R.sub.8. In certain embodiments, non-hydrogen
substituents are selected from halogen, cyano, alkyl, alkenyl,
alkynyl, aryl, nitro, thiol, imino, amido, carbonyl, carboxyl,
anhydride, thioether, alkylsulfonyl, arylsulfonyl, ketone,
aldehyde, and ester. In certain embodiments, non-hydrogen
substituents are selected from halogen, cyano, alkyl, alkenyl,
alkynyl, nitro, amido, carboxyl, anhydride, alkylsulfonyl, ketone,
aldehyde, and ester.
[0327] In certain embodiments, X can be selected from
--N(R.sub.8)--, --O--, --S--, a direct bond, and a heterocycle, Y
can be selected from --C(.dbd.O)--, --C(.dbd.S)--, and
--S(O.sub.2)--, and Z can be selected from --N(R.sub.8)--, --O--,
--S--, a direct bond, and a heterocycle. In certain such
embodiments, at least one of Z and X is present.
[0328] In certain related embodiments, X-Y-Z taken together
represents a urea (NC(O)N) or an amide (NC(O) or C(O)N).
[0329] In certain embodiments, W is a substituted or unsubstituted
benzene ring.
[0330] In certain embodiments, X represents a diazacarbocycle, such
as a piperazine, e.g., substituted or unsubstituted.
[0331] In certain embodiments, X can be selected from
--N(R.sub.8)--, --O--, --S--, and a direct bond, Y can be selected
from --C(.dbd.O)--, --C(.dbd.S)--, and --S(O.sub.2)--, and Z can be
selected from --N(R.sub.8)--, --O--, --S--, and a direct bond, such
that at least one of X and Z is present.
[0332] In certain embodiments R.sub.8 represents H, lower alkyl,
aralkyl, heteroaralkyl, aryl, or heteroaryl, e.g., H or lower
alkyl.
[0333] In certain embodiments, X represents --NH--.
[0334] In certain embodiments, -L-X-- represents -(unbranched lower
alkyl)-NH--, e.g., --CH.sub.2--NH--, --CH.sub.2CH.sub.2--NH--,
etc.
[0335] In certain embodiments, the subject antagonists can be
chosen on the basis of their selectively for the hedgehog pathway.
This selectivity can be for the hedgehog pathway versus other
pathways, or for selectivity between particular hedgehog pathways,
e.g., e.g., ptc-1, ptc-2, etc.
[0336] In certain preferred embodiments, the subject inhibitors
inhibit hedgehog-mediated signal transduction with an ED.sub.50 of
1 mM or less, more preferably of 1 .mu.M or less, and even more
preferably of 1 nM or less.
[0337] In particular embodiments, the small molecule is chosen for
use because it is more selective for one patched isoform over the
next, e.g., 10 fold, and more preferably at least 100 or even 1000
fold more selective for one patched pathway (ptc-1, ptc-2) over
another.
[0338] In certain embodiments, a compound which is an antagonist of
the hedgehog pathway is chosen to selectively antagonize hedgehog
activity over protein kinases other than PKA, such as PKC, e.g.,
the compound modulates the activity of the hedgehog pathway at
least an order of magnitude more strongly than it modulates the
activity of another protein kinase, preferably at least two orders
of magnitude more strongly, even more preferably at least three
orders of magnitude more strongly. Thus, for example, a preferred
inhibitor of the hedgehog pathway may inhibit hedgehog activity
with a K.sub.i at least an order of magnitude lower than its
K.sub.i for inhibition of PKC, preferably at least two orders of
magnitude lower, even more preferably at least three orders of
magnitude lower. In certain embodiments, the K.sub.i for PKA
inhibition is less than 10 nM, preferably less than 1 nM, even more
preferably less than 0.1 nM.
[0339] In certain embodiments, compounds useful in the present
invention may be represented by general formula (IV): 46
[0340] wherein, as valence and stability permit,
[0341] R.sub.1 and R.sub.2, independently for each occurrence,
represent H, substituted or unsubstituted lower alkyl, alkenyl, or
alkynyl, --(CH.sub.2).sub.ncycloalkyl (e.g., substituted or
unsubstituted), --(CH.sub.2).sub.naryl (e.g., substituted or
unsubstituted), or --(CH.sub.2).sub.nheterocyclyl (e.g.,
substituted or unsubstituted);
[0342] L, independently for each occurrence, is absent or
represents --(CH.sub.2).sub.n-alkyl, -alkenyl-, -alkynyl-,
--(CH.sub.2).sub.nalkenyl- -, --(CH.sub.2).sub.nalkynyl-,
--(CH.sub.2).sub.nO(CH.sub.2).sub.p--,
--(CH.sub.2).sub.nNR.sub.2(CH.sub.2).sub.p--,
--(CH.sub.2).sub.nS(CH.sub.- 2).sub.p--,
--(CH.sub.2).sub.nalkenyl(CH.sub.2).sub.p--,
--(CH.sub.2).sub.nalkynyl(CH.sub.2).sub.p', --O(CH.sub.2).sub.n--,
--NR.sub.2(CH.sub.2).sub.n--, or --S(CH.sub.2).sub.n--;
[0343] X and Z, independently, can be selected from --CH--,
--N(R.sub.8)--, --O--, --S--, or --Se--;
[0344] Y can be selected from --C(.dbd.O)--, --C(.dbd.S)--,
--S(O.sub.2)--, --S(O)--, --C(.dbd.NCN)--, or
--P(.dbd.O)(OR.sub.2)--;
[0345] R.sub.8, independently for each occurrence, represents H,
substituted or unsubstituted lower alkyl,
--(CH.sub.2).sub.ncycloalkyl (e.g., substituted or unsubstituted),
--(CH.sub.2).sub.naryl (e.g., substituted or unsubstituted),
--(CH.sub.2).sub.nheterocyclyl (e.g., substituted or
unsubstituted), or two R.sub.8 taken together may form a 4- to
8-membered ring, e.g., with X.sub.1 and Z.sub.1 or X.sub.2 and
Z.sub.1, which ring may include one or more carbonyls;
[0346] R.sub.3 and R.sub.4, independently represent from 1-4
substituents on the ring to which they are attached, selected from,
independently for each occurrence, hydrogen, halogens, alkyls,
alkenyls, alkynyls, aryls, hydroxyl, .dbd.O, .dbd.S, alkoxyl,
silyloxy, amino, nitro, thiol, amines, imines, amides, phosphoryls,
phosphonates, phosphines, carbonyls, carboxyls, carboxamides,
anhydrides, silyls, ethers, thioethers, alkylsulfonyls,
arylsulfonyls, selenoethers, ketones, aldehydes, esters, or
--(CH.sub.2).sub.m--R.sub.8;
[0347] p represents, independently for each occurrence, an integer
from 0 to 10, preferably from 0 to 3; and
[0348] n, individually for each occurrence, represents an integer
from 0 to 10, preferably from 0 to 5.
[0349] In certain embodiments, R.sub.1 and R.sub.2 are
independently selected from substituted or unsubstituted aryl,
heterocyclyl, branched or unbranched alkyl, or cycloalkyl. In
embodiments wherein R.sub.1 or R.sub.2 is aryl or heterocyclyl,
substituents are preferably selected from H, alkyl, acyl, carboxy,
ester, amide, cyano, ether, thioether, amino, halogen, nitro, and
trihalomethyl.
[0350] In certain embodiments, R.sub.3 is absent or represents one
or two substituents selected from alkyl, acyl, carboxy, ester,
amide, cyano, ether, thioether, amino, acyl, halogen, nitro, and
trihalomethyl.
[0351] In certain embodiments, R.sub.4 is absent or represents one
or two substituents selected from ether, amino, thioether, alkyl,
aryl, (.dbd.O), or carbonyl (e.g., carboxy, ester, ketone,
aldehyde, etc.).
[0352] In certain embodiments, L is absent for each occurrence, or
represents --CH.sub.2-- or --CH.sub.2CH.sub.2--.
[0353] In certain embodiments, X represents NR.sub.8. R.sub.8
preferably represents H.
[0354] In certain embodiments, Z represents NR.sub.8. R.sub.8
preferably represents H.
[0355] In certain embodiments, Y represents --C(.dbd.O)--,
--C(.dbd.S)--, or --S(O.sub.2)--.
[0356] Exemplary Compounds Part 4
[0357] As described in further detail below, it is contemplated
that the subject methods can be carried out using a variety of
different small molecules which can be readily identified, for
example, by such drug screening assays as described herein. For
example, compounds useful in the subject methods include compounds
may be represented by general formula (XVIII): 47
[0358] wherein, as valence and stability permit,
[0359] R.sub.1, R.sub.2, R.sub.3, and R.sub.4, independently for
each occurrence, represent H, lower alkyl, --(CH.sub.2).sub.naryl
(e.g., substituted or unsubstituted), or
--(CH.sub.2).sub.nheteroaryl (e.g., substituted or
unsubstituted);
[0360] L, independently for each occurrence, is absent or
represents --(CH.sub.2).sub.n--, -alkenyl-, -alkynyl-,
--(CH.sub.2).sub.nalkenyl-, --(CH.sub.2).sub.nalkynyl-,
--(CH.sub.2).sub.nO(CH.sub.2).sub.p--,
--(CH.sub.2).sub.nNR.sub.8(CH.sub.2).sub.p--,
--(CH.sub.2).sub.nS(CH.sub.- 2).sub.p--,
--(CH.sub.2).sub.nalkenyl(CH.sub.2).sub.p--,
--(CH.sub.2).sub.nalkynyl(CH.sub.2).sub.p--, --O(CH.sub.2).sub.n--,
--NR.sub.8(CH.sub.2).sub.n--, or --S(CH.sub.2).sub.n--;
[0361] X and D, independently, can be selected from --N(R.sub.8)--,
--O--, --S--, --(R.sub.8)N--N(R.sub.8)--, --ON(R.sub.8)--, or a
direct bond;
[0362] Y and Z, independently, can be selected from O or S;
[0363] E represents O, S, or NR.sub.5, wherein R.sub.5 represents
LR.sub.8 or --(C.dbd.O)LR.sub.8.
[0364] R.sub.8, independently for each occurrence, represents H,
lower alkyl, --(CH.sub.2).sub.naryl (e.g., substituted or
unsubstituted), --(CH.sub.2).sub.nheteroaryl (e.g., substituted or
unsubstituted), or two R.sub.8 taken together may form a 4- to
8-membered ring;
[0365] p represents, independently for each occurrence, an integer
from 0 to 10, preferably from 0 to 3;
[0366] n, individually for each occurrence, represents an integer
from 0 to 10, preferably from 0 to 5; and
[0367] q and r represent, independently for each occurrence, an
integer from 0-2.
[0368] In certain embodiments, D does not represent N-lower alkyl.
In certain embodiments, D represents an aralkyl- or
heteroaralkyl-substitute- d amine.
[0369] In certain embodiments, R.sub.1 represents a lower alkyl
group, such as a branched alkyl, a cycloalkyl, or a
cycloalkylalkyl, for example, cyclopropyl, cyclopropylmethyl,
neopentyl, cyclobutyl, isobutyl, isopropyl, sec-butyl,
cyclobutylmethyl, etc.
[0370] In certain embodiments, Y and Z are O.
[0371] In certain embodiments, the sum of q and r is less than 4,
e.g., is 2 or 3.
[0372] In certain embodiments, XLR.sub.4, taken together, include a
cyclic amine, such as a piperazine, a morpholine, a piperidine, a
pyrrolidine, etc.
[0373] In certain embodiments, at least one of R.sub.1, R.sub.2,
and R.sub.3 includes an aryl or heteroaryl group. In certain
related embodiments, at least two of R.sub.1, R.sub.2, and R.sub.3
include an aryl or heteroaryl group. In certain embodiments,
R.sub.1 is lower alkyl.
[0374] In certain embodiments, L attached to R.sub.1 represents O,
S, or NR.sub.8, such as NH.
[0375] In certain embodiments, E is NR.sub.8. In certain
embodiments, E represents an aralkyl- or heteroaralkyl-substituted
amine, e.g., including polycyclic R.sub.8.
[0376] In certain embodiments, X is not NH. In certain embodiments,
X is included in a ring, or, taken together with --C(.dbd.Y)--,
represents a tertiary amide.
[0377] In certain embodiments, compounds useful in the present
invention may be represented by general formula (XIX): 48
[0378] wherein, as valence and stability permit,
[0379] R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.8, L, X, Y, Z, n,
p, q, and r are as defined above;
[0380] M is absent or represents L, --SO.sub.2L-, or --(C.dbd.O)L-;
and
[0381] s represents, independently for each occurrence, an integer
from 0-2.
[0382] In certain embodiments, Y and Z are O.
[0383] In certain embodiments, R.sub.1 represents a lower alkyl
group, such as a branched alkyl, a cycloalkyl, or a
cycloalkylalkyl, for example, cyclopropyl, cyclopropylmethyl,
neopentyl, cyclobutyl, isobutyl, isopropyl, sec-butyl,
cyclobutylmethyl, etc.
[0384] In certain embodiments, the sum of q, r, and s is less than
5, e.g., is 2, 3, or 4.
[0385] In certain embodiments, XLR.sub.4, taken together, include a
cyclic amine, such as a piperazine, a morpholine, a piperidine, a
pyrrolidine, etc.
[0386] In certain embodiments, L attached to R.sub.1 represents O,
S, or NR.sub.8, such as NH.
[0387] In certain embodiments, at least one of R.sub.1, R.sub.2,
and R.sub.3 includes an aryl or heteroaryl group. In certain
related embodiments, at least two of R.sub.1, R.sub.2, and R.sub.3
include an aryl or heteroaryl group.
[0388] In certain embodiments, M is absent.
[0389] In certain embodiments, X is not NH. In certain embodiments,
X is included in a ring, or, taken together with --C(.dbd.Y)--,
represents a tertiary amide.
[0390] In certain embodiments, compounds useful in the present
invention may be represented by general formula (XX): 49
[0391] wherein, as valence and stability permit,
[0392] R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.8, L, M, X, Y, Z,
n, p, q, and r are as defined above.
[0393] In certain embodiments, Y and Z are O.
[0394] In certain embodiments, R.sub.1 represents a lower alkyl
group, preferably a branched alkyl, a cycloalkyl, or a
cycloalkylalkyl, for example, cyclopropyl, cyclopropylmethyl,
neopentyl, cyclobutyl, isobutyl, isopropyl, sec-butyl,
cyclobutylmethyl, etc.
[0395] In certain embodiments, the sum of q and r is less than 4,
e.g., is 2 or 3.
[0396] In certain embodiments, XLR.sub.4, taken together, include a
cyclic amine, such as a piperazine, a morpholine, a piperidine, a
pyrrolidine, etc.
[0397] In certain embodiments, at least one of R.sub.1, R.sub.2,
and R.sub.3 includes an aryl or heteroaryl group. In certain
related embodiments, at least two of R.sub.1, R.sub.2, and R.sub.3
include an aryl or heteroaryl group. In certain embodiments,
R.sub.1 is lower alkyl.
[0398] In certain embodiments, L attached to R.sub.1 represents O,
S, or NR.sub.8, such as NH.
[0399] In certain embodiments, M is absent.
[0400] In certain embodiments, X is not NH. In certain embodiments,
X is included in a ring, or, taken together with --C(.dbd.Y)--,
represents a tertiary amide.
[0401] In certain embodiments, compounds useful in the present
invention may be represented by general formula (XXI): 50
[0402] wherein, as valence and stability permit,
[0403] R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.8, L, M, X, n, and
p are as defined above.
[0404] In certain embodiments, XLR.sub.4, taken together, include a
cyclic amine, such as a piperazine, a morpholine, a piperidine, a
pyrrolidine, etc.
[0405] In certain embodiments, R.sub.1 represents a lower alkyl
group, preferably a branched alkyl, a cycloalkyl, or a
cycloalkylalkyl, for example, cyclopropyl, cyclopropylmethyl,
neopentyl, cyclobutyl, isobutyl, isopropyl, sec-butyl,
cyclobutylmethyl, etc.
[0406] In certain embodiments, at least one of R.sub.1, R.sub.2,
and R.sub.3 includes an aryl or heteroaryl group. In certain
related embodiments, at least two of R.sub.1, R.sub.2, and R.sub.3
include an aryl or heteroaryl group. In certain embodiments,
R.sub.1 is lower alkyl.
[0407] In certain embodiments, L attached to R.sub.1 represents O,
S, or NR.sub.8, such as NH.
[0408] In certain embodiments, M is absent.
[0409] In certain embodiments, X is not NH. In certain embodiments,
X is included in a ring, or, taken together with --C(.dbd.Y)--,
represents a tertiary amide.
[0410] In certain embodiments L represents a direct bond for all
occurrences.
[0411] In certain embodiments, compounds useful in the present
invention may be represented by general formula (XXII): 51
[0412] wherein, as valence and stability permit,
[0413] Y, n, p, q, and r are as defined above;
[0414] Z' represents --C(.dbd.O)--, --C(.dbd.S)--, --C(.dbd.NH)--,
SO.sub.2, or SO, preferably --C(.dbd.O)--, --C(.dbd.S)--;
[0415] V is absent or represents O, S, or NR.sub.8;
[0416] G is absent or represents --C(.dbd.O)-- or --SO.sub.2--;
[0417] J, independently for each occurrence, represents H or
substituted or unsubstituted lower alkyl or alkylene, such as
methyl, ethyl, methylene, ethylene, etc., attached to NC(.dbd.Y),
such that both occurrences of N adjacent to J are linked through at
least one occurrence of J, and
[0418] R.sub.9, independently for each occurrence, is absent or
represents H or lower alkyl, or two occurrences of J or one
occurrence of J taken together with one occurrence of R.sub.9,
forms a ring of from 5 to 7 members, which ring includes one or
both occurrences of N;
[0419] R.sub.5 represents substituted or unsubstituted alkyl (e.g.,
branched or unbranched), alkenyl (e.g., branched or unbranched),
alkynyl (e.g., branched or unbranched), cycloalkyl, or
cycloalkylalkyl;
[0420] R.sub.6 represents substituted or unsubstituted aryl,
aralkyl, heteroaryl, heteroaralkyl, heterocyclyl,
heterocyclylalkyl, cycloalkyl, or cycloalkylalkyl, including
polycyclic groups; and
[0421] R.sub.7 represents substituted or unsubstituted aryl,
aralkyl, heteroaryl, or heteroaralkyl.
[0422] In certain embodiments, Y is O. In certain embodiments, Z'
represents SO.sub.2, --C(.dbd.O)--, or --C(.dbd.S)--.
[0423] In certain embodiments, the sum of q and r is less than
4.
[0424] In certain embodiments, NJ.sub.2N, taken together, represent
a cyclic diamine, such as a piperazine, etc., which may be
substituted or unsubstituted, e.g., with one or more substitutents
such as oxo, lower alkyl, lower alkyl ether, etc. In certain other
embodiments, NJ.sub.2 or NJR.sub.9 taken together represent a
substituted or unsubstituted heterocyclic ring to which the other
occurrence of N is attached. In certain embodiments, one or both
occurrences of J are substituted with one or more of lower alkyl,
lower alkyl ether, lower alkyl thioether, amido, oxo, etc. In
certain embodiments, a heterocyclic ring that comprises an
occurrence of J has from 5 to 8 members.
[0425] In certain embodiments, R.sub.5 represents a branched alkyl,
cycloalkyl, or cycloalkylalkyl.
[0426] In certain embodiments, R.sub.6 includes at least one
heterocyclic ring, such as a thiophene, furan, oxazole,
benzodioxane, benzodioxole, pyrrole, indole, etc.
[0427] In certain embodiments, R.sub.7 represents a phenyl alkyl,
such as a benzyl group, optionally substituted with halogen,
hydroxyl, lower alkyl, nitro, cyano, lower alkyl ether (e.g.,
optionally substituted, such as CHF.sub.2CF.sub.2O), or lower alkyl
thioether (e.g., optionally substituted, such as CF.sub.3S).
[0428] In certain embodiments, R.sub.8, when it occurs in V,
represents H or lower alkyl, preferably H.
[0429] In certain embodiments, compounds useful in the present
invention may be represented by general formula (XXIII): 52
[0430] wherein, as valence and stability permit,
[0431] R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9, R.sub.10, G, J,
V, Y, Z', n, and p are as defined above.
[0432] In certain embodiments, Y is O. In certain embodiments, Z'
represents SO.sub.2, --C(.dbd.O)--, or --C(.dbd.S)--.
[0433] In certain embodiments, NJ.sub.2N, taken together, represent
a heterocyclic ring, such as a piperazine, etc., which may be
substituted or unsubstituted, e.g., with one or more substitutents
such as oxo, lower alkyl, lower alkyl ether, etc. In certain other
embodiments, NJ.sub.2 or NJR.sub.9 taken together represent a
substituted or unsubstituted heterocyclic ring to which the other
occurrence of N is attached. In certain embodiments, one or both
occurrences of J are substituted with one or more of lower alkyl,
lower alkyl ether, lower alkyl thioether, amido, oxo, etc. In
certain embodiments, a heterocyclic ring that comprises an
occurrence of J has from 5 to 8 members.
[0434] In certain embodiments, R.sub.5 represents a branched alkyl,
cycloalkyl, or cycloalkylalkyl.
[0435] In certain embodiments, R.sub.6 includes at least one
heterocyclic ring, such as a thiophene, furan, oxazole,
benzodioxane, benzodioxole, pyrrole, indole, etc.
[0436] In certain embodiments, R.sub.7 represents a phenyl alkyl,
such as a benzyl group, optionally substituted with halogen,
hydroxyl, lower alkyl, nitro, cyano, lower alkyl ether (e.g.,
optionally substituted, such as CHF.sub.2CF.sub.2O), or lower alkyl
thioether (e.g., optionally substituted, such as CF.sub.3S).
[0437] In certain embodiments, R.sub.8, when it occurs in V,
represents H or lower alkyl, preferably H.
[0438] Exemplary Compounds Part 5
[0439] As described in further detail below, it is contemplated
that the subject methods can be carried out using a variety of
different small molecules which can be readily identified, for
example, by such drug screening assays as described herein. For
example, compounds useful in the subject methods include compounds
may be represented by general formula (XXIV): 53
[0440] wherein, as valence and stability permit,
[0441] X and Z, independently, represent --N(R.sub.7)--, --O--,
--S--, --(R.sub.7)N--N(R.sub.7)--, --ON(R.sub.7)--, or a direct
bond, preferably --N(R.sub.7)--, --O--, --S--, or a direct
bond;
[0442] Y represents --C(.dbd.O)--, --C(.dbd.S)--,
--C(.dbd.NR.sub.7)--, SO.sub.2, or SO, preferably --C(.dbd.O)--,
SO.sub.2, or --C(.dbd.S)--;
[0443] A represents O, S, or NR.sub.7, preferably O or NH, and most
preferably NH;
[0444] G represents a cycloalkyl, heterocyclyl, aryl, or heteroaryl
ring fused to the ring to which it is attached, preferably an aryl
or heteroaryl ring.
[0445] Ar represents a substituted or unsubstituted aryl or
heteroaryl ring, such as a substituted or unsubstituted phenyl
ring;
[0446] R.sub.1 represents H or substituted or unsubstituted alkyl,
alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl,
including polycyclic groups;
[0447] R.sub.2 represents from 0-4 substituents on the ring to
which it is attached, such as halogen, lower alkyl, lower alkenyl,
aryl, heteroaryl, carbonyl group (e.g., ester, carboxyl, or
formyl), thiocarbonyl (e.g., thioester, thiocarboxylate, or
thioformate), ketone, aldehyde, amino, acylamino, amido, amidino,
cyano, nitro, azido, sulfonyl, sulfoxido, sulfate, sulfonate,
sulfamoyl, sulfonamido, phosphoryl, phosphonate, phosphinate,
J-R.sub.8, J-OH, J-lower alkyl, J-lower alkenyl, J-R.sub.8, J-SH,
J-NH.sub.2, protected forms of the above, or any two R.sub.2, when
occurring more than once in a cyclic or polycyclic structure, can
be taken together form a 4- to 8-membered cycloalkyl, aryl, or
heteroaryl;
[0448] R.sub.7, independently for each occurrence, represents H,
lower alkyl (e.g., substituted or unsubstituted), J-cycloalkyl
(e.g., substituted or unsubstituted), J-heterocyclyl (e.g.,
substituted or unsubstituted), J-aryl (e.g., substituted or
unsubstituted), J-heteroaryl (e.g., substituted or
unsubstituted);
[0449] R.sub.8, independently for each occurrence, represents H,
lower alkyl (e.g., substituted or unsubstituted), cycloalkyl (e.g.,
substituted or unsubstituted), heterocyclyl (e.g., substituted or
unsubstituted), aryl (e.g., substituted or unsubstituted), or
heteroaryl (e.g., substituted or unsubstituted); and
[0450] J represents, independently for each occurrence, a chain
having from 0-8 (preferably from 0-4) units selected from CK.sub.2,
NK, O, and S, wherein K represents, independently for each
occurrence, H or lower alkyl.
[0451] In certain embodiments, at least one of Z and X is not a
direct bond. In certain embodiments, X-Y-Z includes an amide, urea,
or sulfonamide. In certain embodiments, X is selected from
--N(R.sub.8)--, --O--, --S--, and preferably represents NH.
[0452] In certain embodiments, R.sub.1 includes an aryl or
heteroaryl ring, optionally substituted with from 1-5 substituents,
such as nitro, halogen, cyano, lower alkyl, acylamino (e.g.,
R.sub.8--C(.dbd.O)NH--), alkoxy, alkylamino, a substituted or
unsubstituted cycloalkyl, heterocyclyl, aryl, or heteroaryl fused
to the aryl or heteroaryl ring.
[0453] In certain embodiments, X and the ring comprising A are
disposed on Ar in a meta (i.e., 1,3) relationship.
[0454] In certain embodiments, G represents a phenyl or piperidine
ring.
[0455] In certain embodiments, J is absent.
[0456] In certain embodiments, R.sub.2 represents from 1-4
substituents selected from halogen, cyano, nitro, alkoxy, amino,
acylamino (e.g., R.sub.8--C(.dbd.O)NH--), a substituted or
unsubstituted cycloalkyl, heterocyclyl, aryl, or heteroaryl fused
to G, and substituted or unsubstituted lower alkyl.
[0457] In certain embodiments, compounds useful in the present
invention may be represented by general formula (XXV): 54
[0458] wherein, as valence and stability permit,
[0459] X and Z, independently, represent --N(R.sub.7)--, --O--,
--S--, --(R.sub.7)N--N(R.sub.7)--, --ON(R.sub.7)--, or a direct
bond, preferably --N(R.sub.7)--, --O--, --S--, or a direct
bond;
[0460] Y represents --C(.dbd.O)--, --C(.dbd.S)--,
--C(.dbd.NR.sub.7)--, SO.sub.2, or SO, preferably --C(.dbd.O)--,
SO.sub.2, or --C(.dbd.S)--;
[0461] A represents O, S, or NR.sub.7, preferably O or NH, and most
preferably NH;
[0462] G represents a cycloalkyl, heterocyclyl, aryl, or heteroaryl
ring fused to the ring to which it is attached, preferably an aryl
or heteroaryl ring.
[0463] R.sub.1 represents H or substituted or unsubstituted alkyl,
alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, or cycloalkyl,
including polycyclic groups;
[0464] R.sub.2 represents from 0-4 substituents on the ring to
which it is attached, such as halogen, lower alkyl, lower alkenyl,
aryl, heteroaryl, carbonyl group (e.g., ester, carboxyl, or
formyl), thiocarbonyl (e.g., thioester, thiocarboxylate, or
thioformate), ketone, aldehyde, amino, acylamino, amido, amidino,
cyano, nitro, azido, sulfonyl, sulfoxido, sulfate, sulfonate,
sulfamoyl, sulfonamido, phosphoryl, phosphonate, phosphinate,
J-R.sub.8, J-OH, J-lower alkyl, J-lower alkenyl, J-R.sub.8, J-SH,
J-NH.sub.2, protected forms of the above, or any two R.sub.2, when
occurring more than once in a cyclic or polycyclic structure, can
be taken together form a 4- to 8-membered cycloalkyl, aryl, or
heteroaryl;
[0465] R.sub.3 represents from 0-4 substituents on the ring to
which it is attached, such as halogen, hydroxyl, alkoxy, amino,
alkylamino, cyano, nitro, substituted or unsubstituted lower alkyl,
and acyl, preferably halogen or substituted or unsubstituted lower
alkyl;
[0466] R.sub.7, independently for each occurrence, represents H,
lower alkyl (e.g., substituted or unsubstituted), J-cycloalkyl
(e.g., substituted or unsubstituted), J-heterocyclyl (e.g.,
substituted or unsubstituted), J-aryl (e.g., substituted or
unsubstituted), J-heteroaryl (e.g., substituted or
unsubstituted);
[0467] R.sub.8, independently for each occurrence, represents H,
lower alkyl (e.g., substituted or unsubstituted), cycloalkyl (e.g.,
substituted or unsubstituted), heterocyclyl (e.g., substituted or
unsubstituted), aryl (e.g., substituted or unsubstituted), or
heteroaryl (e.g., substituted or unsubstituted); and
[0468] J represents, independently for each occurrence, a chain
having from 0-8 (preferably from 0-4) units selected from CK.sub.2,
NK, O, and S, wherein K represents, independently for each
occurrence, H or lower alkyl.
[0469] In certain embodiments, at least one of Z and X is not a
direct bond. In certain embodiments, X-Y-Z includes an amide, urea,
or sulfonamide. In certain embodiments, X is selected from
--N(R.sub.8)--, --O--, --S--, and preferably represents NH.
[0470] In certain embodiments, R.sub.1 includes an aryl or
heteroaryl ring, optionally substituted with from 1-5 substituents,
such as nitro, halogen, cyano, lower alkyl, acylamino (e.g.,
R.sub.8--C(.dbd.O)NH--), alkoxy, alkylamino, a substituted or
unsubstituted cycloalkyl, heterocyclyl, aryl, or heteroaryl fused
to the aryl or heteroaryl ring.
[0471] In certain embodiments, G represents a phenyl or piperidine
ring.
[0472] In certain embodiments, J is absent.
[0473] In certain embodiments, R.sub.2 represents from 1-4
substituents selected from halogen, cyano, nitro, alkoxy, amino,
acylamino (e.g., R.sub.8--C(.dbd.O)NH--), a substituted or
unsubstituted cycloalkyl, heterocyclyl, aryl, or heteroaryl fused
to G, and substituted or unsubstituted lower alkyl.
[0474] In certain embodiments, R.sub.3 includes a substituent, such
as a substituted or unsubstituted alkyl or a halogen, at a position
para either to X or to the ring including A.
[0475] In certain embodiments, the subject antagonists can be
chosen on the basis of their selectivity for the hedgehog pathway.
This selectivity can be for the hedgehog pathway versus other
pathways, or for selectivity between particular hedgehog pathways,
e.g., ptc-1, ptc-2, etc.
[0476] In certain preferred embodiments, the subject inhibitors
inhibit ptc loss-of-function, hedgehog gain-of-function, or
smoothened gain-of-function mediated signal transduction with an
ED.sub.50 of 1 mM or less, more preferably of 1 .mu.M or less, and
even more preferably of 1 nM or less. Similarly, in certain
preferred embodiments, the subject inhibitors inhibit activity of
the hedgehog pathway with a K.sub.i less than 10 nM, preferably
less than 1 nM, even more preferably less than 0.1 nM.
[0477] In particular embodiments, the small molecule is chosen for
use because it is more selective for one patched isoform over the
next, e.g., 10-fold, and more preferably at least 100- or even
1000-fold more selective for one patched pathway (ptc- 1, ptc-2)
over another.
[0478] In certain embodiments, a compound which is an antagonist of
the hedgehog pathway is chosen to selectively antagonize hedgehog
activity over protein kinases other than PKA, such as PKC, e.g.,
the compound modulates the activity of the hedgehog pathway at
least an order of magnitude more strongly than it modulates the
activity of another protein kinase, preferably at least two orders
of magnitude more strongly, even more preferably at least three
orders of magnitude more strongly. Thus, for example, a preferred
inhibitor of the hedgehog pathway may inhibit hedgehog activity
with a K.sub.i at least an order of magnitude lower than its
K.sub.i for inhibition of PKC, preferably at least two orders of
magnitude lower, even more preferably at least three orders of
magnitude lower. In certain embodiments, the K.sub.i for PKA
inhibition is less than 10 nM, preferably less than 1 nM, even more
preferably less than 0.1 nM.
[0479] In certain preferred embodiments, hedgehog antagonists
include AY.sub.9944, triparanol, jervine, cyclopamine and
tomatidine (see FIG. 1), compound A (see FIG. 2) and compound B
(see FIG. 3).
[0480] Antagonist Hedgehog Mutants
[0481] It is anticipated that certain mutant forms of a hedgehog
protein may act as hedgehog antagonists. While not wishing to be
bound to any particular theory, it is well known that mutant forms
of protein signaling factors are capable of binding to the
appropriate receptor and yet not capable of activating the
receptor. Such mutant proteins act as antagonists by displacing the
wild-type proteins and blocking the normal receptor activation.
Mutant hedgehog proteins may behave similarly. Alternatively,
altered hedgehog proteins may bind directly to and inhibit the
wild-type forms of hedgehog and so act as antagonists. There are
many well known methods for obtaining mutants with a desired
activity.
[0482] Antagonist forms of hedgehog may be identified by using a
hedgehog sensitive screening system. For example, a cell line
transfected with a gli-1-lacZ reporter gene construct could be
monitored for beta-galactosidase activity. Gli-1 is a reporter for
activation of the hedgehog signaling pathway and hedgehog mutants
that inhibit gli-1-driven reporter gene expression would be
hedgehog antagonists. Any number of reporter genes may be used,
including luciferase, green fluorescent protein (and variants
including yellow, red, blue and cyan), GUS, and other fluorescent
or chromogenic proteins.
[0483] Methods for generating large pools of mutant proteins are
well known in the art. In one embodiment, the invention
contemplates using hedgehog polypeptides generated by combinatorial
mutagenesis. Such methods, as are known in the art, are convenient
for generating both point and truncation mutants, and can be
especially useful for identifying potential variant sequences
(e.g., homologs) that are functional in binding to a receptor for
hedgehog proteins. The purpose of screening such combinatorial
libraries is to generate, for example, novel hedgehog homologs that
can act as either agonists or antagonists. To illustrate, hedgehog
homologs can be engineered by the present method to provide more
efficient binding to a cognate receptor, such as patched, yet still
retain at least a portion of an activity associated with hedgehog.
Thus, combinatorially derived homologs can be generated to have an
increased potency relative to a naturally occurring form of the
protein. Likewise, hedgehog homologs can be generated by the
present combinatorial approach to act as antagonists, in that they
are able to mimic, for example, binding to other extracellular
matrix components (such as receptors), yet not induce any
biological response, thereby inhibiting the action of authentic
hedgehog or hedgehog agonists. Moreover, manipulation of certain
domains of hedgehog by the present method can provide domains more
suitable for use in fusion proteins, such as one that incorporates
portions of other proteins which are derived from the extracellular
matrix and/or which bind extracellular matrix components.
[0484] To further illustrate the state of the art of combinatorial
mutagenesis, it is noted that the review article of Gallop et al.
(1994) J Med Chem 37:1233 describes the general state of the art of
combinatorial libraries as of the earlier 1990's. In particular,
Gallop et al state at page 1239 "[s]creening the analog libraries
aids in determining the minimum size of the active sequence and in
identifying those residues critical for binding and intolerant of
substitution". In addition, the Ladner et al. PCT publication
WO90/02809, the Goeddel et al. U.S. Pat. No. 5,223,408, and the
Markland et al. PCT publication WO92/15679 illustrate specific
techniques which one skilled in the art could utilize to generate
libraries of hedgehog variants which can be rapidly screened to
identify variants/fragments which retained a particular activity of
the hedgehog polypeptides. These techniques are exemplary of the
art and demonstrate that large libraries of related
variants/truncants can be generated and assayed to isolate
particular variants without undue experimentation. Gustin et al.
(1993) Virology 193:653, and Bass et al. (1990) Proteins:
Structure, Function and Genetics 8:309-314 also describe other
exemplary techniques from the art which can be adapted as means for
generating mutagenic variants of hedgehog polypeptides.
[0485] Indeed, it is plain from the combinatorial mutagenesis art
that large scale mutagenesis of hedgehog proteins, without any
preconceived ideas of which residues were critical to the
biological function, can generate wide arrays of variants having
equivalent biological activity. Indeed, it is the ability of
combinatorial techniques to screen billions of different variants
by high throughout analysis that removes any requirement of a
priori understanding or knowledge of critical residues.
[0486] To illustrate, the amino acid sequences for a population of
hedgehog homologs or other related proteins are aligned, preferably
to promote the highest homology possible. Such a population of
variants can include, for example, hedgehog homologs from one or
more species. Amino acids that appear at each position of the
aligned sequences are selected to create a degenerate set of
combinatorial sequences. In a preferred embodiment, the variegated
library of hedgehog variants is generated by combinatorial
mutagenesis at the nucleic acid level, and is encoded by a
variegated gene library. For instance, a mixture of synthetic
oligonucleotides can be enzymatically ligated into gene sequences
such that the degenerate set of potential hedgehog sequences are
expressible as individual polypeptides, or alternatively, as a set
of larger fusion proteins (e.g., for phage display) containing the
set of hedgehog sequences therein.
[0487] As illustrated in PCT publication WO 95/18856, to analyze
the sequences of a population of variants, the amino acid sequences
of interest can be aligned relative to sequence homology. The
presence or absence of amino acids from an aligned sequence of a
particular variant is relative to a chosen consensus length of a
reference sequence, which can be real or artificial.
[0488] In an illustrative embodiment, alignment of exons 1, 2 and a
portion of exon 3 encoded sequences (e.g., the N-terminal
approximately 221 residues of the mature protein) of each of the
Shh clones produces a degenerate set of Shh polypeptides
represented by the general formula:
1 (SEQ ID No: 21 C-G-P-G-R-X(1)-G-X(2)-R-R-H-P-K-K-L-T-P-L--
A-Y-K-Q-F-I-P-N-V-A-E-K-T-L-G-A-S-
G-R-Y-E-G-K-I-X(3)-R-N-S-E-R-F-K-E-L-T-P-N-Y-N-P-D-I-I-F-K-D-E-E-N-T-G-A--
D-R-L- M-T-Q-R-C-K-D-K-L-N-X(4)-L-A-I-S-V-M-N-X(5)-W-P-G-V-
-X(6)-L-R-V-T-E-G-W-D-E-D- G-H-H-X(7)-E-E-S-L-H-Y-E-G-R-A--
V-D-I-T-T-S-D-R-D-X(8)-S-K-Y-G-X-(9)-L-X(10)-R-L-
A-V-E-A-G-F-D-W-V-Y-Y-E-S-K-A-H-I-H-C-S-V-K-A-E-N-S-V-A-A-K-S-G-G-C-F-P-G-
-S- A-X(11)-V-X(12)-L-X(13)-X(14)-G-G-X(15)-K-X-(16)-V-K-D-
-L-X(17)-P-G-D-X(18)-V-L-A- A-D-X(19)-X(20)-G-X(21)-L-X(22-
)-X(23)-S-D-F-X(24)-X(25)-F-X(26)-D-R
[0489] wherein each of the degenerate positions "X" can be an amino
acid which occurs in that position in one of the human, mouse,
chicken or zebrafish Shh clones, or, to expand the library, each X
can also be selected from amongst amino acid residue which would be
conservative substitutions for the amino acids which appear
naturally in each of those positions. For instance, Xaa(l)
represents Gly, Ala, Val, Leu, Ile, Phe, Tyr or Trp; Xaa(2)
represents Arg, His or Lys; Xaa(3) represents Gly, Ala, Val, Leu,
Ile, Ser or Thr; Xaa(4) represents Gly, Ala, Val, Leu, Ile, Ser or
Thr; Xaa(5) represents Lys, Arg, His, Asn or Gln; Xaa(6) represents
Lys, Arg or His; Xaa(7) represents Ser, Thr, Tyr, Trp or Phe;
Xaa(8) represents Lys, Arg or His; Xaa(9) represents Met, Cys, Ser
or Thr; Xaa(10) represents Gly, Ala, Val, Leu, Ile, Ser or Thr;
Xaa(11) represents Leu, Val, Met, Thr or Ser; Xaa(12) represents
His, Phe, Tyr, Ser, Thr, Met or Cys; Xaa(13) represents Gln, Asn,
Glu, or Asp; Xaa(14) represents His, Phe, Tyr, Thr, Gln, Asn, Glu
or Asp; Xaa(15) represents Gln, Asn, Glu, Asp, Thr, Ser, Met or
Cys; Xaa(16) represents Ala, Gly, Cys, Leu, Val or Met; Xaa(17)
represents Arg, Lys, Met, Ile, Asn, Asp, Glu, Gln, Ser, Thr or Cys;
Xaa(18) represents Arg, Lys, Met or Ile; Xaa(19) represents Ala,
Gly, Cys, Asp, Glu, Gln, Asn, Ser, Thr or Met; Xaa(20) represents
Ala, Gly, Cys, Asp, Asn, Glu or Gln; Xaa(21) represents Arg, Lys,
Met, Ile, Asn, Asp, Glu or Gln; Xaa(22) represent Leu, Val, Met or
Ile; Xaa(23) represents Phe, Tyr, Thr, His or Trp; Xaa(24)
represents Ile, Val, Leu or Met; .Xaa(25) represents Met, Cys, Ile,
Leu, Val, Thr or Ser; Xaa(26) represents Leu, Val, Met, Thr or Ser.
In an even more expansive library, each X can be selected from any
amino acid.
[0490] In similar fashion, alignment of each of the human, mouse,
chicken and zebrafish hedgehog clones, can provide a degenerate
polypeptide sequence represented by the general formula:
2 (SEQ ID No:22) C-G-P-G-R-G-X(1)-X(2)-X(3)-R-R-X(4)-X(5)-X-
(6)-P-K-X(7)-L-X(8)-P-L-X(9)-Y-K-Q-F-
X(10)-P-X(11)-X(12)-X(13)-E-X(14)-T-L-G-A-S-G-X(15)-X(16)-E-G-X(17)-X(18)-
-X(19)-R- X(20)-S-E-R-F-X(21)-X(22)-L-T-P-N-Y-N-P-D-I-I-F--
K-D-E-E-N-X(23)-G-A-D-R-L-M-T- X(24)-R-C-K-X(25)-X(26)-X(2-
7)-N-X(28)-L-A-I-S-V-M-N-X(29)-W-P-G-V-X(30)-L-R-V-T-
E-G-X(31)-D-E-D-G-H-H-X(32)-X(33)-X(34)-S-L-H-Y-E-G-R-A-X(35)-D-I-T-T-S-D-
-R-D- X(36)-X(37)-K-Y-G-X(38)-L-X(39)-R-L-A-V-E-A-G-F-D-W--
V-Y-Y-E-S-X(40)-X(41)-H- X(42)-H-X(43)-S-V-K-X(44)-X(45)
[0491] wherein, as above, each of the degenerate positions "X" can
be an amino acid which occurs in a corresponding position in one of
the wild-type clones, and may also include amino acid residue which
would be conservative substitutions, or each X can be any amino
acid residue. In an exemplary embodiment, Xaa(1) represents Gly,
Ala, Val, Leu, Ile, Pro, Phe or Tyr; Xaa(2) represents Gly, Ala,
Val, Leu or Ile; Xaa(3) represents Gly, Ala, Val, Leu, Ile, Lys,
His or Arg; Xaa(4) represents Lys, Arg or His; Xaa(5) represents
Phe, Trp, Tyr or an amino acid gap; Xaa(6) represents Gly, Ala,
Val, Leu, Ile or an amino acid gap; Xaa(7) represents Asn, Gln,
His, Arg or Lys; Xaa(8) represents Gly, Ala, Val, Leu, Ile, Ser or
Thr; Xaa(9) represents Gly, Ala, Val, Leu, Ile, Ser or Thr; Xaa(10)
represents Gly, Ala, Val, Leu, Ile, Ser or Thr; Xaa(11) represents
Ser, Thr, Gln or Asn; Xaa(12) represents Met, Cys, Gly, Ala, Val,
Leu, Ile, Ser or Thr; Xaa(13) represents Gly, Ala, Val, Leu, Ile or
Pro; Xaa(14) represents Arg, His or Lys; Xaa(15) represents Gly,
Ala, Val, Leu, Ile, Pro, Arg, His or Lys; Xaa(16) represents Gly,
Ala, Val, Leu, Ile, Phe or Tyr; Xaa(17) represents Arg, His or Lys;
Xaa(18) represents Gly, Ala, Val, Leu, Ile, Ser or Thr; Xaa(19)
represents Thr or Ser; Xaa(20) represents Gly, Ala, Val, Leu, Ile,
Asn or Gln; Xaa(21) represents Arg, His or Lys; Xaa(22) represents
Asp or Glu; Xaa(23) represents Ser or Thr; Xaa(24) represents Glu,
Asp, Gln or Asn; Xaa(25) represents Glu or Asp; Xaa(26) represents
Arg, His or Lys; Xaa(27) represents Gly, Ala, Val, Leu or Ile;
Xaa(28) represents Gly, Ala, Val, Leu, Ile, Thr or Ser; Xaa(29)
represents Met, Cys, Gln, Asn, Arg, Lys or His; Xaa(30) represents
Arg, His or Lys; Xaa(31) represents Trp, Phe, Tyr, Arg, His or Lys;
Xaa(32) represents Gly, Ala, Val, Leu, Ile, Ser, Thr, Tyr or Phe;
Xaa(33) represents Gln, Asn, Asp or Glu; Xaa(34) represents Asp or
Glu; Xaa(35) represents Gly, Ala, Val, Leu, or Ile; Xaa(36)
represents Arg, His or Lys; Xaa(37) represents Asn, Gln, Thr or
Ser; Xaa(38) represents Gly, Ala, Val, Leu, Ile, Ser, Thr, Met or
Cys; Xaa(39) represents Gly, Ala, Val, Leu, Ile, Thr or Ser;
Xaa(40) represents Arg, His or Lys; Xaa(41) represents Asn, Gln,
Gly, Ala, Val, Leu or Ile; Xaa(42) represents Gly, Ala, Val, Leu or
Ile; Xaa(43) represents Gly, Ala, Val, Leu, Ile, Ser, Thr or Cys;
Xaa(44) represents Gly, Ala, Val, Leu, Ile, Thr or Ser; and Xaa(45)
represents Asp or Glu.
[0492] There are many ways by which the library of potential
hedgehog homologs can be generated from a degenerate
oligonucleotide sequence. Chemical synthesis of a degenerate gene
sequence can be carried out in an automatic DNA synthesizer, and
the synthetic genes then ligated into an appropriate expression
vector. The purpose of a degenerate set of genes is to provide, in
one mixture, all of the sequences encoding the desired set of
potential hedgehog sequences. The synthesis of degenerate
oligonucleotides is well known in the art (see for example, Narang,
S A (1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA,
Proc 3rd Cleveland Sympos. Macromolecules, ed. A G Walton,
Amsterdam: Elsevier pp273-289; Itakura et al. (1984) Annu. Rev.
Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al.
(1983) Nucleic Acid Res. 11:477. Such techniques have been employed
in the directed evolution of other proteins (see, for example,
Scott et al. (1990) Science 249:386-390; Roberts et al. (1992) PNAS
89:2429-2433; Devlin et al. (1990) Science 249: 404-406; Cwirla et
al. (1990) PNAS 87: 6378-6382; as well as U.S. Pat. Nos. 5,223,409,
5,198,346, and 5,096,815).
[0493] A wide range of techniques are known in the art for
screening gene products of combinatorial libraries made by point
mutations, and for screening cDNA libraries for gene products
having a certain property. Such techniques will be generally
adaptable for rapid screening of the gene libraries generated by
the combinatorial mutagenesis of hedgehog homologs. The most widely
used techniques for screening large gene libraries typically
comprises cloning the gene library into replicable expression
vectors, transforming appropriate cells with the resulting library
of vectors, and expressing the combinatorial genes under conditions
in which detection of a desired activity facilitates relatively
easy isolation of the vector encoding the gene whose product was
detected. Each of the illustrative assays described below are
amenable to high through-put analysis as necessary to screen large
numbers of degenerate hedgehog sequences created by combinatorial
mutagenesis techniques.
[0494] In yet another screening assay, the candidate hedgehog gene
products are displayed on the surface of a cell or viral particle,
and the ability of particular cells or viral particles to associate
with a hedgehog-binding moiety (such as the patched protein or
other hedgehog receptor) via this gene product is detected in a
"panning assay". Such panning steps can be carried out on cells
cultured from embryos. For instance, the gene library can be cloned
into the gene for a surface membrane protein of a bacterial cell,
and the resulting fusion protein detected by panning (Ladner et
al., WO 88/06630; Fuchs et al. (1991) Bio/Technology 9:1370-1371;
and Goward et al. (1992) TIBS 18:136-140). In a similar fashion,
fluorescently labeled molecules that bind hedgehog can be used to
score for potentially functional hedgehog homologs. Cells can be
visually inspected and separated under a fluorescence microscope,
or, where the morphology of the cell permits, separated by a
fluorescence-activated cell sorter.
[0495] In an alternate embodiment, the gene library is expressed as
a fusion protein on the surface of a viral particle. For instance,
in the filamentous phage system, foreign peptide sequences can be
expressed on the surface of infectious phage, thereby conferring
two significant benefits. First, since these phage can be applied
to affinity matrices at very high concentrations, large number of
phage can be screened at one time. Second, since each infectious
phage displays the combinatorial gene product on its surface, if a
particular phage is recovered from an affinity matrix in low yield,
the phage can be amplified by another round of infection. The group
of almost identical E. coli filamentous phages M13, fd, and fl are
most often used in phage display libraries, as either of the phage
gIII or gVIII coat proteins can be used to generate fusion proteins
without disrupting the ultimate packaging of the viral particle
(Ladner et al. PCT publication WO 90/02909; Garrard et al., PCT
publication WO 92/09690; Marks et al. (1992) J. Biol. Chem.
267:16007-16010; Griffths et al. (1993) EMBO J 12:725-734; Clackson
et al. (1991) Nature 352:624-628; and Barbas et al. (1992) PNAS
89:4457-4461).
[0496] In an illustrative embodiment, the recombinant phage
antibody system (RPAS, Pharamacia Catalog number 27-9400-01) can be
easily modified for use in expressing and screening hedgehog
combinatorial libraries. For instance, the pCANTAB 5 phagemid of
the RPAS kit contains the gene that encodes the phage gIII coat
protein. The hedgehog combinatorial gene library can be cloned into
the phagemid adjacent to the gIII signal sequence such that it will
be expressed as a gIII fusion protein. After ligation, the phagemid
is used to transform competent E. coli TG1 cells. Transformed cells
are subsequently infected with M13KO7 helper phage to rescue the
phagemid and its candidate hedgehog gene insert. The resulting
recombinant phage contain phagemid DNA encoding a specific
candidate hedgehog, and display one or more copies of the
corresponding fusion coat protein. The phage-displayed candidate
hedgehog proteins that are capable of binding a hedgehog receptor
are selected or enriched by panning. For instance, the phage
library can be applied to cells that express the patched protein
and unbound phage washed away from the cells. The bound phage is
then isolated, and if the recombinant phage express at least one
copy of the wild type gIII coat protein, they will retain their
ability to infect E. coli. Thus, successive rounds of reinfection
of E. coli, and panning will greatly enrich for hedgehog homologs,
which can then be screened for further biological activities in
order to differentiate agonists and antagonists.
[0497] Combinatorial mutagenesis has a potential to generate very
large libraries of mutant proteins, e.g., in the order of 10.sup.26
molecules. Combinatorial libraries of this size may be technically
challenging to screen even with high throughput screening assays
such as phage display. To overcome this problem, a new technique
has been developed recently, recursive ensemble mutagenesis (REM),
which allows one to avoid the very high proportion of
non-functional proteins in a random library and simply enhances the
frequency of functional proteins, thus decreasing the complexity
required to achieve a useful sampling of sequence space. REM is an
algorithm that enhances the frequency of functional mutants in a
library when an appropriate selection or screening method is
employed (Arkin and Yourvan, 1992, PNAS USA 89:7811-7815; Yourvan
et al., 1992, Parallel Problem Solving from Nature, 2., In Maenner
and Manderick, eds., Elsevir Publishing Co., Amsterdam, pp.
401-410; Delgrave et al., 1993, Protein Engineering
6(3):327-331).
[0498] Antibody Antagonists
[0499] It is anticipated that antibodies can act as hedgehog
antagonists. Antibodies can have extraordinary affinity and
specificity for particular epitopes. Antibodies that bind to any
protein in the hedgehog signaling pathway may have the capacity to
act as antagonists. Antibodies that bind to hedgehog, smoothened or
gli-1 may act by simply sterically hindering the proper
protein-protein interactions or occupying active sites. Antibodies
that bind to patched proteins may act as antagonists if they cause
hyperactivation of the patched protein, for example stimulating
patched association with smoothened. Proteins with extracellular
domains are readily bound by exogenously supplied antibodies.
[0500] Antibodies with hedgehog antagonist activity can be
identified in much the same way as other hedgehog antagonists. For
example, candidate antibodies can be administered to cells
expressing a hedgehog reporter gene, and antibodies that cause
decreased reporter gene expression are antagonists.
[0501] In one variation, antibodies of the invention can be single
chain antibodies (scFv), comprising variable antigen binding
domains linked by a polypeptide linker. Single chain antibodies are
expressed as a single polypeptide chain and can be expressed in
bacteria and as part of a phage display library. In this way, phage
that express the appropriate scFv will have hedgehog antagonist
activity. The nucleic acid encoding the single chain antibody can
then be recovered from the phage and used to produce large
quantities of the scFv. Construction and screening of scFv
libraries is extensively described in various publications (U.S.
Pat. Nos. 5,258,498; 5,482,858; 5,091,513; 4,946,778; 5,969,108;
5,871,907; 5,223,409; 5,225,539).
[0502] Antisense, Ribozyme and Triplex Techniques
[0503] Another aspect of the invention relates to the use of the
isolated nucleic acid in "antisense" therapy. As used herein,
"antisense" therapy refers to administration or in situ generation
of oligonucleotide molecules or their derivatives which
specifically hybridize (e.g., bind) under cellular conditions, with
the cellular mRNA and/or genomic DNA encoding one or more of the
subject hedgehog pathway proteins so as to inhibit expression of
that protein, e.g., by inhibiting transcription and/or translation.
The binding may be by conventional base pair complementarity, or,
for example, in the case of binding to DNA duplexes, through
specific interactions in the major groove of the double helix. In
general, "antisense" therapy refers to the range of techniques
generally employed in the art, and includes any therapy that relies
on specific binding to oligonucleotide sequences.
[0504] An antisense construct of the present invention can be
delivered, for example, as an expression plasmid which, when
transcribed in the cell, produces RNA which is complementary to at
least a unique portion of the cellular mRNA which encodes a
hedgehog signaling protein. Alternatively, the antisense construct
is an oligonucleotide probe that is generated ex vivo and which,
when introduced into the cell causes inhibition of expression by
hybridizing with the mRNA and/or genomic sequences of a hedgehog
signaling gene. Such oligonucleotide probes are preferably modified
oligonucleotides that are resistant to endogenous nucleases, e.g.,
exonucleases and/or endonucleases, and are therefore stable in
vivo. Exemplary nucleic acid molecules for use as antisense
oligonucleotides are phosphoramidate, phosphothioate and
methylphosphonate analogs of DNA (see also U.S. Pat. Nos.
5,176,996; 5,264,564; and 5,256,775). Additionally, general
approaches to constructing oligomers useful in antisense therapy
have been reviewed, for example, by Van der Krol et al. (1988)
BioTechniques 6:958-976; and Stein et al. (1988) Cancer Res
48:2659-2668. With respect to antisense DNA,
oligodeoxyribonucleotides derived from the translation initiation
site, e.g., between the -10 and +10 regions of the hedgehog
signaling gene nucleotide sequence of interest, are preferred.
[0505] Antisense approaches involve the design of oligonucleotides
(either DNA or RNA) that are complementary to mRNA encoding a
hedgehog signaling protein. The antisense oligonucleotides will
bind to the mRNA transcripts and prevent translation. Absolute
complementarity, although preferred, is not required. In the case
of double-stranded antisense nucleic acids, a single strand of the
duplex DNA may thus be tested, or triplex formation may be assayed.
The ability to hybridize will depend on both the degree of
complementarity and the length of the antisense nucleic acid.
Generally, the longer the hybridizing nucleic acid, the more base
mismatches with an RNA it may contain and still form a stable
duplex (or triplex, as the case may be). One skilled in the art can
ascertain a tolerable degree of mismatch by use of standard
procedures to determine the melting point of the hybridized
complex.
[0506] Oligonucleotides that are complementary to the 5' end of the
mRNA, e.g., the 5' untranslated sequence up to and including the
AUG initiation codon, should work most efficiently at inhibiting
translation. However, sequences complementary to the 3'
untranslated sequences of mRNAs have recently been shown to be
effective at inhibiting translation of mRNAs as well. (Wagner, R.
1994. Nature 372:333). Therefore, oligonucleotides complementary to
either the 5' or 3' untranslated, non-coding regions of a gene
could be used in an antisense approach to inhibit translation of
that mRNA. Oligonucleotides complementary to the 5' untranslated
region of the mRNA should include the complement of the AUG start
codon. Antisense oligonucleotides complementary to mRNA coding
regions are less efficient inhibitors of translation but could also
be used in accordance with the invention. Whether designed to
hybridize to the 5', 3' or coding region of mRNA, antisense nucleic
acids should be at least six nucleotides in length, and are
preferably less that about 100 and more preferably less than about
50, 25, 17 or 10 nucleotides in length.
[0507] Regardless of the choice of target sequence, it is preferred
that in vitro studies are first performed to quantitate the ability
of the antisense oligonucleotide to quantitate the ability of the
antisense oligonucleotide to inhibit gene expression. It is
preferred that these studies utilize controls that distinguish
between antisense gene inhibition and nonspecific biological
effects of oligonucleotides. It is also preferred that these
studies compare levels of the target RNA or protein with that of an
internal control RNA or protein. Additionally, it is envisioned
that results obtained using the antisense oligonucleotide are
compared with those obtained using a control oligonucleotide. It is
preferred that the control oligonucleotide is of approximately the
same length as the test oligonucleotide and that the nucleotide
sequence of the oligonucleotide differs from the antisense sequence
no more than is necessary to prevent specific hybridization to the
target sequence.
[0508] The oligonucleotides can be DNA or RNA or chimeric mixtures
or derivatives or modified versions thereof, single-stranded or
double-stranded. The oligonucleotide can be modified at the base
moiety, sugar moiety, or phosphate backbone, for example, to
improve stability of the molecule, hybridization, etc. The
oligonucleotide may include other appended groups such as peptides
(e.g., for targeting host cell receptors), or agents facilitating
transport across the cell membrane (see, e.g., Letsinger et al.,
1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre et al.,
1987, Proc. Natl. Acad. Sci. 84:648-652; PCT Publication No.
WO88/09810, published Dec. 15, 1988) or the blood- brain barrier
(see, e.g., PCT Publication No. WO89/10134, published Apr. 25,
1988), hybridization-triggered cleavage agents. (See, e.g., Krol et
al., 1988, BioTechniques 6:958- 976) or intercalating agents. (See,
e.g., Zon, 1988, Pharm. Res. 5:539-549). To this end, the
oligonucleotide may be conjugated to another molecule, e.g., a
peptide, hybridization triggered cross-linking agent, transport
agent, hybridization-triggered cleavage agent, etc.
[0509] The antisense oligonucleotide may comprise at least one
modified base moiety which is selected from the group including but
not limited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil,
5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,
5-(carboxyhydroxytriethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomet-
hyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine,
N6-isopentenyladenine, 1-methylguanine, 1-methylinosine,
2,2-dimethylguanine, 2-methyladenine, 2-methylguanine,
3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopenten- yladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methyl ester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine.
[0510] The antisense oligonucleotide may also comprise at least one
modified sugar moiety selected from the group including but not
limited to arabinose, 2-fluoroarabinose, xylulose, and hexose.
[0511] The antisense oligonucleotide can also contain a neutral
peptide-like backbone. Such molecules are termed peptide nucleic
acid (PNA)-oligomers and are described, e.g., in Perry-O'Keefe et
al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93:14670 and in Eglom et
al. (1993) Nature 365:566. One advantage of PNA oligomers is their
capability to bind to complementary DNA essentially independently
from the ionic strength of the medium due to the neutral backbone
of the DNA. In yet another embodiment, the antisense
oligonucleotide comprises at least one modified phosphate backbone
selected from the group consisting of a phosphorothioate, a
phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a
phosphordiamidate, a methylphosphonate, an alkyl phosphotriester,
and a formacetal or analog thereof.
[0512] In yet a further embodiment, the antisense oligonucleotide
is an -anomeric oligonucleotide. An -anomeric oligonucleotide forms
specific double-stranded hybrids with complementary RNA in which,
contrary to the usual -units, the strands run parallel to each
other (Gautier et al., 1987, Nucl. Acids Res. 15:6625-6641). The
oligonucleotide is a 2'-O-methylribonucleotide (Inoue et al., 1987,
Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNA analogue
(Inoue et al., 1987, FEBS Lett. 215:327-330).
[0513] Oligonucleotides of the invention may be synthesized by
standard methods known in the art, e.g., by use of an automated DNA
synthesizer (such as are commercially available from Biosearch,
Applied Biosystems, etc.). As examples, phosphorothioate
oligonucleotides may be synthesized by the method of Stein et al.
(1988, Nucl. Acids Res. 16:3209), methylphosphonate
oligonucleotides can be prepared by use of controlled pore glass
polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A.
85:7448-7451), etc.
[0514] While antisense nucleotides complementary to the coding
region of an mRNA sequence can be used, those complementary to the
transcribed untranslated region and to the region comprising the
initiating methionine are most preferred.
[0515] The antisense molecules can be delivered to cells that
express hedgehog signaling genes in vivo. A number of methods have
been developed for delivering antisense DNA or RNA to cells; e.g.,
antisense molecules can be injected directly into the tissue site,
or modified antisense molecules, designed to target the desired
cells (e.g., antisense linked to peptides or antibodies that
specifically bind receptors or antigens expressed on the target
cell surface) can be administered systematically.
[0516] However, it may be difficult to achieve intracellular
concentrations of the antisense sufficient to suppress translation
on endogenous mRNAs in certain instances. Therefore a preferred
approach utilizes a recombinant DNA construct in which the
antisense oligonucleotide is placed under the control of a strong
pol III or pol II promoter. The use of such a construct to
transfect target cells in the patient will result in the
transcription of sufficient amounts of single stranded RNAs that
will form complementary base pairs with the endogenous hedgehog
signaling transcripts and thereby prevent translation. For example,
a vector can be introduced in vivo such that it is taken up by a
cell and directs the transcription of an antisense RNA. Such a
vector can remain episomal or become chromosomally integrated, as
long as it can be transcribed to produce the desired antisense RNA.
Such vectors can be constructed by recombinant DNA technology
methods standard in the art. Vectors can be plasmid, viral, or
others known in the art, used for replication and expression in
mammalian cells. Expression of the sequence encoding the antisense
RNA can be by any promoter known in the art to act in mammalian,
preferably human cells. Such promoters can be inducible or
constitutive. Such promoters include but are not limited to: the
SV40 early promoter region (Bernoist and Chambon, 1981, Nature
290:304-310), the promoter contained in the 3' long terminal repeat
of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797), the
herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl.
Acad. Sci. U.S.A. 78:1441-1445), the regulatory sequences of the
metallothionein gene (Brinster et al, 1982, Nature 296:39-42), etc.
Any type of plasmid, cosmid, YAC or viral vector can be used to
prepare the recombinant DNA construct that can be introduced
directly into the tissue site. Alternatively, viral vectors can be
used which selectively infect the desired tissue, in which case
administration may be accomplished by another route (e.g.,
systematically).
[0517] Ribozyme molecules designed to catalytically cleave hedgehog
signaling mRNA transcripts can also be used to prevent translation
of mRNA (See, e.g., PCT International Publication WO90/11364,
published Oct. 4, 1990; Sarver et al., 1990, Science 247:1222-1225
and U.S. Pat. No. 5,093,246). While ribozymes that cleave mRNA at
site-specific recognition sequences can be used to destroy
particular mRNAs, the use of hammerhead ribozymes is preferred.
Hammerhead ribozymes cleave mRNAs at locations dictated by flanking
regions that form complementary base pairs with the target mRNA.
The sole requirement is that the target mRNA have the following
sequence of two bases: 5'-UG-3'. The construction and production of
hammerhead ribozymes is well known in the art and is described more
fully in Haseloff and Gerlach, 1988, Nature, 334:585-591.
[0518] The ribozymes of the present invention also include RNA
endoribonucleases (hereinafter "Cech-type ribozymes") such as the
one which occurs naturally in Tetrahyrnena thermophila (known as
the IVS, or L-19 IVS RNA) and which has been extensively described
by Thomas Cech and collaborators (Zaug, et al., 1984, Science,
224:574-578; Zaug and Cech, 1986, Science, 231:470-475; Zaug, et
al., 1986, Nature, 324:429-433; published International patent
application No. WO88/04300 by University Patents Inc.; Been and
Cech, 1986, Cell, 47:207-216). The Cech-type ribozymes have an
eight base pair active site that hybridizes to a target RNA
sequence whereafter cleavage of the target RNA takes place. The
invention encompasses those Cech-type ribozymes that target eight
base-pair active site sequences.
[0519] As in the antisense approach, the ribozymes can be composed
of modified oligonucleotides (e.g., for improved stability,
targeting, etc.) and should be delivered to cells that express
hedgehog signaling genes in vivo. A preferred method of delivery
involves using a DNA construct "encoding" the ribozyme under the
control of a strong constitutive pol III or pol II promoter, so
that transfected cells will produce sufficient quantities of the
ribozyme to destroy targeted messages and inhibit translation.
Because ribozymes unlike antisense molecules, are catalytic, a
lower intracellular concentration is required for efficiency.
[0520] Alternatively, endogenous hedgehog signaling gene expression
can be reduced by targeting deoxyribonucleotide sequences
complementary to the regulatory region of the gene (i.e., the
promoter and/or enhancers) to form triple helical structures that
prevent transcription of the gene in target cells in the body. (See
generally, Helene, C. 1991, Anticancer Drug Des., 6(6):569-84;
Helene, C., et al., 1992, Ann. N.Y. Acad. Sci., 660:27-36; and
Maher, L. J., 1992, Bioassays 14(12):807-15).
[0521] Nucleic acid molecules to be used in triple helix formation
for the inhibition of transcription are preferably single stranded
and composed of deoxyribonucleotides. The base composition of these
oligonucleotides should promote triple helix formation via
Hoogsteen base pairing rules, which generally require sizable
stretches of either purines or pyrimidines to be present on one
strand of a duplex. Nucleotide sequences may be pyrimidine-based,
which will result in TAT and CGC triplets across the three
associated strands of the resulting triple helix. The
pyrimidine-rich molecules provide base complementarity to a
purine-rich region of a single strand of the duplex in a parallel
orientation to that strand. In addition, nucleic acid molecules may
be chosen that are purine-rich, for example, containing a stretch
of G residues. These molecules will form a triple helix with a DNA
duplex that is rich in GC pairs, in which the majority of the
purine residues are located on a single strand of the targeted
duplex, resulting in CGC triplets across the three strands in the
triplex.
[0522] Alternatively, the potential sequences that can be targeted
for triple helix formation may be increased by creating a so-called
"switchback" nucleic acid molecule. Switchback molecules are
synthesized in an alternating 5'-3', 3'-5' manner, such that they
base pair with first one strand of a duplex and then the other,
eliminating the necessity for a sizable stretch of either purines
or pyrimidines to be present on one strand of a duplex.
[0523] Antisense RNA and DNA, ribozyme, and triple helix molecules
of the invention may be prepared by any method known in the art for
the synthesis of DNA and RNA molecules. These include techniques
for chemically synthesizing oligodeoxyribonucleotides and
oligoribonucleotides well known in the art such as for example
solid phase phosphoramidite chemical synthesis. Alternatively, RNA
molecules may be generated by in vitro and in vivo transcription of
DNA sequences encoding the antisense RNA molecule. Such DNA
sequences may be incorporated into a wide variety of vectors that
incorporate suitable RNA polymerase promoters such as the T7 or SP6
polymerase promoters. Alternatively, antisense cDNA constructs that
synthesize antisense RNA constitutively or inducibly, depending on
the promoter used, can be introduced stably into cell lines.
[0524] Moreover, various well-known modifications to nucleic acid
molecules may 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 oligodeoxyribonucleotide
backbone.
[0525] In general, it is anticipated that methods decreasing the
presence or translation of hedgehog, smoothened or gli-1 mRNA will
act as hedgehog antagonists, while methods that decrease the
production of patched will have an agonist effect.
[0526] In certain embodiments, the subject antagonists can be
chosen on the basis of their selectively for the hedgehog pathway.
This selectivity can be for the hedgehog pathway versus other
pathways, or for selectivity between particular hedgehog pathways,
e.g., e.g., ptc-1, ptc-2, etc.
[0527] In certain preferred embodiments, the subject inhibitors
inhibit hedgehog-mediated signal transduction with an ED.sub.50 of
1 mM or less, more preferably of 1 .mu.M or less, and even more
preferably of 1 nM or less.
[0528] In particular embodiments, the small molecule is chosen for
use because it is more selective for one patched isoform over the
next, e.g., 10 fold, and more preferably at least 100 or even 1000
fold more selective for one patched pathway (ptc-1, ptc-2) over
another.
[0529] In certain embodiments, a compound which is an antagonist of
the hedgehog pathway is chosen to selectively antagonize hedgehog
activity over protein kinases other than PKA, such as PKC, e.g.,
the compound modulates the activity of the hedgehog pathway at
least an order of magnitude more strongly than it modulates the
activity of another protein kinase, preferably at least two orders
of magnitude more strongly, even more preferably at least three
orders of magnitude more strongly. Thus, for example, a preferred
inhibitor of the hedgehog pathway may inhibit hedgehog activity
with a K.sub.i at least an order of magnitude lower than its
K.sub.i for inhibition of PKC, preferably at least two orders of
magnitude lower, even more preferably at least three orders of
magnitude lower. In certain embodiments, the K.sub.i for PKA
inhibition is less than 10 nM, preferably less than 1 nM, even more
preferably less than 0.1 nM.
[0530] IV. Exemplary Applications of Method and Compositions
[0531] Another aspect of the present invention relates to methods
of modulating a differentiated state, survival, and/or
proliferation of a cell.
[0532] For example, it is contemplated that the subject method
could be used to inhibit angiogenesis. Hedgehog is known to
stimulate angiogenesis. Matrigel plugs impregnated with hedgehog
protein and inserted into mice evince substantial
neovascularization, whereas Matrigel plugs not carrying hedgehog
show comparatively little vascularization. Hedgehog protein is also
capable of increasing vascularization of the normally avascular
mouse cornea. The ptc-1 gene is expressed in normal vascular
tissues, including the endothelial cells of the aorta, vascular
smooth muscle cells, adventitial fibroblasts of the aorta, the
coronary vasculature and cardiomyocytes of the atria and
ventricles. These tissues are also sensitive to hedgehog protein.
Treatment with exogenous hedgehog causes upregulation of ptc-1
expression. In addition, hedgehog proteins stimulate proliferation
of vascular smooth muscle cells in vivo. Hedgehog proteins also
cause fibroblasts to increase expression of angiogenic growth
factors such as VEGF, bFGF, Ang-1 and Ang-2. Lastly, hedgehog
proteins are known to stimulate recovery from ischemic injury and
stimulate formation of collateral vessels.
[0533] Given that hedgehog promotes angiogenesis, hedgehog
antagonists are expected to act as angiogenesis inhibitors,
particularly in situations where some level of hedgehog signaling
is necessary for angiogenesis.
[0534] Angiogenesis is fundamental to many disorders. Persistent,
unregulated angiogenesis occurs in a range of disease states, tumor
metastases and abnormal growths by endothelial cells. The
vasculature created as a result of angiogenic processes supports
the pathological damage seen in these conditions. The diverse
pathological states created due to unregulated angiogenesis have
been grouped together as angiogenic dependent or angiogenic
associated diseases. Therapies directed at control of the
angiogenic processes could lead to the abrogation or mitigation of
these diseases.
[0535] Diseases caused by, supported by or associated with
angiogenesis include ocular neovascular disease, age-related
macular degeneration, diabetic retinopathy, retinopathy of
prematurity, corneal graft rejection, neovascular glaucoma,
retrolental fibroplasia, epidemnic keratoconjunctivitis, Vitamin A
deficiency, contact lens overwear, atopic keratitis, superior
limbic keratitis, pterygium keratitis sicca, sjogrens, acne
rosacea, phylectenulosis, syphilis, Mycobacteria infections, lipid
degeneration, chemical burns, bacterial ulcers, fungal ulcers,
Herpes simplex infections, Herpes zoster infections, protozoan
infections, Kaposi sarcoma, Mooren ulcer, Terrien's marginal
degeneration, mariginal keratolysis, rheumatoid arthritis, systemic
lupus, polyarteritis, trauma, Wegeners sarcoidosis, Scleritis,
Steven's Johnson disease, periphigoid radial keratotomy, corneal
graph rejection, rheumatoid arthritis, osteoarthritis chronic
inflammation (e.g., ulcerative colitis or Crohn's disease),
hemangioma, Osler-Weber-Rendu disease, and hereditary hemorrhagic
telangiectasia.
[0536] In addition, angiogenesis plays a critical role in cancer. A
tumor cannot expand without a blood supply to provide nutrients and
remove cellular wastes. Tumors in which angiogenesis is important
include solid tumors such as rhabdomyosarcomas, retinoblastoma,
Ewing sarcoma, neuroblastoma, and osteosarcoma, and benign tumors
such as acoustic neuroma, neurofibroma, trachoma and pyogenic
granulomas. Angiogenic factors have been found associated with
several solid tumors . Prevention of angiogenesis could halt the
growth of these tumors and the resultant damage to the animal due
to the presence of the tumor. Angiogenesis is also associated with
blood-born tumors such as leukemias, any of various acute or
chronic neoplastic diseases of the bone marrow in which
unrestrained proliferation of white blood cells occurs, usually
accompanied by anemia, impaired blood clotting, and enlargement of
the lymph nodes, liver, and spleen. It is believed that
angiogenesis plays a role in the abnormalities in the bone marrow
that give rise to leukemia-like tumors.
[0537] In addition to tumor growth, angiogenesis is important in
metastasis. Initially, angiogenesis is important is in the
vascularization of the tumor which allows cancerous cells to enter
the blood stream and to circulate throughout the body. After the
tumor cells have left the primary site, and have settled into the
secondary, metastasis site, angiogenesis must occur before the new
tumor can grow and expand. Therefore, prevention of angiogenesis
could lead to the prevention of metastasis of tumors and possibly
contain the neoplastic growth at the primary site.
[0538] Angiogenesis is also involved in normal physiological
processes such as reproduction and wound healing. Angiogenesis is
an important step in ovulation and also in implantation of the
blastula after fertilization. Prevention of angiogenesis could be
used to induce amenorrhea, to block ovulation or to prevent
implantation by the blastula.
[0539] It is anticipated that the invention will be useful for the
treatment and/or prevention of respiratory distress syndrome or
other disorders resulting from inappropriate lung surface tension.
Respiratory distress syndrome results from insufficient surfactant
in the alveolae of the lungs. The lungs of vertebrates contain
surfactant, a complex mixture of lipids and protein that causes
surface tension to rise during lung inflation and decrease during
lung deflation. During lung deflation, surfactant decreases such
that there are no surface forces that would otherwise promote
alveolar collapse. Aerated alveoli that have not collapsed during
expiration permit continuous oxygen and carbon dioxide transport
between blood and alveolar gas and require much less force to
inflate during the subsequent inspiration. During inflation, lung
surfactant increases surface tension as the alveolar surface area
increases. A rising surface tension in expanding alveoli opposes
over-inflation in those airspaces and tends to divert inspired air
to less well-aerated alveoli, thereby facilitating even lung
aeration.
[0540] Respiratory distress syndrome is particularly prevalent
among premature infants. Lung surfactant is normally synthesized at
a very low rate until the last six weeks of fetal life. Human
infants born more than six weeks before the normal term of a
pregnancy have a high risk of being born with inadequate amounts of
lung surfactant and inadequate rates of surfactant synthesis. The
more prematurely an infant is born, the more severe the surfactant
deficiency is likely to be. Severe surfactant deficiency can lead
to respiratory failure within a few minutes or hours of birth. The
surfactant deficiency produces progressive collapse of alveoli
(atelectasis) because of the decreasing ability of the lung to
expand despite maximum inspiratory effort. As a result, inadequate
amounts of oxygen reach the infant's blood. RDS can occur in adults
as well, typically as a consequence of failure in surfactant
biosynthesis.
[0541] Lung tissue of premature infants shows high activity of the
hedgehog signaling pathway. Inhibition of this pathway using
hedgehog antagonists increases the formation of lamellated bodies
and increases the expression of genes involved in surfactant
biosynthesis. Lamellar bodies are subcellular structures associated
with surfactant biosynthesis. For these reasons, treatment of
premature infants with a hedgehog antagonist should stimulate
surfactant biosynthesis and ameliorate RDS. In cases where adult
RDS is associated with hedgehog pathway activation, treatment with
hedgehog antagonists should also be effective.
[0542] It is further contemplated that the use of hedgehog
antagonists may be specifically targeted to disorders where the
affected tissue and/or cells evince high hedgehog pathway
activation. Expression of gli genes is activated by the hedgehog
signaling pathway, including gli-1, gli-2 and gli-3. gli-1
expression is most consistently correlated with hedgehog signaling
activity across a wide range of tissues and disorders, while gli-3
is somewhat less so. The gli genes encode transcription factors
that activate expression of many genes needed to elicit the full
effects of hedgehog signaling. However, the Gli-3 transcription
factor can also act as a repressor of hedgehog effector genes, and
therefore, expression of gli-3 can cause a decreased effect of the
hedgehog signaling pathway. Whether Gli-3 acts as a transcriptional
activator or repressor depends on post-translational events, and
therefore it is expected that methods for detecting the activating
form (versus the repressing form) of Gli-3 protein would also be a
reliable measure of hedgehog pathway activation. gli-2 gene
expression is expected to provide a reliable marker for hedgehog
pathway activation. The gli-1 gene is strongly expressed in a wide
array of cancers, hyperplasias and immature lungs, and serves as a
marker for the relative activation of the hedgehog pathway. In
addition, tissues, such as immature lung, that have high gli gene
expression are strongly affected by hedgehog inhibitors.
Accordingly, it is contemplated that the detection of gli gene
expression may be used as a powerful predictive tool to identify
tissues and disorders that will particularly benefit from treatment
with a hedgehog antagonist.
[0543] In preferred embodiments, gli-1 expression levels are
detected, either by direct detection of the transcript or by
detection of protein levels or activity. Transcripts may be
detected using any of a wide range of techniques that depend
primarily on hybridization of probes to the gli-1 transcripts or to
cDNAs synthesized therefrom. Well known techniques include Northern
blotting, reverse-transcriptase PCR and microarray analysis of
transcript levels. Methods for detecting Gli protein levels include
Western blotting, immunoprecipitation, two-dimensional
polyacrylamide gel electrophoresis (2D SDS-PAGE) (preferably
compared against a standard wherein the position of the Gli
proteins has been determined), and mass spectroscopy. Mass
spectroscopy may be coupled with a series of purification steps to
allow high-throughput identification of many different protein
levels in a particular sample. Mass spectroscopy and 2D SDS-PAGE
can also be used to identify post-transcriptional modifications to
proteins including proteolytic events, ubiquitination,
phosphorylation, lipid modification etc. Gli activity may also be
assessed by analyzing binding to substrate DNA or in vitro
transcriptional activation of target promoters. Gel shift assays,
DNA footprinting assays and DNA-protein crosslinking assays are all
methods that may be used to assess the presence of a protein
capable of binding to Gli binding sites on DNA. (J Mol Med 1999
June; 77(6):459-68; Cell 2000 Feb. 18;100(4):423-34; Development
2000; 127(19):4293-4301)
[0544] In preferred embodiments, gli transcript levels are measured
and diseased or disordered tissues showing abnormally high gli
levels are treated with a hedgehog antagonist. Premature lung
tissue, lung cancers (e.g., adenocarcinomas, broncho-alveolar
adenocarcinomas, small cell carcinomas), breast cancers (e.g.,
inferior ductal carcinomas, inferior lobular carcinomas, tubular
carcinomas), prostate cancers (e.g., adenocarcinomas), and benign
prostatic hyperplasias all show strongly elevated gli-1 expression
levels in certain cases. Accordingly, gli-1 expression levels are a
powerful diagnostic device to determine which of these tissues
should be treated with a hedgehog antagonist. In addition, there is
substantial correlative evidence that cancers of urothelial cells
(e.g., bladder cancer, other urogenital cancers) will also have
elevated gli-1 levels in certain cases. For example, it is known
that loss of heterozygosity on chromosome 9q22 is common in bladder
cancers. The ptc-1 gene is located at this position and ptc-1 loss
of function is probably a partial cause of hyperproliferation, as
in many other cancer types. Accordingly, such cancers would also
show high gli expression and would be particularly amenable to
treatment with a hedgehog antagonist.
[0545] Expression of ptc-1 and ptc-2 is also activated by the
hedgehog signaling pathway, but these genes are inferior to the gli
genes as markers of hedgehog pathway activation. In certain tissues
only one of ptc-1 or ptc-2 is expressed although the hedgehog
pathway is highly active. For example, in testicular development,
indian hedgehog plays an important role and the hedgehog pathway is
activated, but only ptc-2 is expressed. Accordingly, these genes
are individually unreliable as markers for hedgehog pathway
activation, although simultaneous measurement of both genes is
contemplated as a useful indicator for tissues to be treated with a
hedgehog antagonist.
[0546] It is anticipated that any degree of gli overexpression may
be useful in determining that a hedgehog antagonist will be an
effective therapeutic. In preferred embodiments, gli should be
expressed at a level at least twice as high as normal. In
particularly preferred embodiments, expression is four, six, eight
or ten times as high as normal.
[0547] For instance, it is contemplated by the invention that, in
light of the findings of an apparently broad involvement of
hedgehog, ptc, and smoothened in the formation of ordered spatial
arrangements of differentiated tissues in vertebrates, the subject
method could be used as part of a process for generating and/or
maintaining an array of different vertebrate tissue both in vitro
and in vivo. The hedgehog antagonist, whether inductive or
anti-inductive with respect to proliferation or differentiation of
a given tissue, can be, as appropriate, any of the preparations
described above.
[0548] For example, the present method is applicable to cell
culture techniques wherein it is desirable to reduce the level of
hedgehog signaling. In vitro neuronal culture systems have proved
to be fundamental and indispensable tools for the study of neural
development, as well as the identification of neurotrophic factors
such as nerve growth factor (NGF), ciliary trophic factors (CNTF),
and brain derived neurotrophic factor (BDNF). One use of the
present method may be in cultures of neuronal stem cells, such as
in the use of such cultures for the generation of new neurons and
glia. In such embodiments of the subject method, the cultured cells
can be contacted with a hedgehog antagonist of the present
invention in order to alter the rate of proliferation of neuronal
stem cells in the culture and/or alter the rate of differentiation,
or to maintain the integrity of a culture of certain terminally
differentiated neuronal cells. In an exemplary embodiment, the
subject method can be used to culture, for example, sensory neurons
or, alternatively, motor neurons. Such neuronal cultures can be
used as convenient assay systems as well as sources of implantable
cells for therapeutic treatments.
[0549] To further illustrate other uses of the subject hedgehog
antagonists, it is noted that intracerebral grafting has emerged as
an additional approach to central nervous system therapies. For
example, one approach to repairing damaged brain tissues involves
the transplantation of cells from fetal or neonatal animals into
the adult brain (Dunnett et al. (1987) J Exp Biol 123:265-289; and
Freund et al. (1985) J Neurosci 5:603-616). Fetal neurons from a
variety of brain regions can be successfully incorporated into the
adult brain, and such grafts can alleviate behavioral defects. For
example, movement disorder induced by lesions of dopaminergic
projections to the basal ganglia can be prevented by grafts of
embryonic dopaminergic neurons. Complex cognitive functions that
are impaired after lesions of the neocortex can also be partially
restored by grafts of embryonic cortical cells. The subject method
can be used to regulate the growth state in the culture, or where
fetal tissue is used, especially neuronal stem cells, can be used
to regulate the rate of differentiation of the stem cells.
[0550] Stem cells useful in the present invention are generally
known. For example, several neural crest cells have been
identified, some of which are multipotent and likely represent
uncommitted neural crest cells, and others of which can generate
only one type of cell, such as sensory neurons, and likely
represent committed progenitor cells. The role of hedgehog
antagonists employed in the present method to culture such stem
cells can be to regulate differentiation of the uncommitted
progenitor, or to regulate further restriction of the developmental
fate of a committed progenitor cell towards becoming a terminally
differentiated neuronal cell. For example, the present method can
be used in vitro to regulate the differentiation of neural crest
cells into glial cells, schwann cells, chromaffin cells,
cholinergic sympathetic or parasympathetic neurons, as well as
peptidergic and serotonergic neurons. The hedgehog antagonists can
be used alone, or can be used in combination with other
neurotrophic factors that act to more particularly enhance a
particular differentiation fate of the neuronal progenitor
cell.
[0551] In addition to the implantation of cells cultured in the
presence of the subject hedgehog antagonists, yet another aspect of
the present invention concerns the therapeutic application of a
hedgehog antagonist to regulate the growth state of neurons and
other neuronal cells in both the central nervous system and the
peripheral nervous system. The ability of ptc, hedgehog, and
smoothened to regulate neuronal differentiation during development
of the nervous system and also presumably in the adult state
indicates that, in certain instances, the subject hedgehog
antagonists can be expected to facilitate control of adult neurons
with regard to maintenance, functional performance, and aging of
normal cells; repair and regeneration processes in chemically or
mechanically lesioned cells; and treatment of degeneration in
certain pathological conditions. In light of this understanding,
the present invention specifically contemplates applications of the
subject method to the treatment protocol of (prevention and/or
reduction of the severity of) neurological conditions deriving
from: (i) acute, subacute, or chronic injury to the nervous system,
including traumatic injury, chemical injury, vascular injury and
deficits (such as the ischemia resulting from stroke), together
with infectious/inflammatory and tumor-induced injury; (ii) aging
of the nervous system including Alzheimer's disease; (iii) chronic
neurodegenerative diseases of the nervous system, including
Parkinson's disease, Huntington's chorea, amyotrophic lateral
sclerosis and the like, as well as spinocerebellar degenerations;
and (iv) chronic immunological diseases of the nervous system or
affecting the nervous system, including multiple sclerosis.
[0552] As appropriate, the subject method can also be used in
generating nerve prostheses for the repair of central and
peripheral nerve damage. In particular, where a crushed or severed
axon is intubulated by use of a prosthetic device, hedgehog
antagonists can be added to the prosthetic device to regulate the
rate of growth and regeneration of the dendritic processes.
Exemplary nerve guidance channels are described in U.S. Pat. Nos.
5,092,871 and 4,955,892.
[0553] In another embodiment, the subject method can be used in the
treatment of neoplastic or hyperplastic transformations such as may
occur in the central nervous system. For instance, the hedgehog
antagonists can be utilized to cause such transformed cells to
become either post-mitotic or apoptotic. The present method may,
therefore, be used as part of a treatment for, e.g., malignant
gliomas, meningiomas, medulloblastomas, neuroectodermal tumors, and
ependymomas.
[0554] In a preferred embodiment, the subject method can be used as
part of a treatment regimen for malignant medulloblastoma and other
primary CNS malignant neuroectodermal tumors.
[0555] In certain embodiments, the subject method is used as part
of treatment program for medulloblastoma. Medulloblastoma, a
primary brain tumor, is the most common brain tumor in children. A
medulloblastoma is a primitive neuroectodermal tumor arising in the
posterior fossa. They account for approximately 25% of all
pediatric brain tumors (Miller). Histologically, they are small
round cell tumors commonly arranged in true rosettes, but may
display some differentiation to astrocytes, ependymal cells or
neurons (Rorke; Kleihues). PNET's may arise in other areas of the
brain including the pineal gland (pineoblastoma) and cerebrum.
Those arising in the supratentorial region generally fare worse
than their PF counterparts.
[0556] Medulloblastoma/PNET's are known to recur anywhere in the
CNS after resection, and can even metastasize to bone. Pretreatment
evaluation should therefore include an examination of the spinal
cord to exclude the possibility of "dropped metastases".
Gadolinium-enhanced MRI has largely replaced myelography for this
purpose, and CSF cytology is obtained postoperatively as a routine
procedure.
[0557] In other embodiments, the subject method is used as part of
treatment program for ependymomas. Ependymomas account for
approximately 10% of the pediatric brain tumors in children.
Grossly, they are tumors that arise from the ependymal lining of
the ventricles and microscopically form rosettes, canals, and
perivascular rosettes. In the CHOP series of 51 children reported
with ependymomas, 3/4 were histologically benign. Approximately 2/3
arose from the region of the 4th ventricle. One third presented in
the supratentorial region. Age at presentation peaks between birth
and 4 years, as demonstrated by SEER data as well as data from
CHOP. The median age is about 5 years. Because so many children
with this disease are babies, they often require multimodal
therapy.
[0558] Yet another aspect of the present invention concerns the
observation in the art that ptc, hedgehog, and/or smoothened are
involved in morphogenic signals involved in other vertebrate
organogenic pathways in addition to neuronal differentiation as
described above, having apparent roles in other endodermal
patterning, as well as both mesodermal and endodermal
differentiation processes. Thus, it is contemplated by the
invention that compositions comprising hedgehog antagonists can
also be utilized for both cell culture and therapeutic methods
involving generation and maintenance of non-neuronal tissue.
[0559] In one embodiment, the present invention makes use of the
discovery that ptc, hedgehog, and smoothened are apparently
involved in controlling the development of stem cells responsible
for formation of the digestive tract, liver, lungs, and other
organs which derive from the primitive gut. Shh serves as an
inductive signal from the endoderm to the mesoderm, which is
critical to gut morphogenesis. Therefore, for example, hedgehog
antagonists of the instant method can be employed for regulating
the development and maintenance of an artificial liver that can
have multiple metabolic functions of a normal liver. In an
exemplary embodiment, the subject method can be used to regulate
the proliferation and differentiation of digestive tube stem cells
to form hepatocyte cultures which can be used to populate
extracellular matrices, or which can be encapsulated in
biocompatible polymers, to form both implantable and extracorporeal
artificial livers.
[0560] In another embodiment, therapeutic compositions of hedgehog
antagonists can be utilized in conjunction with transplantation of
such artificial livers, as well as embryonic liver structures, to
regulate uptake of intraperitoneal implantation, vascularization,
and in vivo differentiation and maintenance of the engrafted liver
tissue.
[0561] In yet another embodiment, the subject method can be
employed therapeutically to regulate such organs after physical,
chemical or pathological insult. For instance, therapeutic
compositions comprising hedgehog antagonists can be utilized in
liver repair subsequent to a partial hepatectomy.
[0562] The generation of the pancreas and small intestine from the
embryonic gut depends on intercellular signalling between the
endodermal and mesodermal cells of the gut. In particular, the
differentiation of intestinal mesoderm into smooth muscle has been
suggested to depend on signals from adjacent endodermal cells. One
candidate mediator of endodermally derived signals in the embryonic
hindgut is Sonic hedgehog. See, for example, Apelqvist et al.
(1997) Curr Biol 7:801-4. The Shh gene is expressed throughout the
embryonic gut endoderm with the exception of the pancreatic bud
endoderm, which instead expresses high levels of the homeodomain
protein Ipf1/Pdx1 (insulin promoter factor 1/pancreatic and
duodenal homeobox 1), an essential regulator of early pancreatic
development. Apelqvist et al., supra, have examined whether the
differential expression of Shh in the embryonic gut tube controls
the differentiation of the surrounding mesoderm into specialised
mesoderm derivatives of the small intestine and pancreas. To test
this, they used the promoter of the Ipf1/Pdx1 gene to selectively
express Shh in the developing pancreatic epithelium. In
Ipf1/Pdx1-Shh transgenic mice, the pancreatic mesoderm developed
into smooth muscle and interstitial cells of Cajal, characteristic
of the intestine, rather than into pancreatic mesenchyme and
spleen. Also, pancreatic explants exposed to Shh underwent a
similar program of intestinal differentiation. These results
provide evidence that the differential expression of endodermally
derived Shh controls the fate of adjacent mesoderm at different
regions of the gut tube.
[0563] In the context of the present invention, it is contemplated
therefore that the subject hedgehog antagonists can be used to
control or regulate the proliferation and/or differentiation of
pancreatic tissue both in vivo and in vitro.
[0564] In another embodiment, hedgehog antagonists are used to
generate endodermal tissue from non-endodermal stem cells including
mesenchymal stem cells and stem cells derived from mesodermal
tissues. Exemplary mesodermal tissues from which stem cells may be
isolated include skeletal muscle, cardiac muscle, kidney, bone,
cartilage, and fat.
[0565] There are a wide variety of pathological cell proliferative
and differentiative conditions for which the inhibitors of the
present invention may provide therapeutic benefits, with the
general strategy being, for example, the correction of aberrant
insulin expression, or modulation of differentiation. More
generally, however, the present invention relates to a method of
inducing and/or maintaining a differentiated state, enhancing
survival and/or affecting proliferation of pancreatic cells, by
contacting the cells with the subject inhibitors. For instance, it
is contemplated by the invention that, in light of the apparent
involvement of ptc, hedgehog, and smoothened in the formation of
ordered spatial arrangements of pancreatic tissues, the subject
method could be used as part of a technique to generate and/or
maintain such tissue both in vitro and in vivo. For instance,
modulation of the function of hedgehog can be employed in both cell
culture and therapeutic methods involving generation and
maintenance of .beta.-cells and possibly also for non-pancreatic
tissue, such as in controlling the development and maintenance of
tissue from the digestive tract, spleen, lungs, urogenital organs
(e.g., bladder), and other organs which derive from the primitive
gut.
[0566] In an exemplary embodiment, the present method can be used
in the treatment of hyperplastic and neoplastic disorders effecting
pancreatic tissue, particularly those characterized by aberrant
proliferation of pancreatic cells. For instance, pancreatic cancers
are marked by abnormal proliferation of pancreatic cells, which can
result in alterations of insulin secretory capacity of the
pancreas. For instance, certain pancreatic hyperplasias, such as
pancreatic carcinomas, can result in hypoinsulinemia due to
dysfunction of .beta.-cells or decreased islet cell mass.
[0567] Moreover, manipulation of hedgehog signaling properties at
different points may be useful as part of a strategy for
reshaping/repairing pancreatic tissue both in vivo and in vitro. In
one embodiment, the present invention makes use of the apparent
involvement of ptc, hedgehog, and smoothened in regulating the
development of pancreatic tissue. In general, the subject method
can be employed therapeutically to regulate the pancreas after
physical, chemical or pathological insult. In yet another
embodiment, the subject method can be applied to cell culture
techniques, and in particular, may be employed to enhance the
initial generation of prosthetic pancreatic tissue devices.
Manipulation of proliferation and differentiation of pancreatic
tissue, for example, by altering hedgehog activity, can provide a
means for more carefully controlling the characteristics of a
cultured tissue. In an exemplary embodiment, the subject method can
be used to augment production of prosthetic devices which require
.beta.-islet cells, such as may be used in the encapsulation
devices described in, for example, the Aebischer et al. U.S. Pat.
No. 4,892,538, the Aebischer et al. U.S. Pat. No. 5,106,627, the
Lim U.S. Pat. No. 4,391,909, and the Sefton U.S. Pat. No.
4,353,888. Early progenitor cells to the pancreatic islets are
multipotential, and apparently coactivate all the islet-specific
genes from the time they first appear. As development proceeds,
expression of islet-specific hormones, such as insulin, becomes
restricted to the pattern of expression characteristic of mature
islet cells. The phenotype of mature islet cells, however, is not
stable in culture, as reappearance of embryonal traits in mature
.beta.-cells can be observed. By utilizing the subject hedgehog
antagonists, the differentiation path or proliferative index of the
cells can be regulated.
[0568] Furthermore, manipulation of the differentiative state of
pancreatic tissue can be utilized in conjunction with
transplantation of artificial pancreas. For instance, manipulation
of hedgehog function to affect tissue differentiation can be
utilized as a means of maintaining graft viability.
[0569] Bellusci et al. (1997) Development 124:53 report that Sonic
hedgehog regulates lung mesenchymal cell proliferation in vivo.
Accordingly, the present method can be used to regulate
regeneration of lung tissue, e.g., in the treatment of
emphysema.
[0570] Fujita et al. (1997) Biochem Biophys Res Commun 238:658
reported that Sonic hedgehog is expressed in human lung squamous
carcinoma and adenocarcinoma cells. The expression of Sonic
hedgehog was also detected in the human lung squamous carcinoma
tissues, but not in the normal lung tissue of the same patient.
They also observed that Sonic hedgehog stimulates the incorporation
of BrdU into the carcinoma cells and stimulates their cell growth,
while anti-Shh-N inhibited their cell growth. These results suggest
that a ptc, hedgehog, and/or smoothened is involved in the cell
growth of such transformed lung tissue and therefore indicates that
the subject method can be used as part of a treatment of lung
carcinoma and adenocarcinomas, and other proliferative disorders
involving the lung epithelia.
[0571] Many other tumors may, based on evidence such as involvement
of the hedgehog pathway in these tumors, or detected expression of
hedgehog or its receptor in these tissues during development, be
affected by treatment with the subject compounds. Such tumors
include, but are by no means limited to, tumors related to Gorlin's
syndrome (e.g., medulloblastoma, meningioma, etc.), tumors
evidenced in ptc knock-out mice (e.g., hemangioma,
rhabdomyosarcoma, etc.), tumors resulting from gli-1 amplification
(e.g., glioblastoma, sarcoma, etc.), tumors connected with TRC8, a
ptc homolog (e.g., renal carcinoma, thyroid carcinoma, etc.),
Ext-1-related tumors (e.g., bone cancer, etc.), Shh-induced tumors
(e.g., lung cancer, chondrosarcomas, etc.), and other tumors (e.g.,
breast cancer, urogenital cancer (e.g., kidney, bladder, ureter,
prostate, etc.), adrenal cancer, gastrointestinal cancer (e.g.,
stomach, intestine, etc.), etc.).
[0572] In still another embodiment of the present invention,
compositions comprising hedgehog antagonists can be used in the in
vitro generation of skeletal tissue, such as from skeletogenic stem
cells, as well as the in vivo treatment of skeletal tissue
deficiencies. The present invention particularly contemplates the
use of hedgehog antagonists to regulate the rate of chondrogenesis
and/or osteogenesis. By "skeletal tissue deficiency", it is meant a
deficiency in bone or other skeletal connective tissue at any site
where it is desired to restore the bone or connective tissue, no
matter how the deficiency originated, e.g., whether as a result of
surgical intervention, removal of tumor, ulceration, implant,
fracture, or other traumatic or degenerative conditions.
[0573] For instance, the method of the present invention can be
used as part of a regimen for restoring cartilage function to a
connective tissue. Such methods are useful in, for example, the
repair of defects or lesions in cartilage tissue which is the
result of degenerative wear such as that which results in
arthritis, as well as other mechanical derangements which may be
caused by trauma to the tissue, such as a displacement of torn
meniscus tissue, meniscectomy, a laxation of a joint by a torn
ligament, malignment of joints, bone fracture, or by hereditary
disease. The present reparative method is also useful for
remodeling cartilage matrix, such as in plastic or reconstructive
surgery, as well as periodontal surgery. The present method may
also be applied to improving a previous reparative procedure, for
example, following surgical repair of a meniscus, ligament, or
cartilage. Furthermore, it may prevent the onset or exacerbation of
degenerative disease if applied early enough after trauma.
[0574] In one embodiment of the present invention, the subject
method comprises treating the afflicted connective tissue with a
therapeutically sufficient amount of a hedgehog antagonist,
particularly an antagonist selective for India hedgehog signal
transduction, to regulate a cartilage repair response in the
connective tissue by managing the rate of differentiation and/or
proliferation of chondrocytes embedded in the tissue. Such
connective tissues as articular cartilage, interarticular cartilage
(menisci), costal cartilage (connecting the true ribs and the
sternum), ligaments, and tendons are particularly amenable to
treatment in reconstructive and/or regenerative therapies using the
subject method. As used herein, regenerative therapies include
treatment of degenerative states which have progressed to the point
of which impairment of the tissue is obviously manifest, as well as
preventive treatments of tissue where degeneration is in its
earliest stages or imminent.
[0575] In an illustrative embodiment, the subject method can be
used as part of a therapeutic intervention in the treatment of
cartilage of a diarthroidal joint, such as a knee, an ankle, an
elbow, a hip, a wrist, a knuckle of either a finger or toe, or a
tempomandibular joint. The treatment can be directed to the
meniscus of the joint, to the articular cartilage of the joint, or
both. To further illustrate, the subject method can be used to
treat a degenerative disorder of a knee, such as which might be the
result of traumatic injury (e.g., a sports injury or excessive
wear) or osteoarthritis. The subject antagonists may be
administered as an injection into the joint with, for instance, an
arthroscopic needle. In some instances, the injected agent can be
in the form of a hydrogel or other slow release vehicle described
above in order to permit a more extended and regular contact of the
agent with the treated tissue.
[0576] The present invention further contemplates the use of the
subject method in the field of cartilage transplantation and
prosthetic device therapies. However, problems arise, for instance,
because the characteristics of cartilage and fibrocartilage varies
between different tissue: such as between articular, meniscal
cartilage, ligaments, and tendons, between the two ends of the same
ligament or tendon, and between the superficial and deep parts of
the tissue. The zonal arrangement of these tissues may reflect a
gradual change in mechanical properties, and failure occurs when
implanted tissue, which has not differentiated under those
conditions, lacks the ability to appropriately respond. For
instance, when meniscal cartilage is used to repair anterior
cruciate ligaments, the tissue undergoes a metaplasia to pure
fibrous tissue. By regulating the rate of chondrogenesis, the
subject method can be used to particularly address this problem, by
helping to adaptively control the implanted cells in the new
environment and effectively resemble hypertrophic chondrocytes of
an earlier developmental stage of the tissue.
[0577] In similar fashion, the subject method can be applied to
enhancing both the generation of prosthetic cartilage devices and
to their implantation. The need for improved treatment has
motivated research aimed at creating new cartilage that is based on
collagen-glycosaminoglyc- an templates (Stone et al. (1990) Clin
Orthop Relat Red 252:129), isolated chondrocytes (Grande et al.
(1989) J Orthop Res 7:208; and Takigawa et al. (1987) Bone Miner
2:449), and chondrocytes attached to natural or synthetic polymers
(Walitani et al. (1989) J Bone Jt Surg 71B:74; Vacanti et al.
(1991) Plast Reconstr Surg 88:753; von Schroeder et al. (1991) J
Biomed Mater Res 25:329; Freed et al. (1993) J Biomed Mater Res
27:11; and the Vacanti et al. U.S. Pat. No. 5,041,138). For
example, chondrocytes can be grown in culture on biodegradable,
biocompatible highly porous scaffolds formed from polymers such as
polyglycolic acid, polylactic acid, agarose gel, or other polymers
that degrade over time as function of hydrolysis of the polymer
backbone into innocuous monomers. The matrices are designed to
allow adequate nutrient and gas exchange to the cells until
engraftment occurs. The cells can be cultured in vitro until
adequate cell volume and density has developed for the cells to be
implanted. One advantage of the matrices is that they can be cast
or molded into a desired shape on an individual basis, so that the
final product closely resembles the patient's own ear or nose (by
way of example), or flexible matrices can be used which allow for
manipulation at the time of implantation, as in a joint.
[0578] In one embodiment of the subject method, the implants are
contacted with a hedgehog antagonist during certain stages of the
culturing process in order to manage the rate of differentiation of
chondrocytes and the formation of hypertrophic chrondrocytes in the
culture.
[0579] In another embodiment, the implanted device is treated with
a hedgehog antagonist in order to actively remodel the implanted
matrix and to make it more suitable for its intended function. As
set out above with respect to tissue transplants, the artificial
transplants suffer from the same deficiency of not being derived in
a setting which is comparable to the actual mechanical environment
in which the matrix is implanted. The ability to regulate the
chondrocytes in the matrix by the subject method can allow the
implant to acquire characteristics similar to the tissue for which
it is intended to replace.
[0580] In yet another embodiment, the subject method is used to
enhance attachment of prosthetic devices. To illustrate, the
subject method can be used in the implantation of a periodontal
prosthesis, wherein the treatment of the surrounding connective
tissue stimulates formation of periodontal ligament about the
prosthesis.
[0581] In still further embodiments, the subject method can be
employed as part of a regimen for the generation of bone
(osteogenesis) at a site in the animal where such skeletal tissue
is deficient. India hedgehog is particularly associated with the
hypertrophic chondrocytes that are ultimately replaced by
osteoblasts. For instance, administration of a hedgehog antagonists
of the present invention can be employed as part of a method for
regulating the rate of bone loss in a subject. For example,
preparations comprising hedgehog antagonists can be employed, for
example, to control endochondral ossification in the formation of a
"model" for ossification.
[0582] In yet another embodiment of the present invention, a
hedgehog antagonist can be used to regulate spermatogenesis. The
hedgehog proteins, particularly Dhh, have been shown to be involved
in the differentiation and/or proliferation and maintenance of
testicular germ cells. Dhh expression is initiated in Sertoli cell
precursors shortly after the activation of Sry (testicular
determining gene) and persists in the testis into the adult. Males
are viable but infertile, owing to a complete absence of mature
sperm. Examination of the developing testis in different genetic
backgrounds suggests that Dhh regulates both early and late stages
of spermatogenesis. Bitgood et al. (1996) Curr Biol 6:298. In a
preferred embodiment, the hedgehog antagonist can be used as a
contraceptive. In similar fashion, hedgehog antagonists of the
subject method are potentially useful for modulating normal ovarian
function.
[0583] The subject method also has wide applicability to the
treatment or prophylaxis of disorders afflicting epithelial tissue,
as well as in cosmetic uses. In general, the method can be
characterized as including a step of administering to an animal an
amount of a hedgehog antagonist effective to alter the growth state
of a treated epithelial tissue. The mode of administration and
dosage regimens will vary depending on the epithelial tissue(s)
that is to be treated. For example, topical formulations will be
preferred where the treated tissue is epidermal tissue, such as
dermal or mucosal tissues.
[0584] A method that "promotes the healing of a wound" results in
the wound healing more quickly as a result of the treatment than a
similar wound heals in the absence of the treatment. "Promotion of
wound healing" can also mean that the method regulates the
proliferation and/or growth of, inter alia, keratinocytes, or that
the wound heals with less scarring, less wound contraction, less
collagen deposition and more superficial surface area. In certain
instances, "promotion of wound healing" can also mean that certain
methods of wound healing have improved success rates, (e.g., the
take rates of skin grafts,) when used together with the method of
the present invention.
[0585] Despite significant progress in reconstructive surgical
techniques, scarring can be an important obstacle in regaining
normal function and appearance of healed skin. This is particularly
true when pathologic scarring such as keloids or hypertrophic scars
of the hands or face causes functional disability or physical
deformity. In the severest circumstances, such scarring may
precipitate psychosocial distress and a life of economic
deprivation. Wound repair includes the stages of hemostasis,
inflammation, proliferation, and remodeling. The proliferative
stage involves multiplication of fibroblasts and endothelial and
epithelial cells. Through the use of the subject method, the rate
of proliferation of epithelial cells in and proximal to the wound
can be controlled in order to accelerate closure of the wound
and/or minimize the formation of scar tissue.
[0586] The present treatment can also be effective as part of a
therapeutic regimen for treating oral and paraoral ulcers, e.g.,
resulting from radiation and/or chemotherapy. Such ulcers commonly
develop within days after chemotherapy or radiation therapy. These
ulcers usually begin as small, painful irregularly shaped lesions
usually covered by a delicate gray necrotic membrane and surrounded
by inflammatory tissue. In many instances, lack of treatment
results in proliferation of tissue around the periphery of the
lesion on an inflammatory basis. For instance, the epithelium
bordering the ulcer usually demonstrates proliferative activity,
resulting in loss of continuity of surface epithelium. These
lesions, because of their size and loss of epithelial integrity,
dispose the body to potential secondary infection. Routine
ingestion of food and water becomes a very painful event and, if
the ulcers proliferate throughout the alimentary canal, diarrhea
usually is evident with all its complicating factors. According to
the present invention, a treatment for such ulcers that includes
application of a hedgehog antagonist can reduce the abnormal
proliferation and differentiation of the affected epithelium,
helping to reduce the severity of subsequent inflammatory
events.
[0587] The subject method and compositions can also be used to
treat wounds resulting from dermatological diseases, such as
lesions resulting from autoimmune disorders such as psoriasis.
Atopic dermititis refers to skin trauma resulting from allergies
associated with an immune response caused by allergens such as
pollens, foods, dander, insect venoms and plant toxins.
[0588] In other embodiments, antiproliferative preparations of
hedgehog antagonists can be used to inhibit lens epithelial cell
proliferation to prevent post-operative complications of
extracapsular cataract extraction. Cataract is an intractable eye
disease and various studies on a treatment of cataract have been
made. But at present, the treatment of cataract is attained by
surgical operations. Cataract surgery has been applied for a long
time and various operative methods have been examined.
Extracapsular lens extraction has become the method of choice for
removing cataracts. The major medical advantages of this technique
over intracapsular extraction are lower incidence of aphakic
cystoid macular edema and retinal detachment. Extracapsular
extraction is also required for implantation of posterior
chamber-type intraocular lenses, which are now considered to be the
lenses of choice in most cases.
[0589] However, a disadvantage of extracapsular cataract extraction
is the high incidence of posterior lens capsule opacification,
often called after-cataract, which can occur in up to 50% of cases
within three years after surgery. After-cataract is caused by
proliferation of equatorial and anterior capsule lens epithelial
cells that remain after extracapsular lens extraction. These cells
proliferate to cause Sommerling rings, and along with fibroblasts,
which also deposit and occur on the posterior capsule, cause
opacification of the posterior capsule, which interferes with
vision. Prevention of after-cataract would be preferable to
treatment. To inhibit secondary cataract formation, the subject
method provides a means for inhibiting proliferation of the
remaining lens epithelial cells. For example, such cells can be
induced to remain quiescent by instilling a solution containing a
hedgehog antagonist preparation into the anterior chamber of the
eye after lens removal. Furthermore, the solution can be
osmotically balanced to provide minimal effective dosage when
instilled into the anterior chamber of the eye, thereby inhibiting
subcapsular epithelial growth with some specificity.
[0590] The subject method can also be used in the treatment of
corneopathies marked by corneal epithelial cell proliferation, as
for example in ocular epithelial disorders such as epithelial
downgrowth or squamous cell carcinomas of the ocular surface.
[0591] Levine et al. (1997) J Neurosci 17:6277 show that hedgehog
proteins can regulate mitogenesis and photoreceptor differentiation
in the vertebrate retina, and Ihh is a candidate factor from the
pigmented epithelium to promote retinal progenitor proliferation
and photoreceptor differentiation. Likewise, Jensen et al. (1997)
Development 124:363 demonstrated that treatment of cultures of
perinatal mouse retinal cells with the amino-terminal fragment of
Sonic hedgehog protein results in an increase in the proportion of
cells that incorporate bromodeoxyuridine, in total cell numbers,
and in rod photoreceptors, amacrine cells and Muller glial cells,
suggesting that Sonic hedgehog promotes the proliferation of
retinal precursor cells. Thus, the subject method can be used in
the treatment of proliferative diseases of retinal cells and
regulate photoreceptor differentiation.
[0592] Yet another aspect of the present invention relates to the
use of the subject method to control hair growth. Hair is basically
composed of keratin, a tough and insoluble protein; its chief
strength lies in its disulfide bond of cystine. Each individual
hair comprises a cylindrical shaft and a root, and is contained in
a follicle, a flask-like depression in the skin. The bottom of the
follicle contains a finger-like projection termed the papilla,
which consists of connective tissue from which hair grows, and
through which blood vessels supply the cells with nourishment. The
shaft is the part that extends outwards from the skin surface,
whilst the root has been described as the buried part of the hair.
The base of the root expands into the hair bulb, which rests upon
the papilla. Cells from which the hair is produced grow in the bulb
of the follicle; they are extruded in the form of fibers as the
cells proliferate in the follicle. Hair "growth" refers to the
formation and elongation of the hair fiber by the dividing
cells.
[0593] As is well known in the art, the common hair cycle is
divided into three stages: anagen, catagen and telogen. During the
active phase (anagen), the epidermal stem cells of the dermal
papilla divide rapidly. Daughter cells move upward and
differentiate to form the concentric layers of the hair itself. The
transitional stage, catagen, is marked by the cessation of mitosis
of the stem cells in the follicle. The resting stage is known as
telogen, where the hair is retained within the scalp for several
weeks before an emerging new hair developing below it dislodges the
telogen-phase shaft from its follicle. From this model it has
become clear that the larger the pool of dividing stem cells that
differentiate into hair cells, the more hair growth occurs.
Accordingly, methods for increasing or reducing hair growth can be
carried out by potentiating or inhibiting, respectively, the
proliferation of these stem cells.
[0594] In certain embodiments, the subject method can be employed
as a way of reducing the growth of human hair as opposed to its
conventional removal by cutting, shaving, or depilation. For
instance, the present method can be used in the treatment of
trichosis characterized by abnormally rapid or dense growth of
hair, e.g., hypertrichosis. In an exemplary embodiment, hedgehog
antagonists can be used to manage hirsutism, a disorder marked by
abnormal hairiness. The subject method can also provide a process
for extending the duration of depilation.
[0595] Moreover, because a hedgehog antagonist will often be
cytostatic to epithelial cells, rather than cytotoxic, such agents
can be used to protect hair follicle cells from cytotoxic agents
that require progression into S-phase of the cell-cycle for
efficacy, e.g., radiation-induced death. Treatment by the subject
method can provide protection by causing the hair follicle cells to
become quiescent, e.g., by inhibiting the cells from entering S
phase, and thereby preventing the follicle cells from undergoing
mitotic catastrophe or programmed cell death. For instance,
hedgehog antagonists can be used for patients undergoing chemo- or
radiation-therapies that ordinarily result in hair loss. By
inhibiting cell-cycle progression during such therapies, the
subject treatment can protect hair follicle cells from death, which
might otherwise result from activation of cell death programs.
After the therapy has concluded, the instant method can also be
removed with concomitant relief of the inhibition of follicle cell
proliferation.
[0596] The subject method can also be used in the treatment of
folliculitis, such as folliculitis decalvans, folliculitis
ulerythematosa reticulata or keloid folliculitis. For example, a
cosmetic preparation of a hedgehog antagonist can be applied
topically in the treatment of pseudofolliculitis, a chronic
disorder occurring most often in the submandibular region of the
neck and associated with shaving, the characteristic lesions of
which are erythematous papules and pustules containing buried
hairs.
[0597] In another aspect of the invention, the subject method can
be used to induce differentiation and/or inhibit proliferation of
epithelially derived tissue. Such forms of these molecules can
provide a basis for differentiation therapy for the treatment of
hyperplastic and/or neoplastic conditions involving epithelial
tissue. For example, such preparations can be used for the
treatment of cutaneous diseases in which there is abnormal
proliferation or growth of cells of the skin.
[0598] For instance, the pharmaceutical preparations of the
invention are intended for the treatment of hyperplastic epidermal
conditions, such as keratosis, as well as for the treatment of
neoplastic epidermal conditions such as those characterized by a
high proliferation rate for various skin cancers, as for example
squamous cell carcinoma. The subject method can also be used in the
treatment of autoimmune diseases affecting the skin, in particular,
of dermatological diseases involving morbid proliferation and/or
keratinization of the epidermis, as for example, caused by
psoriasis or atopic dermatosis.
[0599] Many common diseases of the skin, such as psoriasis,
squamous cell carcinoma, keratoacanthoma and actinic keratosis are
characterized by localized abnormal proliferation and growth. For
example, in psoriasis, which is characterized by scaly, red,
elevated plaques on the skin, the keratinocytes are known to
proliferate much more rapidly than normal and to differentiate less
completely.
[0600] In one embodiment, the preparations of the present invention
are suitable for the treatment of dermatological ailments linked to
keratinization disorders causing abnormal proliferation of skin
cells, which disorders may be marked by either inflammatory or
non-inflammatory components. To illustrate, therapeutic
preparations of a hedgehog antagonist, e.g., which promotes
quiescence or differentiation can be used to treat varying forms of
psoriasis, be they cutaneous, mucosal or ungual. Psoriasis, as
described above, is typically characterized by epidermal
keratinocytes that display marked proliferative activation and
differentiation along a "regenerative" pathway. Treatment with an
antiproliferative embodiment of the subject method can be used to
reverse the pathological epidermal activation and can provide a
basis for sustained remission of the disease.
[0601] A variety of other keratotic lesions are also candidates for
treatment with the subject method. Actinic keratoses, for example,
are superficial inflammatory premalignant tumors arising on
sun-exposed and irradiated skin. The lesions are erythematous to
brown with variable scaling. Current therapies include excisional
and cryosurgery. These treatments are painful, however, and often
produce cosmetically unacceptable scarring. Accordingly, treatment
of keratosis, such as actinic keratosis, can include application,
preferably topical, of a hedgehog antagonist composition in amounts
sufficient to inhibit hyperproliferation of epidermal/epidermoid
cells of the lesion.
[0602] Acne represents yet another dermatologic ailment which may
be treated by the subject method. Acne vulgaris, for instance, is a
multifactor disease most commonly occurring in teenagers and young
adults, and is characterized by the appearance of inflammatory and
noninflammatory lesions on the face and upper trunk. The basic
defect which gives rise to acne vulgaris is hypercornification of
the duct of a hyperactive sebaceous gland. Hypercornification
blocks the normal mobility of skin and follicle microorganisms, and
in so doing, stimulates the release of lipases by Propinobacterium
acnes and Staphylococcus epidermidis bacteria and Pitrosporum
ovale, a yeast. Treatment with an antiproliferative hedgehog
antagonist, particularly topical preparations, may be useful for
preventing the transitional features of the ducts, e.g.,
hypercornification, which lead to lesion formation. The subject
treatment may further include, for example, antibiotics, retinoids
and antiandrogens.
[0603] The present invention also provides a method for treating
various forms of dermatitis. Dermatitis is a descriptive term
referring to poorly demarcated lesions that are either pruritic,
erythematous, scaly, blistered, weeping, fissured or crusted. These
lesions arise from any of a wide variety of causes. The most common
types of dermatitis are atopic, contact and diaper dermatitis. For
instance, seborrheic dermatitis is a chronic, usually pruritic,
dermatitis with erythema, dry, moist, or greasy scaling, and
yellow-crusted patches on various areas, especially the scalp, with
exfoliation of an excessive amount of dry scales. The subject
method can also be used in the treatment of stasis dermatitis, an
often chronic, usually eczematous dermatitis. Actinic dermatitis is
dermatitis that due to exposure to actinic radiation such as that
from the sun, ultraviolet waves, or x- or gamma-radiation.
According to the present invention, the subject method can be used
in the treatment and/or prevention of certain symptoms of
dermatitis caused by unwanted proliferation of epithelial cells.
Such therapies for these various forms of dermatitis can also
include topical and systemic corticosteroids, antipruritics, and
antibiotics.
[0604] Ailments that may be treated by the subject method are
disorders specific to non-humans, such as mange.
[0605] In still another embodiment, the subject method can be used
in the treatment of human cancers, such as tumors of epithelial
tissues such as the skin. For example, hedgehog antagonists can be
employed in the subject method as part of a treatment for human
carcinomas, adenocarcinomas, sarcomas and the like.
[0606] In another aspect, the present invention provides
pharmaceutical preparations comprising hedgehog antagonists. The
hedgehog antagonists for use in the subject method may be
conveniently formulated for administration with a biologically
acceptable medium, such as water, buffered saline, polyol (for
example, glycerol, propylene glycol, liquid polyethylene glycol and
the like) or suitable mixtures thereof. The optimum concentration
of the active ingredient(s) in the chosen medium can be determined
empirically, according to procedures well known to medicinal
chemists. As used herein, "biologically acceptable medium" includes
any and all solvents, dispersion media, and the like which may be
appropriate for the desired route of administration of the
pharmaceutical preparation. The use of such media for
pharmaceutically active substances is known in the art. Except
insofar as any conventional media or agent is incompatible with the
activity of the hedgehog antagonist, its use in the pharmaceutical
preparation of the invention is contemplated. Suitable vehicles and
their formulation inclusive of other proteins are described, for
example, in the book Remington's Pharmaceutical Sciences
(Remington's Pharmaceutical Sciences. Mack Publishing Company,
Easton, Pa., USA 1985). These vehicles include injectable "deposit
formulations".
[0607] Pharmaceutical formulations of the present invention can
also include veterinary compositions, e.g., pharmaceutical
preparations of the hedgehog antagonists suitable for veterinary
uses, e.g., for the treatment of livestock or domestic animals,
e.g., dogs.
[0608] Methods of introduction may also be provided by rechargeable
or biodegradable devices. Various slow release polymeric devices
have been developed and tested in vivo in recent years for the
controlled delivery of drugs, including proteinaceous
biopharmaceuticals. A variety of biocompatible polymers (including
hydrogels), including both biodegradable and non-degradable
polymers, can be used to form an implant for the sustained release
of a hedgehog antagonist at a particular target site.
[0609] The preparations of the present invention may be given
orally, parenterally, topically, or rectally. They are, of course,
given by forms suitable for each administration route. For example,
they are administered in tablets or capsule form, by injection,
inhalation, eye lotion, ointment, suppository, controlled release
patch, etc. administration by injection, infusion or inhalation;
topical by lotion or ointment; and rectal by suppositories. Oral
and topical administrations are preferred.
[0610] The phrases "parenteral administration" and "administered
parenterally" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticular, subcapsular,
subarachnoid, intraspinal and intrastemal injection and
infusion.
[0611] The phrases "systemic administration," "administered
systemically," "peripheral administration" and "administered
peripherally" as used herein mean the administration of a compound,
drug or other material other than directly into the central nervous
system, such that it enters the patient's system and, thus, is
subject to metabolism and other like processes, for example,
subcutaneous administration.
[0612] These compounds may be administered to humans and other
animals for therapy by any suitable route of administration,
including orally, nasally, as by, for example, a spray, rectally,
intravaginally, parenterally, intracisternally and topically, as by
powders, ointments or drops, including buccally and
sublingually.
[0613] Regardless of the route of administration selected, the
compounds of the present invention, which may be used in a suitable
hydrated form, and/or the pharmaceutical compositions of the
present invention, are formulated into pharmaceutically acceptable
dosage forms such as described below or by other conventional
methods known to those of skill in the art.
[0614] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of this invention may be varied so as
to obtain an amount of the active ingredient that is effective to
achieve the desired therapeutic response for a particular patient,
composition, and mode of administration, without being toxic to the
patient.
[0615] The selected dosage level will depend upon a variety of
factors including the activity of the particular compound of the
present invention employed, or the ester, salt or amide thereof,
the route of administration, the time of administration, the rate
of excretion of the particular compound being employed, the
duration of the treatment, other drugs, compounds and/or materials
used in combination with the particular hedgehog antagonist
employed, the age, sex, weight, condition, general health and prior
medical history of the patient being treated, and like factors well
known in the medical arts.
[0616] A physician or veterinarian having ordinary skill in the art
can readily determine and prescribe the effective amount of the
pharmaceutical composition required. For example, the physician or
veterinarian could start doses of the compounds of the invention
employed in the pharmaceutical composition at levels lower than
that required in order to achieve the desired therapeutic effect
and gradually increase the dosage until the desired effect is
achieved.
[0617] In general, a suitable daily dose of a compound of the
invention will be that amount of the compound that is the lowest
dose effective to produce a therapeutic effect. Such an effective
dose will generally depend upon the factors described above.
Generally, intravenous, intracerebroventricular and subcutaneous
doses of the compounds of this invention for a patient will range
from about 0.0001 to about 100 mg per kilogram of body weight per
day.
[0618] If desired, the effective daily dose of the active compound
may be administered as two, three, four, five, six or more
sub-doses administered separately at appropriate intervals
throughout the day, optionally, in unit dosage forms.
[0619] The term "treatment" is intended to encompass also
prophylaxis, therapy and cure.
[0620] The patient receiving this treatment is any animal in need,
including primates, in particular humans, and other mammals such as
equines, cattle, swine and sheep; and poultry and pets in
general.
[0621] The compound of the invention can be administered as such or
in admixtures with pharmaceutically acceptable and/or sterile
carriers and can also be administered in conjunction with other
antimicrobial agents such as penicillins, cephalosporins,
aminoglycosides and glycopeptides. Conjunctive therapy thus
includes sequential, simultaneous and separate administration of
the active compound in a way that the therapeutic effects of the
first administered one is not entirely disappeared when the
subsequent is administered.
[0622] V. Pharmacogenomics
[0623] The ability to rapidly assess gene expression in patients
promises to radically change the means by which a physician selects
an appropriate pharmaceutical for treating a particular disease.
Gene expression profiles of diseased tissue can be obtained and
therapeutic measures can be selected based on the gene expression
profile. This methodology is particularly effective when the
molecular mechanism of action for a given therapeutic is known. In
other words, if an anti-tumor agent acts by inhibiting a particular
oncoprotein, it is desirable to know whether a particular cancer
expresses that oncogene before attempting to treat the cancer with
the anti-tumor agent. As expression profiling becomes faster,
cheaper and more reliable, such information may become a routine
part of treatment selection, minimizing fruitless treatment
protocols and allowing the more rapid application of appropriate
therapeutics.
[0624] In addition, if a pool of patients suffering from a certain
type of disorder can be segregated into subgroups based on gene
expression profiles, drugs can be re-tested for their ability to
affect these defined subgroups of patients. Thus drugs that
appeared useless in the patient group as a whole may now be found
to be useful for patient subgroups. This type of screening may
allow the resurrection of failed compounds, the identification of
new compounds and the identification of new uses for well-known
compounds.
[0625] The expression of a particular gene can be assessed in many
ways. The level of gene transcript or the level of encoded protein
may be determined. The presence of a protein may be determined
directly, through methods such as antibody binding, mass
spectroscopy and two-dimensional gel electrophoresis, or
indirectly, by detecting an activity of the protein, be it a
biochemical activity or an effect on the levels of another protein
or expression of one or more genes.
[0626] Methods for measuring levels of gene transcripts are well
known in the art and depend for the most part on hybridization of a
single stranded probe to the transcript in question (or a cDNA
thereof). Such methods include Northern blotting, using a labeled
probe, or PCR amplification of the cDNA (also known as RT-PCR).
mRNAs and cDNAs may be labeled according to various methods and
hybridized to an oligonucleotide array. Such arrays may contain
ordered probes corresponding to one or more genes, and in preferred
embodiments, the array contains probes corresponding to all the
genes in the genome of the organism from which the RNA was
obtained.
[0627] A number of methodologies are currently used for the
measurement of gene expression. The most sensitive of these
methodologies utilizes the polymerase chain reaction (PCR)
technique, the details of which are provided in U.S. Pat. No.
4,683,195, U.S. Pat. No. 4,683,202, and U.S. Pat. No. 4,965,188,
all to Mullis et al., all of which are specifically incorporated
herein by reference. The details of PCR technology, thus, are not
included herein. Recently, additional technologies for the
amplification of nucleic acids have been described, most of which
are based upon isothermal amplification strategies as opposed to
the temperature cycling required for PCR. These strategies include,
for example, Strand Displacement Amplification (SDA)(U.S. Pat. Nos.
5,455,166 and 5,457,027 both to Walker; Walker et al. (1992) PNAS
89:392; each of which is specifically incorporated herein by
reference) and Nucleic Acid Sequence-Based Amplification
(NASBA)(U.S. Pat. No. 5,130,238 to Malek et al.; European Patent
525882 to Kievits et al.; both specifically incorporated herein by
reference). Each of these amplification technologies are similar in
that they employ the use of short, deoxyribonucleic acid primers to
define the region of amplification, regardless of the enzymes or
specific conditions used.
[0628] Until recently, RNA amplification required a separate,
additional step and the use of non-thermostable reverse
transcriptase enzymes to generate a cDNA capable of being amplified
by a thermostable DNA polymerase, such as Taq. The discovery of a
recombinant thermostable enzyme (rTth) capable of coupling reverse
transcription of the RNA with DNA amplification in a single
enzyme:single reaction procedure greatly simplified and enhanced
RNA amplification (see, Myers & Gelfand (1991) Biochemistry
30:7661-7666; U.S. Pat. No. 5,407,800 to Gelfand and Myers, both
incorporated herein by reference).
[0629] In gene expression analysis with microarrays, an array of
"probe" oligonucleotides is contacted with a nucleic acid sample of
interest, i.e., target, such as polyA mRNA from a particular tissue
type. Contact is carried out under hybridization conditions and
unbound nucleic acid is then removed. The resultant pattern of
hybridized nucleic acid provides information regarding the genetic
profile of the sample tested. Gene expression analysis finds use in
a variety of applications, including: the identification of novel
expression of genes, the correlation of gene expression to a
particular phenotype, screening for disease predisposition,
identifying the effect of a particular agent on cellular gene
expression, such as in toxicity testing; among other applications.
Detailed methods for analyzing transcript levels are described in
the following patents: U.S. Pat. No. 5,082,830 and WO 97/27317.
[0630] Other references of interest include: Schena et al., Science
(1995) 467-470; Schena et al., P.N.A.S. U.S.A. (1996) 93:
10614-10616; Pietu et al., Genome Res. (June 1996) 6: 492-503; Zhao
et al., Gene (Apr. 24, 1995) 156: 207-213; Soares, Curr. Opin.
Biotechnol. (October 1997) 8: 542-546; Raval, J. Pharmacol Toxicol
Methods (November 1994) 32: 125-127; Chalifour et al., Anal.
Biochem (Feb. 1, 1994) 216: 299-304; Stolz & Tuan, Mol.
Biotechnol. (December 19960 6: 225-230; Hong et al., Bioscience
Reports (1982) 2: 907; and McGraw, Anal. Biochem. (1984)143:
298.
[0631] VI. Pharmaceutical Compositions
[0632] While it is possible for a compound of the present invention
to be administered alone, it is preferable to administer the
compound as a pharmaceutical formulation (composition). The
hedgehog antagonists according to the invention may be formulated
for administration in any convenient way for use in human or
veterinary medicine. In certain embodiments, the compound included
in the pharmaceutical preparation may be active itself, or may be a
prodrug, e.g., capable of being converted to an active compound in
a physiological setting.
[0633] Thus, another aspect of the present invention provides
pharmaceutically acceptable compositions comprising a
therapeutically effective amount of one or more of the compounds
described above, formulated together with one or more
pharmaceutically acceptable carriers (additives) and/or diluents.
As described in detail below, the pharmaceutical compositions of
the present invention may be specially formulated for
administration in solid or liquid form, including those adapted for
the following: (1) oral administration, for example, drenches
(aqueous or non-aqueous solutions or suspensions), tablets,
boluses, powders, granules, pastes for application to the tongue;
(2) parenteral administration, for example, by subcutaneous,
intramuscular or intravenous injection as, for example, a sterile
solution or suspension; (3) topical application, for example, as a
cream, ointment or spray applied to the skin; or (4) intravaginally
or intrarectally, for example, as a pessary, cream or foam.
However, in certain embodiments the subject compounds may be simply
dissolved or suspended in sterile water. In certain embodiments,
the pharmaceutical preparation is non-pyrogenic, i.e., does not
elevate the body temperature of a patient.
[0634] The phrase "therapeutically effective amount" as used herein
means that amount of a compound, material, or composition
comprising a compound of the present invention which is effective
for producing some desired therapeutic effect by overcoming a
hedgehog gain-of-function phenotype in at least a sub-population of
cells in an animal and thereby blocking the biological consequences
of that pathway in the treated cells, at a reasonable benefit/risk
ratio applicable to any medical treatment.
[0635] The phrase "pharmaceutically acceptable" is employed herein
to refer to those compounds, materials, compositions, and/or dosage
forms which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of human beings and
animals without excessive toxicity, irritation, allergic response,
or other problem or complication, commensurate with a reasonable
benefit/risk ratio.
[0636] The phrase "pharmaceutically acceptable carrier" as used
herein means a pharmaceutically acceptable material, composition or
vehicle, such as a liquid or solid filler, diluent, excipient,
solvent or encapsulating material, involved in carrying or
transporting the subject antagonists from one organ, or portion of
the body, to another organ, or portion of the body. Each carrier
must be "acceptable" in the sense of being compatible with the
other ingredients of the formulation and not injurious to the
patient. Some examples of materials which can serve as
pharmaceutically acceptable carriers include: (1) sugars, such as
lactose, glucose and sucrose; (2) starches, such as corn starch and
potato starch; (3) cellulose, and its derivatives, such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)
powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8)
excipients, such as cocoa butter and suppository waxes; (9) oils,
such as peanut oil, cottonseed oil, safflower oil, sesame oil,
olive oil, corn oil and soybean oil; (10) glycols, such as
propylene glycol; (11) polyols, such as glycerin, sorbitol,
mannitol and polyethylene glycol; (12) esters, such as ethyl oleate
and ethyl laurate; (13) agar; (14) buffering agents, such as
magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)
pyrogen-free water; (17) isotonic saline; (18) Ringer's solution;
(19). ethyl alcohol; (20) phosphate buffer solutions; and (21)
other non-toxic compatible substances employed in pharmaceutical
formulations.
[0637] As set out above, certain embodiments of the present
hedgehog antagonists may contain a basic functional group, such as
amino or alkylamino, and are, thus, capable of forming
pharmaceutically acceptable salts with pharmaceutically acceptable
acids. The term "pharmaceutically acceptable salts" in this
respect, refers to the relatively non-toxic, inorganic and organic
acid addition salts of compounds of the present invention. These
salts can be prepared in situ during the final isolation and
purification of the compounds of the invention, or by separately
reacting a purified compound of the invention in its free base form
with a suitable organic or inorganic acid, and isolating the salt
thus formed. Representative salts include the hydrobromide,
hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate,
valerate, oleate, palmitate, stearate, laurate, benzoate, lactate,
phosphate, tosylate, citrate, maleate, fumarate, succinate,
tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, and
laurylsulphonate salts and the like. (See, for example, Berge et
al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:1-19)
[0638] The pharmaceutically acceptable salts of the subject
compounds include the conventional nontoxic salts or quaternary
ammonium salts of the compounds, e.g., from non-toxic organic or
inorganic acids. For example, such conventional nontoxic salts
include those derived from inorganic acids such as hydrochloride,
hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like;
and the salts prepared from organic acids such as acetic,
propionic, succinic, glycolic, stearic, lactic, malic, tartaric,
citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic,
glutamic, benzoic, salicyclic, sulfanilic, 2-acetoxybenzoic,
fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic,
oxalic, isothionic, and the like.
[0639] In other cases, the compounds of the present invention may
contain one or more acidic functional groups and, thus, are capable
of forming pharmaceutically acceptable salts with pharmaceutically
acceptable bases. The term "pharmaceutically acceptable salts" in
these instances refers to the relatively non-toxic, inorganic and
organic base addition salts of compounds of the present invention.
These salts can likewise be prepared in situ during the final
isolation and purification of the compounds, or by separately
reacting the purified compound in its free acid form with a
suitable base, such as the hydroxide, carbonate or bicarbonate of a
pharmaceutically acceptable metal cation, with ammonia, or with a
pharmaceutically acceptable organic primary, secondary or tertiary
amine. Representative alkali or alkaline earth salts include the
lithium, sodium, potassium, calcium, magnesium, and aluminum salts
and the like. Representative organic amines useful for the
formation of base addition salts include ethylamine, diethylamine,
ethylenediamine, ethanolamine, diethanolamine, piperazine and the
like. (See, for example, Berge et al., supra)
[0640] Wetting agents, emulsifiers and lubricants, such as sodium
lauryl sulfate and magnesium stearate, as well as coloring agents,
release agents, coating agents, sweetening, flavoring and perfuming
agents, preservatives and antioxidants can also be present in the
compositions.
[0641] Examples of pharmaceutically acceptable antioxidants
include: (1) water soluble antioxidants, such as ascorbic acid,
cysteine hydrochloride, sodium bisulfate, sodium metabisulfite,
sodium sulfite and the like; (2) oil-soluble antioxidants, such as
ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol,
and the like; and (3) metal chelating agents, such as citric acid,
ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,
phosphoric acid, and the like.
[0642] Formulations of the present invention include those suitable
for oral, nasal, topical (including buccal and sublingual), rectal,
vaginal and/or parenteral administration. The formulations may
conveniently be presented in unit dosage form and may be prepared
by any methods well known in the art of pharmacy. The amount of
active ingredient which can be combined with a carrier material to
produce a single dosage form will vary depending upon the host
being treated, the particular mode of administration. The amount of
active ingredient that can be combined with a carrier material to
produce a single dosage form will generally be that amount of the
compound that produces a therapeutic effect. Generally, out of one
hundred per cent, this amount will range from about 1 per cent to
about ninety-nine percent of active ingredient, preferably from
about 5 per cent to about 70 per cent, most preferably from about
10 per cent to about 30 per cent.
[0643] Methods of preparing these formulations or compositions
include the step of bringing into association a compound of the
present invention with the carrier and, optionally, one or more
accessory ingredients. In general, the formulations are prepared by
uniformly and intimately bringing into association a compound of
the present invention with liquid carriers, or finely divided solid
carriers, or both, and then, if necessary, shaping the product.
[0644] Formulations of the invention suitable for oral
administration may be in the form of capsules, cachets, pills,
tablets, lozenges (using a flavored basis, usually sucrose and
acacia or tragacanth), powders, granules, or as a solution or a
suspension in an aqueous or non-aqueous liquid, or as an
oil-in-water or water-in-oil liquid emulsion, or as an elixir or
syrup, or as pastilles (using an inert base, such as gelatin and
glycerin, or sucrose and acacia) and/or as mouth washes and the
like, each containing a predetermined amount of a compound of the
present invention as an active ingredient. A compound of the
present invention may also be administered as a bolus, electuary or
paste.
[0645] In solid dosage forms of the invention for oral
administration (capsules, tablets, pills, dragees, powders,
granules and the like), the active ingredient is mixed with one or
more pharmaceutically acceptable carriers, such as sodium citrate
or dicalcium phosphate, and/or any of the following: (1) fillers or
extenders, such as starches, lactose, sucrose, glucose, mannitol,
and/or silicic acid; (2) binders, such as, for example,
carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,
sucrose and/or acacia; (3) humectants, such as glycerol; (4)
disintegrating agents, such as agar-agar, calcium carbonate, potato
or tapioca starch, alginic acid, certain silicates, and sodium
carbonate; (5) solution retarding agents, such as paraffin; (6)
absorption accelerators, such as quaternary ammonium compounds; (7)
wetting agents, such as, for example, cetyl alcohol and glycerol
monostearate; (8) absorbents, such as kaolin and bentonite clay;
(9) lubricants, such a talc, calcium stearate, magnesium stearate,
solid polyethylene glycols, sodium lauryl sulfate, and mixtures
thereof; and (10) coloring agents. In the case of capsules, tablets
and pills, the pharmaceutical compositions may also comprise
buffering agents. Solid compositions of a similar type may also be
employed as fillers in soft and hard-filled gelatin capsules using
such excipients as lactose or milk sugars, as well as high
molecular weight polyethylene glycols and the like.
[0646] A tablet may be made by compression or molding, optionally
with one or more accessory ingredients. Compressed tablets may be
prepared using binder (for example, gelatin or hydroxypropylmethyl
cellulose), lubricant, inert diluent, preservative, disintegrant
(for example, sodium starch glycolate or cross-linked sodium
carboxymethyl cellulose), surface-active or dispersing agent.
Molded tablets may be made by molding in a suitable machine a
mixture of the powdered compound moistened with an inert liquid
diluent.
[0647] The tablets, and other solid dosage forms of the
pharmaceutical compositions of the present invention, such as
dragees, capsules, pills and granules, may optionally be scored or
prepared with coatings and shells, such as enteric coatings and
other coatings well known in the pharmaceutical-formulating art.
They may also be formulated so as to provide slow or controlled
release of the active ingredient therein using, for example,
hydroxypropylmethyl cellulose in varying proportions to provide the
desired release profile, other polymer matrices, liposomes and/or
microspheres. They may be sterilized by, for example, filtration
through a bacteria-retaining filter, or by incorporating
sterilizing agents in the form of sterile solid compositions that
can be dissolved in sterile water, or some other sterile injectable
medium immediately before use. These compositions may also
optionally contain opacifying agents and may be of a composition
that they release the active ingredient(s) only, or preferentially,
in a certain portion of the gastrointestinal tract, optionally, in
a delayed manner. Examples of embedding compositions that can be
used include polymeric substances and waxes. The active ingredient
can also be in micro-encapsulated form, if appropriate, with one or
more of the above-described excipients.
[0648] Liquid dosage forms for oral administration of the compounds
of the invention include pharmaceutically acceptable emulsions,
microemulsions, solutions, suspensions, syrups and elixirs. In
addition to the active ingredient, the liquid dosage forms may
contain inert diluents commonly used in the art, such as, for
example, water or other solvents, solubilizing agents and
emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, oils (in particular,
cottonseed, groundnut, corn, germ, olive, castor and sesame oils),
glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty
acid esters of sorbitan, and mixtures thereof.
[0649] Besides inert diluents, the oral compositions can also
include adjuvants such as wetting agents, emulsifying and
suspending agents, sweetening, flavoring, coloring, perfuming and
preservative agents.
[0650] Suspensions, in addition to the active compounds, may
contain suspending agents as, for example, ethoxylated isostearyl
alcohols, polyoxyethylene sorbitol and sorbitan esters,
microcrystalline cellulose, aluminum metahydroxide, bentonite,
agar-agar and tragacanth, and mixtures thereof.
[0651] It is known that sterols, such as cholesterol, will form
complexes with cyclodextrins. Thus, in preferred embodiments, where
the inhibitor is a steroidal alkaloid, it may be formulated with
cyclodextrins, such as .alpha.-, .beta.- and .gamma.-cyclodextrin,
dimethyl-.beta.cyclodextrin and
2-hydroxypropyl-.beta.-cyclodextrin.
[0652] Formulations of the pharmaceutical compositions of the
invention for rectal or vaginal administration may be presented as
a suppository, which may be prepared by mixing one or more
compounds of the invention with one or more suitable nonirritating
excipients or carriers comprising, for example, cocoa butter,
polyethylene glycol, a suppository wax or a salicylate, and which
is solid at room temperature, but liquid at body temperature and,
therefore, will melt in the rectum or vaginal cavity and release
the active hedgehog antagonist.
[0653] Formulations of the present invention which are suitable for
vaginal administration also include pessaries, tampons, creams,
gels, pastes, foams or spray formulations containing such carriers
as are known in the art to be appropriate.
[0654] Dosage forms for the topical or transdermal administration
of a compound of this invention include powders, sprays, ointments,
pastes, creams, lotions, gels, solutions, patches and inhalants.
The active compound may be mixed under sterile conditions with a
pharmaceutically acceptable carrier, and with any preservatives,
buffers, or propellants that may be required.
[0655] The ointments, pastes, creams and gels may contain, in
addition to an active compound of this invention, excipients, such
as animal and vegetable fats, oils, waxes, paraffins, starch,
tragaeanth, cellulose derivatives, polyethylene glycols, silicones,
bentonites, silicic acid, talc and zinc oxide, or mixtures
thereof.
[0656] Powders and sprays can contain, in addition to a compound of
this invention, excipients such as lactose, talc, silicic acid,
aluminum hydroxide, calcium silicates and polyamide powder, or
mixtures of these substances. Sprays can additionally contain
customary propellants, such as chlorofluorohydrocarbons and
volatile unsubstituted hydrocarbons, such as butane and
propane.
[0657] Transdermal patches have the added advantage of providing
controlled delivery of a compound of the present invention to the
body. Such dosage forms can be made by dissolving or dispersing the
hedgehog antagonists in the proper medium. Absorption enhancers can
also be used to increase the flux of the hedgehog antagonists
across the skin. The rate of such flux can be controlled by either
providing a rate controlling membrane or dispersing the compound in
a polymer matrix or gel.
[0658] Ophthalmic formulations, eye ointments, powders, solutions
and the like, are also contemplated as being within the scope of
this invention.
[0659] Pharmaceutical compositions of this invention suitable for
parenteral administration comprise one or more compounds of the
invention in combination with one or more pharmaceutically
acceptable sterile isotonic aqueous or nonaqueous solutions,
dispersions, suspensions or emulsions, or sterile powders which may
be reconstituted into sterile injectable solutions or dispersions
just prior to use, which may contain antioxidants, buffers,
bacteriostats, solutes which render the formulation isotonic with
the blood of the intended recipient or suspending or thickening
agents.
[0660] Examples of suitable aqueous and nonaqueous carriers that
may be employed in the pharmaceutical compositions of the invention
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity can be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0661] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of the action of microorganisms may be ensured
by the inclusion of various antibacterial and antifungal agents,
for example, paraben, chlorobutanol, phenol sorbic acid, and the
like. It may also be desirable to include isotonic agents, such as
sugars, sodium chloride, and the like into the compositions. In
addition, prolonged absorption of the injectable pharmaceutical
form may be brought about by the inclusion of agents that delay
absorption such as aluminum monostearate and gelatin.
[0662] In some cases, in order to prolong the effect of a drug, it
is desirable to slow the absorption of the drug from subcutaneous
or intramuscular injection. This may be accomplished by the use of
a liquid suspension of crystalline or amorphous material having
poor water solubility. The rate of absorption of the drug then
depends upon its rate of dissolution, which, in turn, may depend
upon crystal size and crystalline form. Alternatively, delayed
absorption of a parenterally administered drug form is accomplished
by dissolving or suspending the drug in an oil vehicle.
[0663] Injectable depot forms are made by forming microencapsule
matrices of the subject compounds in biodegradable polymers such as
polylactide-polyglycolide. Depending on the ratio of drug to
polymer, and the nature of the particular polymer employed, the
rate of drug release can be controlled. Examples of other
biodegradable polymers include poly(orthoesters) and
poly(anhydrides). Depot injectable formulations are also prepared
by entrapping the drug in liposomes or microemulsions that are
compatible with body tissue.
[0664] When the compounds of the present invention are administered
as pharmaceuticals, to humans and animals, they can be given per se
or as a pharmaceutical composition containing, for example, 0.1 to
99.5% (more preferably, 0.5 to 90%) of active ingredient in
combination with a pharmaceutically acceptable carrier.
[0665] The addition of the active compound of the invention to
animal feed is preferably accomplished by preparing an appropriate
feed premix containing the active compound in an effective amount
and incorporating the premix into the complete ration.
[0666] Alternatively, an intermediate concentrate or feed
supplement containing the active ingredient can be blended into the
feed. The way in which such feed premixes and complete rations can
be prepared and administered are described in reference books (such
as "Applied Animal Nutrition", W.H. Freedman and CO., San
Francisco, U.S.A., 1969 or "Livestock Feeds and Feeding" O and B
books, Corvallis, Oreg., U.S.A., 1977).
EXAMPLES
[0667] The invention now being generally described, it will be more
readily understood by reference to the following examples, which
are included merely for purposes of illustration of certain aspects
and embodiments of the present invention, and are not intended to
limit the invention.
Example 1
Hedgehog, Lung Development and Surfactant Production
[0668] Respiratory distress syndrome results from insufficient
surfactant in the alveolae of the lungs. The lungs of vertebrates
contain surfactant, a complex mixture of lipids and protein that
causes surface tension to rise during lung inflation and decrease
during lung deflation. During lung deflation, surfactant decreases
such that there are no surface forces that would otherwise promote
alveolar collapse. Aerated alveoli that have not collapsed during
expiration permit continuous oxygen and carbon dioxide transport
between blood and alveolar gas and require much less force to
inflate during the subsequent inspiration. During inflation, lung
surfactant increases surface tension as the alveolar surface area
increases. A rising surface tension in expanding alveoli opposes
over-inflation in those airspaces and tends to divert inspired air
to less well-aerated alveoli, thereby facilitating even lung
aeration.
[0669] Respiratory distress syndrome is particularly prevalent
among premature infants. Lung surfactant is normally synthesized at
a very low rate until the last six weeks of fetal life. Human
infants born more than six weeks before the normal term of a
pregnancy have a high risk of being born with inadequate amounts of
lung surfactant and inadequate rates of surfactant synthesis. The
more prematurely an infant is born, the more severe the surfactant
deficiency is likely to be. Severe surfactant deficiency can lead
to respiratory failure within a few minutes or hours of birth. The
surfactant deficiency produces progressive collapse of alveoli
(atelectasis) because of the decreasing ability of the lung to
expand despite maximum inspiratory effort. As a result, inadequate
amounts of oxygen reach the infant's blood. RDS can occur in adults
as well, typically as a consequence of failure in surfactant
biosynthesis.
[0670] The role of the hedgehog signaling pathway in lung
maturation and surfactant production was investigated, with the
finding that inhibition of the hedgehog signaling pathway
stimulated surfactant production.
[0671] The expression of a hedgehog-regulated gene, Gli-1, was
assessed in embryonic mouse lung tissue. Gli-1 was strongly
expressed in the embryonic lung, however this expression decreases
during lung maturation (FIG. 4). Note that the decline in hedgehog
signaling towards the end of embryogenesis correlates with the
maturation of the distal lung epithelium into respiratory
pneumocytes. Gli-1, a transcription factor indicative of hedgehog
signaling, continues to be expressed in the conducting, but not
respiratory airways in the adult.
[0672] METHODS: Sections of paraformaldehyde-fixed,
paraffin-embedded tissue were cleared, re-hydrated, digested with
proteinase K, acetylated and hybridized with [33P]-labeled sonic
hedgehog and gli-1 RNA probes over night, respectively. After high
stringency post-hybridization washes, slides were dipped in
photo-emulsion, incubated for up to three weeks, developed, and
imaged using dark field illumination. Dark-field signals were
filled in with artificial color (red) and superimposed with
bright-field images.
[0673] To further correlate the decrease in gli-1 expression with
lung maturation, expression of gli-1 was compared to expression of
the lung maturation marker, surfactant type C (Sp-C) (FIG. 5). This
analysis demonstrates that as expression of gli-1 decreases between
E13.5-E16.5, the expression of Sp-C increases.
[0674] METHODS: E13.5 and E16.5 mouse lung explants were dissected
and analyzed by Quantatative Real-Time PCR (Q-RT-PCR). Briefly,
total ribonucleic acid (RNA) is isolated from the tissue and
subjected to reverse transcription to generate DNA. This DNA is
amplified in a polymerase chain reaction using gene-specific
primers as well as primers for the ubiquitously expressed
housekeeping gene GAPDH. The two primer sets are labeled with
different fluorophores, allowing for quantification of both signals
in the same reaction tube in a real-time PCR machine (TaqMan). When
calculating the expression levels of gli-1 and Sp-C, the specific
signal is normalized to the GAPDH signal, which serves as a measure
of the total DNA used in the reaction.
[0675] As Gli-1 expression is a marker for hedgehog signaling, it
appears that the hedgehog signaling pathway is active in immature
lung tissue. Accordingly, it was hypothesized that inhibition of
the hedgehog signaling pathway would permit more rapid lung
maturation and, particularly, stimulate surfactant production.
[0676] Treatment of embryonic mouse lungs with hedgehog antagonist
compound B downregulates Gli-1 expression (FIG. 6). METHODS: E13.5
embryonic mouse lungs were dissected. Explants were grown exposed
to the air-liquid interface in lung explant medium (DMEM based,
additives optimized for the culture of mouse lungs) for 67 hrs.
They were then processed for quantitative real-time PCR (Q-RT-PCR).
Briefly, total ribonucleic acid (RNA) is isolated from the tissue
and subjected to reverse transcription to generate DNA. This DNA is
amplified in a polymerase chain reaction using gene-specific
primers as well as primers for the ubiquitously expressed
housekeeping gene GAPDH. The two primer sets are labeled with
different fluorophores, allowing for quantification of both signals
in the same reaction tube in a real-time PCR machine (TaqMan). When
calculating the expression level of gli-1, the specific signal is
normalized to the GAPDH signal, which serves as a measure of the
total DNA used in the reaction.
[0677] Compound B treatment increases surfactant type C production
in embryonic mouse lungs (FIG. 7). Surfactant production is a
measure of lung maturity, and the inability to produce surfactant
is the primary cause of adult and infant respiratory distress
syndrome. The increase in surfactant type C production was assessed
by measuring expression of Sp-C, which encodes a protein critical
for the production of surfactant.
[0678] METHODS: E13.5 old embryonic mouse lungs were dissected.
Explants were grown submerged in lung explant medium (DMEM based,
additives optimized for the culture of mouse lungs) for 50 hrs.
They were then processed for Q-RT-PCR. Briefly, total ribonucleic
acid (RNA) is isolated from the tissue and subjected to reverse
transcription to generate DNA. This DNA is amplified in a
polymerase chain reaction using gene-specific primers as well as
primers for the ubiquitously expressed housekeeping gene GAPDH. The
two primer sets are labeled with different fluorophores, allowing
for quantification of both signals in the same reaction tube in a
real-time PCR machine (TaqMan). When calculating the expression
level of Sp-C, the specific signal is normalized to the GAPDH
signal, which serves as a measure of the total DNA used in the
reaction.
[0679] Lamellated bodies are subcellular structures found in
surfactin-producing lung cells and are thought to be a site of
surfactin production. Type II pneumocytes in compound B-treated
lungs differentiate prematurely, as evidenced by the presence of
surfactant producing lamellated bodies. No such structures could be
observed in the vehicle-treated controls (FIG. 8).
[0680] METHODS: E13.5 old embryonic mouse lungs were dissected.
Explants were grown exposed to the air-liquid interface in lung
explant medium (DMEM based, additives optimized for the culture of
mouse lungs) for 67 hrs. They were then processed for transmission
electron microscopy and photographed at a magnification of
62,000.
[0681] FIGS. 9 and 10 show similar results as obtained above upon
treatment of embryonic lung cultures with Compound B (FIGS. 9-10).
The increase in Sp-C expression observed following Compound B
treatment is comparable to that observed when embryonic lung
explants are treated with the steroid hormone hydrocortisone.
Steroids are known to increase lung maturation and surfactant
production in animals, including humans.
[0682] The specificity of the effects of hedgehog antagonists on
lung maturation is demonstrated by examining the effects of
agonists of hedgehog signaling on lung maturation. Treatment of
embryonic lung cultures with either a lipid modified sonic hedgehog
or with a hedgehog agonist compound result in increased expression
of gli-1 and decreased expression of Sp-C (FIG. 11).
[0683] In summary, these results demonstrate that hedgehog
inhibitors can stimulate maturation and surfactin production in
immature lung tissue. The hedgehog signaling pathway is active in
immature lung tissues, where surfactins are not produced in
substantial levels, while the hedgehog pathway is relatively
inactive in the adult respiratory airway, where surfactins are
produced. Treatment of immature lung tissue with antagonists of the
hedgehog signaling pathway causes rapid maturation and the
increased presence of molecular and cytological markers associated
with surfactin production. Opposite results obtained upon the
treatment of lung explants with hedgehog antagonists and agonists
demonstrate the specificity of these results.
Example 2
Gli-1 Expression in Human Tumors
[0684] Hedgehog Pathway Activation in Human Tumors
[0685] Hedgehog signaling plays a causative role in the generation
of basal cell carcinoma (BCC). Hedgehog signaling was analyzed to
determine whether this pathway is active in other human tumors,
more specifically prostate, lung and breast cancer, as well as
benign prostate hyperplasia. Hedgehog proteins are known
proliferative agents for a variety of cell types. Since hedgehogs
have a known proliferative effect on a variety of cell types,
hedgehog antagonists may be valuable therapeutics for cancers in
which high level hedgehog signaling is present.
[0686] The question of hedgehog activation in the tumor types was
addressed by conducting radioactive in situ hybridization
experiments with gli-1, a known transcriptional effector gene of
hedgehog signaling.
[0687] Briefly, sections of paraformaldehyde-fixed,
paraffin-embedded tissue were cleared, re-hydrated, digested with
proteinase K, acetylated and hybridized with [33P]-labeled RNA
probes over night. After high stringency post-hybridization washes,
slides were dipped in photo-emulsion, incubated for up to three
weeks, developed, and imaged using dark field illumination.
Dark-field signals were filled in with artificial color (red) and
superimposed with bright-field images. Gli-1 expression was graded
on a scale from "-" to "+" through "++++". Gli-1 expression was
rated "-" when expression was no higher in hyperproliferative cells
than in other non-proliferative cells present in the slide. Ratings
of "+" through "++++" were given for increased expression levels,
with any cell rated "++" or above considered to have substantially
increased gli-1 expression. When the signal was not interpretable,
a sample is indicated as "ND".
[0688] The data for these experiments are summarized in table 1-4
below. In brief, 8 out of 18 breast cancer samples showed
substantially increased gli-1 expression. 7 out of 11 lung cancer
samples, 11 of 19 benign prostatic hypertrophy samples (13PH), and
6 of 15 prostate cancer samples all showed strong gli-1
expression.
3TABLE 1 Results of Gli-1 in situ hybridization in breast cancer
tissue Sample Tissue Diagnosis Number Age/Sex Signal Breast Inf
Ductal Carcinoma 1 93 F ND Breast Inf Ductal Carcinoma 2 37 F +++
Breast Inf Ductal Carcinoma 3 54 F + Breast Inf Ductal Carcinoma 4
39 F ++ Breast Inf Ductal Carcinoma 5 73 F +++ Breast Inf Ductal
Carcinoma 6 65 F ++++ Breast Inf Ductal Carcinoma 7 58 F ND Breast
Inf Ductal Carcinoma 8 48 F + Breast Inf Ductal Carcinoma 9 27 F ++
Breast Inf Ductal Carcinoma 10 NA +++ Breast Inf Ductal Carcinoma
11 34 F + Breast Inf Lobular Carcinoma 12 46 F + Breast Inf Lobular
Carcinoma 13 F - Breast Inf Lobular Carcinoma 14 56 F + Breast Inf
Lobular Carcinoma 15 70 F - Breast Intraductal Carcinoma 16 40 F
+++ Breast Intraductal Carcinoma 17 55 F +++ Breast Medullary
Carcinoma 18 NA + Breast Tubular Carcinoma 19 75 F - Breast Tubular
Carcinoma 20 60 F -
[0689]
4TABLE 2 Results of Gli-1 in situ hybridization in lung cancer
tissue Sample Tissue Diagnosis Number Age/Sex Signal Lung
Adenocarcinoma 1 54 F +++++ Lung Adenocarcinoma 2 61 M ND Lung
Adenocarcinoma 3 61 F ++++ Lung Adenocarcinoma 4 58 F +++ Lung
Adenocarcinoma 5 77 M ND Lung Adenocarcinoma 6 65 M ++ Lung
Adenocarcinoma 7 73 M ND Lung Adenocarcinoma 8 69 M ND Lung
Adenocarcinoma 9 82 M ND Lung Adenocarcinoma 10 NA - Lung
Adenocarcinoma 11 F ND Lung Adenocarcinoma 12 56 F + Lung
Broncho-alveolar adenocar 13 70 F + Lung Broncho-alveolar adenocar
14 76 F - Lung Small Cell Carcinoma 15 68 M ++ Lung Small Cell
Carcinoma 16 61 M ND Lung Small Cell Carcinoma 17 70 M +++++ Lung
Small Cell Carcinoma 18 NA ND Lung SCC 19 60 F ND Lung SCC 20 63 M
+++++
[0690]
5TABLE 3 Results of Gli-1 in situ hybridization in benign prostate
hyperplasia Tissue Diagnosis Sample Number Age/Sex Signal Prostate
BPH 1 65 M + Prostate BPH 2 86 M ++++ Prostate BPH 3 53 M +
Prostate BPH 4 65 M ++++ Prostate BPH 5 68 M ++ Prostate BPH 6 70 M
++ Prostate BPH 7 54 M - Prostate BPH 8 M ++ Prostate BPH 9 69 M -
Prostate BPH 10 M - Prostate BPH 11 73 M +++ Prostate BPH 12 53 M
++++ Prostate BPH 13 84 M - Prostate BPH 14 67 M - Prostate BPH 15
66 M ++ Prostate BPH 16 69 M ++ Prostate BPH 17 72 M ++++ Prostate
BPH 18 M ++ Prostate BPH 19 60 M - Prostate BPH 20 60 M -
[0691]
6TABLE 4 Results of Gli-1 in situ hybridization in prostate cancer
tissue Tissue Diagnosis Sample Number Age/Sex Signal Prostate
Adenocarcinoma 1 79M + Prostate Adenocarcinoma 2 72M + Prostate BPH
next to 3 81M ND Adenocarcinoma Prostate Adenocarcinoma 4 79M ++
Prostate Adenocarcinoma 5 81M ND Prostate Adenocarcinoma 6 73M -
Prostate Adenocarcinoma 7 79M ++ Prostate Adenocarcinoma 8 M +++
Prostate Adenocarcinoma 9 69M ND Prostate Adenocarcinoma 10 53M +++
Prostate Adenocarcinoma 11 65M + Prostate Adenocarcinoma 12 60M ++
Prostate Adenocarcinoma 13 66M ND Prostate Adenocarcinoma 14 66M +
Prostate Adenocarcinoma 15 92M - Prostate Adenocarcinoma 16 80M -
Prostate Adenocarcinoma 17 78M ND Prostate Adenocarcinoma 18 85M -
Prostate Adenocarcinoma 19 78M - Prostate Adenocarcinoma 20 93M
+++
[0692] In summary, high level Gli-1 expression, i.e., hedgehog
signaling activation, can be observed in human prostate cancer and
benign prostatic hyperplasia, lung cancer and breast cancer (FIGS.
12-15). Hedgehog pathway activation in these tumor types has never
before been described. The presence of an exceptionally active
hedgehog pathway in these proliferating cells strongly suggests a
causal link between the hedgehog pathway and hyperproliferation in
these disorders. It is expected that hedgehog antagonists will be
effective as antiproliferative agents in these cancer types.
Example 3
Steroidal Hedgehog Antagonists
[0693] Studies were performed to determine the site in the hedgehog
signaling pathway at which cyclopamine (an alkaloid steroidal
hedgehog antagonist) operates, and therefore better understand the
spectrum of tumors caused by Shh pathway-activating lesions that
could potentially be treated with this compound. These studies are
presented in greater detail in U.S. patent application Beachy et
al. entitled "Hedgehog signaling pathways, compositions and uses
related thereto" filed Oct. 10, 2000, the contents of which are
herein incorporated by reference.
[0694] These studies involve the use of mouse embryonic fibroblasts
(MEFs) that were generated by trypsin digestion of E8.5 embryos
from patched (ptc) +/- matings. The mouse ptc gene was disrupted by
homologous recombination in which part of exon 1 and all of exon 2
were replaced with the bacterial lacZ gene (Goodrich et al, (1997)
Science 277:1109). As Ptc protein suppresses Shh signaling, a loss
of its function activates the Shh signaling pathway. Shh signaling,
through a cascade of events, is mediated by the Gli transcription
factors. One of the target genes of Shh signaling is ptc, through
Gli-binding sites in the ptc promoter region, and this serves as a
feedback mechanism for down regulation of signaling. Thus, in these
ptc -/- embryos, the Shh signaling pathway is activated in many
tissues, and the lacZ gene product .beta.-galactosidase is
expressed in all of those tissues as a report of pathway
activation.
[0695] These MEFs were obtained to determine whether cyclopamine
acts on Ptc or another component of the cascade to inhibit Shh
signaling. If the target of cyclopamine is Ptc, then one would
expect that when the Shh pathway is activated by the loss of ptc
function, it could no longer be inhibited by cyclopamine. The Shh
signaling pathway can be activated in these fibroblasts in cell
culture, and that the level of .beta.-galactosidase activity does
reflect the degree of pathway activation. The MEF line 23-4 is
heterozygous for ptc-lacZ, and thus contains one functional ptc
allele capable of maintaining a repressed state of the pathway, but
will express lacZ when the pathway is activated by addition of Shh
protein.
[0696] In contrast, the .beta.-galactosidase activity in MEFs
homozygous for ptc-lacZ, (cell line 23-1) is markedly elevated,
because in these cells the pathway is constitutively activated by
the loss of a functional ptc allele. When these cells are cultured
with cyclopamine, .beta.-galactosidase activity is decreased,
indicating that when the Shh signaling pathway is unregulated by
Ptc repression, it is still sensitive to cyclopamine inhibition.
The reduction of .beta.-galactosidase activity appears to result
from the specific inhibition of Shh signaling, rather than from
cell toxicity because enzymatic activity is normalized to whole
protein content of the sample. Also, the reduction of
.beta.-galactosidase activity can be obtained with exposure to
cyclopamine over a period of time that is shorter than the average
cell cycle, and so does not appear to be due solely to an
inhibition of cell proliferation.
[0697] A final indication that this represents specific inhibition
of Shh signaling is that it cannot be achieved with a
non-inhibitory, but structurally related compound tomatidine.
Example 4
Lead Compound Discover/High-throughput Screening Assay
[0698] The methodologies described herein can be used to identify a
wide assortment of small molecule hedgehog antagonists.
[0699] Compounds to be tested are dissolved in DMSO to a
concentration of 10 mM, and stored at -20.degree. C. To activate
the Hedgehog pathway in the assay cells, an octylated
(lipid-modified) form of the N-terminal fragment of the Sonic
Hedgehog protein (OCT-SHH) is used. This N-terminal SHH fragment is
produced bacterially.
[0700] Compounds may be tested in the "Gli-Luc" assay below, using
the cell line 10T(s12), wherein the cells contain a
Hedgehog-responsive reporter construct utilizing Luciferase as the
reporter gene. In this way, Hedgehog pathway signaling activity can
be measured via the Gli-Luc response.
[0701] 10t1/2(s12) cells are plated in a 96-well micro-titer plate
(MTP) at 20,000 cells/well in full medium [DMEM with 10% FBS]. Then
plates are placed in the incubator for incubation overnight (O/N),
at 37.degree. C. and 5% CO.sub.2. After 24 h, the medium is
replaced with Luciferase-assay medium (DMEM with 0.5% FBS).
Compounds are thawed and diluted in assay medium at starting
concentration of about 30 .mu.M.
[0702] Subsequently, 150 .mu.l of each 30 .mu.M sample is added to
the first wells (in triplicate). The MTP samples are then diluted
at 3-fold dilutions to a total of seven wells, ultimately resulting
in a regiment of seven dilutions in triplicate, for each compound.
Next, the protein ligand OCT-SHH is diluted in Luciferase-assay
medium and added to each well at a final concentration of 0.3
.mu.g/ml. Plates are then returned to the incubator for further
incubation O/N, at 37.degree. C. and 5% CO.sub.2. After about 24 h,
plates are removed from the incubator and the medium is
aspirated/discarded. Wells are washed once with assay buffer [PBS+1
mM Mg.sup.2+ and 1 mM Ca.sup.2+]. Then 50 .mu.l of assay buffer is
added to each well. The Luciferase assay reagent is prepared as
described by the vendor (LucLite kit from Packard), and 50 .mu.l is
added to each well. Plates are incubated at room temperature (RT)
for about 30 minutes after which the signals are read, again at RT,
on a Topcount (Packard).
[0703] The discovery of compounds that inhibit Shh-induced
Gli-transcription exemplifies the utility of the claims of this
patent. Activities for these compounds are presented in Table 1
below.
7 TABLE 1 Compound IC.sub.50 Compound IC.sub.50 31 <10 .mu.M 55
<5 .mu.M 32 <5 .mu.M 56 <10 .mu.M 34 <5 .mu.M 57 <10
.mu.M 11 <5 .mu.M 58 <5 .mu.M 36 <5 .mu.M 59 <5 .mu.M
38 <5 .mu.M 60 <5 .mu.M 39 <5 .mu.M 61 <1 .mu.M 40
<10 .mu.M 62 <1 .mu.M 41 <10 .mu.M 63 <10 .mu.M 42
<5 .mu.M 64 <10 .mu.M 43 <10 .mu.M 65 <10 .mu.M 44
<1 .mu.M 66 <10 .mu.M 45 <5 .mu.M 67 <5 .mu.M 46
<0.5 .mu.M 68 <1 .mu.M 47 <5 .mu.M 69 <0.5 .mu.M 48
<0.5 .mu.M 5 <0.1 .mu.M 49 <1 .mu.M 71 <10 .mu.M 50
<1 .mu.M 6 <0.5 .mu.M 51 <5 .mu.M 73 <5 .mu.M 52 <1
.mu.M 74 <5 .mu.M 53 <1 .mu.M 75 <5 .mu.M 54 <5
.mu.M
[0704] Mouse #456 is a Ptc-knockout heterozygote that received UV
irradiation for 6 months. The mouse developed many small BCC
lesions, which were blue after X-gal staining. The mouse was
sacrificed and the skin was excised with a 2 mm skin punch. Those
skin punches were then cultured for 6 days. Comparing to vehicle
(DMSO), compound A can decrease the number and size of BCC lesions
(blue spots in the picture). This experiment suggests that compound
A is able to inhibit murine BCC lesions in mouse #456.
[0705] In yet another experiment, E12.5 old ptc-1 (d11) lacZ lungs
were harvested and transgenic embryos identified by lacZ detection
using tails. Lung explants were grown submerged in mouse explant
medium (DMEM based, additives optimized for the culture of mouse
lungs) for 48 hrs, fixed in lacZ fixative, rinsed and stained for
lacZ O/N at 37.degree. C. Control tissue was untreated, while test
tissue was treated with compound A. Strong lacZ expression can be
observed in distal and proximal mesenchyme. Treatment with compound
A leads to significantly decreased reporter gene expression, as
evidenced especially by the weak signal surrounding the distal
branching tops of the growing lung epithelium.
Example 5
Bladder Cancer
[0706] Cytogenetic and Mutational Data Suggest Hedgehog Activation
Plays a Causative Role in Bladder Cancer
[0707] The cytogenetic and molecular alterations found in bladder
cancer are heterogeneous. In establishing the primary, specific
mutations in cancers, it is often useful to examine near-diploid
cancers, which do not yet have complex, multiple chromosome changes
accompanied by hyperdiploidy. Gibas et al., found monosomy of
chromosome 9 in 4 out of 9 cases of transitional cell carcinoma of
the bladder (Gibas et al. (1984) Cancer Research 44:1257-1264). In
three of these, the karyotype was near diploid, and in one,
monosomy 9 was the only abnormality observed. Therefore, monosomy
of chromosome 9 may initiate malignant transformation in a subgroup
of such cancers.
[0708] More evidence that this change appears as an early event was
presented by two other group who reported that deletions of
chromosome 9 are the only genetic changes present frequently in
superficial papillary tumors (Dalbagni et al. (1993) Lancet 342:
469-471). In fact, 9q deletions are estimated to occur in
approximately 60-70 percent of bladder tumors (Cairns et al. (1992)
Oncogene 8: 1083-1085; Dalbagni et al., supra). One study reported
that deletion of 9q22 occurs in 35% of informative cases (Simoneau
et al. 1999). The hedgehog signaling pathway component patched-1 is
located on 9q22.
[0709] LOH of all other chromosomes is infrequent (less than 10%)
in low-grade, non-invasive cancers. Likewise, alteration in
bladder-cancer associated oncogenes (ERBB2, EGFR) are also rare in
superficial, low-grade tumors (Cairns et al., supra).
[0710] On the basis of these cytogenetic findings, the following
model for bladder carcinogenesis has been proposed: Initiation
occurs by deletion of tumor-suppressor genes on chromosome 9,
leading to superficial papillary or occasionally flat tumors, a few
of which may then acquire further mutations (e.g., p53) and
progress to invasion.
[0711] Three groups observed trisomy 7 in a low percentage of
bladder cancers (Sandberg, supra; Berger et al. supra; Smeets et
al., supra). Shh, which according to our own experiments continues
to be expressed in bladder epithelium throughout adult life,
localizes to chromosome 7. Berger et al. also observed deletions of
10q24, the locus of su(fu) (Berger et al (1986) Cancer Genetics and
Cytogenetics 23: 1-24). Likewise, Smeets et al. suggested that 10q
loss may be a primary event in the development of bladder cancer
(Smeets et al. (1987) Cancer Genetics and Cytogenetics 29:
29-41).
[0712] This data suggests mechanisms by which the baseline
expression of hedgehog signaling present in the adult bladder
epithelium may be increased, thus leading to increased
proliferation of urothelial cells. This hypothesis is supported by
the cytological data, as well as by the finding of McGarvey et al.
that described ptc-1, smo and gli-3 expression in normal human
urothelium and two transitional cell carcinoma lines (McGarvey et
al. (1998) Oncogene 17: 1167-1172).
[0713] Hedgehog signaling was examined in the mouse bladder, and
found to be present in normal bladder. In Ptc-lacZ transgenic
newborn mice (ptc-1 (d11) lacZ), LacZ expression can be detected in
the proliferating urothelial cells of the bladder epithelium, and
more weakly, in adjacent mesenchymal cells (FIG. 16A). Additional
in situ hybridization analysis of adult mouse bladder indicates
expression of gli-1 in the bladder epithelium, and specifically in
the proliferating urothelial cells (FIG. 16B).
[0714] METHODS: For lacZ staining, ptc-1 (d11) lacZ bladder was
harvested from the transgenic newborn mouse pups identified by lacZ
detection using tails. Bladders were fixed in lacZ fixative, rinsed
and stained for lacZ O/N at 37.degree. C., then processed for
standard histology. Sections were counter-stained with eosin. For
in situ hybridization, sections of paraformaldehyde-fixed,
paraffin-embedded tissue were cleared, re-hydrated, digested with
proteinase K, acetylated and hybridized with [33P]-labeled gli-1
RNA probe over night. After high stringency post-hybridization
washes, slides were dipped in photo-emulsion, incubated for up to
three weeks, developed, and imaged using dark field illumination.
Dark-field signals were filled in with artificial color (red) and
superimposed with bright-field images.
[0715] Hedgehog Signaling in Bladder Cancer
[0716] Hedgehog signaling and hedgehog pathway gene expression was
analyzed in a human bladder cancer, and in several bladder cancer
cell lines. Gene expression in these tissues was measured using
Quantitative Real-Time PCR (Q-RT-PCR). These results are summarized
in FIGS. 17-19, and demonstrate that hedgehog pathway genes are
expressed in bladder cancer cell lines.
[0717] FIG. 17 demonstrates that shh expression is increased
12-fold and gli-1 expression is increased 2.5 fold in a bladder
tumor sample when compared to normal adult bladder. FIG. 18
examines shh and gli-1 expression in eight human bladder cancer
cell lines, and FIG. 19 examines expression of shh, ptc-1, smo,
gli-1, gli-2, and gli-3 in the same eight human bladder cancer cell
lines. These results indicate that components of the hedgehog
pathway are expressed in eight out of eight cell lines
examined.
[0718] METHODS: Experiment 1 (FIG. 17)--evaluation of hedgehog
signaling in a bladder tumor. For Quantitative Real-Time Polymerase
Chain Reaction (Q-RT-PCR) experiments, commercially available cDNA
(Clontech) was amplified using an ABI Prism 7700 Sequence Detection
System (TaqMan) from Perkin Elmer and gene-specific primers. The
housekeeping gene GAPDH was used to normalize RNA concentration and
PCR efficiency, and GAPDH primers were added to the same reactions.
Since probes for both genes are labeled with different
fluorophores, the specific signal and that of GAPDH can be detected
in the same tube. Signal intensities were calculated using the
algorithms provided in Sequence Detector v1.7, the software
provided by the manufacturer.
[0719] Experiment 2 (FIGS. 18-19)--hedgehog signaling in eight
bladder cancer cell lines. Bladder cancer cell lines were purchased
from ATCC (American Type Culture Collection) and maintained as
recommended in the product description. At confluency, cells were
rinsed and switched to medium containing 1% serum, a treatment that
increases hedgehog signaling. Cells were then grown 2 more days,
collected in Trizol (GIBCO-BRL) and RNA isolated according to the
manufacturer's protocol. The RNA was then transcribed into first
strand cDNA according to standard protocols, and amplified using an
ABI Prism 7700 Sequence Detection System (TaqMan) from Perkin Elmer
and gene-specific primers. The housekeeping gene GAPDH was used to
normalize RNA concentration and PCR efficiency, and GAPDH primers
were added to the same reactions. Since probes for both genes are
labeled with different fluorophores, the specific signal and that
of GAPDH can be detected in the same tube. Signal intensities were
calculated using the algorithms provided in Sequence Detector v1.7,
the software provided by the manufacturer.
[0720] In Vitro Assay to Examine Hedgehog Signaling in Bladder
Cancer Cell Lines
[0721] The expression of components of the hedgehog signaling
pathway in the eight bladder cancer cell lines examined suggested
that hedgehog signaling is active in bladder cancer cells. However
the gene expression observed may not be indicative of functional
signaling. To assess whether functional hedgehog signaling occurs
in bladder cancer cell lines, a gli-Luc in vitro assay was used.
This assay is summarized schematically in FIG. 20. Briefly, 10T 1/2
(S12) fibroblasts expressing a luciferase reporter gene responsive
to hedgehog serve as an indicator of hedgehog signaling. When these
cells are contacted with functional hedgehog protein, the hedgehog
signaling pathway is activated in the S12 cells, and luciferase is
expressed. In the experiments presented here, S12 cells are
co-cultured with bladder cancer cells. If the bladder cancer cell
line secretes functional hedgehog protein, luciferase expression
will be activated in the adjacent S12 cells.
[0722] FIG. 21 shows luciferase induction in S12 cells alone, and
in S12 cells co-cultured with three bladder cancer cell lines. Two
of the three cell lines examined induced expression of luciferase
in S12 cells indicating that these bladder cancer cell lines
secrete functional hedgehog protein.
[0723] To confirm the specificity of this activation of hedgehog
signaling by bladder cancer cell lines, S12/RT-4 co-cultures were
treated with the Shh blocking antibody (5E1). FIG. 22 demonstrates
that 5E1 treatment of co-cultures inhibits expression of luciferase
in S12 cells with an IC.sub.50 of 85 ng/ml and an IC.sub.90 of 500
ng/ml. It should be noted that this model also provides a means for
evaluating the in vitro efficacy of other hedgehog antagonists
including small molecule and polypeptide antagonists.
[0724] Hedgehog Signaling in an In Vivo Mouse Bladder Tumor
Model
[0725] Injection of bladder tumor cells into nude mice induces
tumor formation. Based on the ability of the Shh antibody 5E1 to
inhibit hedgehog signaling in the in vitro gli-Luc assay described
in detail above, the ability of 5E1 to inhibit bladder cell tumor
growth in vivo was examined. Briefly, nude mice were injected
subcutaneously with 10.sup.7 RT-4 cells. The mice were divided into
two groups and treated with either 5E1 or with a control IgG
antibody. FIGS. 23 and 24 show that treatment with 5E1
significantly decreased the size of the tumor in comparison to
treatment with the IgG control. It is important to note that due to
the procedure used in this particular experiment (injection of
tumor cells with Matrigel) the tumors start out with an average
size of 100 mm.sup.3 due to the Matrigel matrix (=100 .mu.l
injection volume). Matrigel is a liquid when kept on wet ice, but
solidifies upon injection. Thus, the average tumor size in the 5E1
group at the end of the experiment is roughly equal to that at the
beginning of treatment. Results are highly statistically
significant (Student's t-test: p=0.017). It should be noted that
this model also provides a means for evaluating the in vivo
efficacy of other hedgehog antagonists including small molecule and
polypeptide antagonists.
[0726] In addition to evaluating the effect of 5E1 treatment on
tumor size, expression of gli-1 in both the RT-4 tumors and in the
surrounding tissue was also evaluated. 5E1 treatment decreased
expression of gli-1 in both the RT-4 tumors and in adjacent tissue
(FIG. 25). This finding is significant because the in vitro
experiments outlined above indicate that these hedgehog-expressing
cells can activate hedgehog signaling in adjacent cell. Given the
complex nature of cancer progression, it is possible that hedgehog
signaling influences cancer both directly and indirectly. The
indirect effects may include the induction of proliferative
factors, angiogenic factors, or anti-apoptotic factors, to name a
few. The induction of such factors may occur within the cancer
cells themselves or in adjacent cells. Thus, the demonstration that
a hedgehog antagonist 5E1 can inhibit hedgehog signaling in both
cancer cells and in surrounding cells has significant
implications.
[0727] METHODS: Exponentially growing RT-4 cultures were
trypsinized, spun down, and resuspended in a small volume of
culture medium. The proportion of viable tumor cells was determined
by trypan blue exclusion. 10.sup.7 cells/animal were resuspended in
100 .mu.l Matrigel (a commercially available preparation of
basement membrane components) and injected subcutaneously in the
right side of the flank of 6-8 week-old athymic male BALB/c nu/nu
nude mice. Treatment was begun the day after injection of the
cells. Mice were divided into two groups containing 16
animals/group. The control group (IgG control antibody) and the
5E1-treated group were injected 3.times./week intraperitoneally
with 10 mg/kg antibody. Tumors were measured 2.times./week by
caliper in 2 dimensions and measurements converted to tumor mass
using the formula for a prolate ellipsoid (axb2x/2). As noted
above, in this particular example the tumors were injected in
combination with Matrigel. Therefore, the tumors have an initial
size of 100 mm.sup.3 and the inhibition of tumor size observed
following 5E1 treatment is nearly a complete inhibition of tumor
growth.
[0728] Expression of gli-1 was measured using Q-RT-PCR as described
throughout the application.
[0729] The inhibition of tumor growth by the hedgehog antagonist
5E1 supports the utility of the claimed invention. It is expected
that antagonism of hedgehog signaling using a range of agents would
have similar effects in decreasing tumor growth, and the efficacy
of any candidate compound could be easily assessed using the in
vitro and in vivo methods described above.
Example 6
Prostate Cancer
[0730] Hedgehog signaling plays an important role in normal
prostate development. Sonic hedgehog is required for prostate
growth, and expression of Shh is strongly correlated with prostate
ductal branching (Podlasek et al. (1999) Developmental Biology 209:
28-39). Recent evidence supporting the essential role of shh in
proper prostate branching demonstrates that treatment of embryonic
prostate with the hedgehog antagonist cyclopamine inhibits growth
and branching (W. Bushman, unpublished result). Additionally, the
maintenance of low levels of hedgehog signaling in the adult mouse
prostate suggests additional roles for hedgehog signaling beyond
this early role in the initial growth and branching of the
embryonic prostate.
[0731] Recent studies have examined the correlation between the
expression of components of the hedgehog pathway and prostate
cancer. These results show a correlation between increased
expression of shh and/or gli-1 and prostate cancer. Additional
cytological data supports the idea that mis-regulation of the
hedgehog pathway plays a role in prostate cancer. Two studies have
described deletions of a fragment of chromosome 10 containing the
Su(fu) locus in prostate cancers (Carter et al. (1990) PNAS 87:
8751-8755; Li et al. (1997) Science 275: 1943-1947). Given the
evidence in the literature suggestive of a role for hedgehog
signaling in prostate cancer, hedgehog signaling in several
prostate cancer cell lines was examined. Additionally, the ability
of hedgehog antagonists to decrease activation of hedgehog
signaling in prostate tumor cell lines was demonstrated. These
results suggest that, like in bladder cancer cells, antagonism of
hedgehog signaling has utility in decreasing growth and
proliferation of prostate cancer cells.
[0732] Hedgehog Signaling in Prostate Cancer
[0733] Expression of shh and gli-1 in both human prostate cancer
samples and in commercially available prostate cancer cell lines
was examined. FIG. 26 shows in situ hybridization analysis of human
prostate cancer samples, and demonstrates the abundant expression
of shh. Similarly, FIG. 27 demonstrates high levels of gli-1
expression in prostate cancer cells as measured by Q-RT-PCR.
Finally, FIG. 28 examined expression of both shh and gli-1 by
Q-RT-PCR in three commercially available prostate cancer cell
lines. These results indicate hedgehog signaling occurs in all
three commercially available cell lines.
[0734] METHODS: In situ hybridization: Paraformaldehyde-fixed
tissue is cryo-sectioned into 30 .mu.m sections, digested with
proteinase K, hybridized overnight with digoxigenin-labeled RNA
probe. After high stringency post-hybridization washes, sections
are incubated with an anti-digoxigenin antibody which is labeled
with alkaline phosphatase. The signal is visualized by addition of
BM purple, a commercially available chromagen solution that reacts
with the alkaline phosphatase to form a purple precipitate.
[0735] Prostate cancer cell lines were purchased from ATCC
(American Type Culture Collection) and maintained as recommended in
the product description. At confluency, cells were rinsed and
switched to medium containing 1% serum, a treatment that increases
hedgehog signaling. Cells were then grown 2 more days, collected in
Trizol (GIBCO-BRL) and RNA isolated according to the manufacturer's
protocol. The RNA was then transcribed into first strand cDNA
according to standard protocols, and amplified using an ABI Prism
7700 Sequence Detection System (TaqMan) from Perkin Elmer and
gene-specific primers. The housekeeping gene GAPDH was used to
normalize RNA concentration and PCR efficiency, and GAPDH primers
were added to the same reactions. Since probes for both genes are
labeled with different fluorophores, the specific signal and that
of GAPDH can be detected in the same tube. Signal intensities were
calculated using the algorithms provided in Sequence Detector v1.7
, the software provided by the manufacturer.
[0736] In Vitro Assay to Examine Hedgehog Signaling in Prostate
Cancer Cell Lines
[0737] The expression of components of the hedgehog signaling
pathway in prostate cancer samples and cell lines suggests that
hedgehog signaling is active in prostate cancer. However the gene
expression observed may not be indicative of functional signaling.
To assess whether functional hedgehog signaling occurs in prostate
cancer cell lines, the gli-Luc in vitro assay was employed. This
assay was summarized above, and is represented schematically in
FIG. 20. Briefly, 10T 1/2 (S12) fibroblasts expressing a luciferase
reporter gene responsive to hedgehog serves as an indicator of
hedgehog signaling. When these cells are contacted with functional
hedgehog protein, the hedgehog signaling pathway is activated in
the S12 cells, and luciferase is expressed. In the experiments
presented here, S12 cells are co-cultured with prostate cancer
cells. If the prostate cancer cell line secretes functional
hedgehog protein, luciferase expression will be activated in the
adjacent S12 cells.
[0738] FIG. 29 shows no induction of luciferase in S12 cells
cultured alone, or in S12 cells cultured with PZ-HPV-7 (normal)
cells. However, luciferase induction is observed when S12 cells are
cultured with any of three prostate cancer cell lines: 22Rv1, PC-3,
or LNCaP. This result indicates that these prostate cancer cell
lines secrete functional hedgehog protein.
[0739] To confirm the specificity of this activation of hedgehog
signaling by prostate cancer cell lines, S12/prostate cancer
co-cultures were treated with the Shh blocking antibody (5E1). FIG.
30 demonstrates that 5E1 treatment of co-cultures inhibits
expression of luciferase in S12 cells.
[0740] METHODS: S12 cultures and co-cultures, and luciferase assays
were performed as detailed above.
Example 7
Benign Prostatic Hyperplasia (BPH)
[0741] As detailed above, hedgehog signaling appears to have both
an important role in early prostate patterning, and a role in
maintenance of the adult prostate. Although prostate cancer is one
potential affect of misregulation of hedgehog signaling in the
adult prostate, another common condition of the prostate that seems
to correlate with hedgehog expression is benign prostatic
hyperplasia (BPH).
[0742] BPH is a disease of the central prostate, and is
characterized by increased smooth muscle around the prostatic
urethra. Interestingly, shh is expressed in a gradient in the adult
prostate with highest expression in the central zone of the
prostate. Additionally, shh is involved in smooth muscle
differentiation in other tissues including the gut and lung
(Apelqvist et al. (1997) Current Biology 7: 801-804; Pepicelli et
al. (1998) Current Biology 8: 1083-1086). This evidence identified
hedgehog signaling as a good candidate for involvement in the
etiology of BPH. Finally, transcription of shh is increased by
exposure to dihydro-testosterone (DHT) (Podlasek et al., supra).
This is significant because the concentration of 5-alpha-reductase,
an enzyme which converts testosterone to DHT, is elevated in BPH
stroma (Wilkin et al. (1980) Acta Endocrinology 94: 284-288). This
data suggests that mis-regulation of hedgehog signaling may be
involved in BPH, and thus that the present invention provides
utility for the treatment of BPH.
[0743] Hedgehog Signaling in BPH
[0744] Expression of sonic hedgehog and gli-1 expression in human
BPH samples was examined. FIGS. 31 and 32 show in situ
hybridization analysis of human BPH samples, and demonstrate that
both shh and gli-1 are abundantly expressed in BPH. Furthermore,
FIG. 33 demonstrates that shh is not ubiquitously expressed
throughout the prostate, but is instead present in a gradient with
the highest level of both hedgehog and ptc-1 transcripts present in
the proximal central zone of the prostate.
[0745] Additionally, the expression of shh and gli-1 by Q-RT-PCR
was analyzed. FIG. 34 shows that both shh and gli-1 are expressed
in BPH samples. Expression of shh and gli-1 in basal cell carcinoma
(BCC) samples is provided for comparison. These results demonstrate
that gli-1 is expressed in BPH samples at a level similar to that
found in a cancer type known to be caused by a hedgehog pathway
mutation. Finally, FIG. 35 shows the expression of shh and gli-1 in
BPH cell lines, and compares expression to that observed in BCC,
prostate cancer cell lines, and normal prostate fibroblasts. Note
that gli-1 is expressed at similar levels in both BPH cell lines
and in BCC samples. These results are suggestive of a role for
hedgehog signaling in BPH and further suggests that antagonism of
hedgehog signaling has significant utility in the treatment of
BPH.
[0746] METHODS: In situ hybridization (FIGS. 31 and 33):
Paraformaldehyde-fixed tissue is cryo-sectioned into 30 .mu.m
sections, digested with proteinase K, hybridized overnight with
digoxigenin-labeled RNA probe. After high stringency
post-hybridization washes, sections are incubated with an
anti-digoxigenin antibody which is labeled with alkaline
phosphatase. The signal is visualized by addition of BM purple, a
commercially available chromagen solution that reacts with the
alkaline phosphatase to form a purple precipitate.
[0747] Radioactive In situ hybridization (FIG. 32): Briefly, 7 mm
sections of paraformaldehyde-fixed, paraffin-embedded tissue
containing large basal cell islands are cleared, re-hydrated,
digested with proteinase K, acetylated and hybridized overnight
with 33P-labeled RNA probes. After high stringency
post-hybridization washes, slides were dipped in photo emulsion and
incubated in the dark for 14 days at 4.degree. C. After developing,
slides were counter-stained with hematoxylin and eosin and imaged
using dark-field illumination. Dark-field images were converted to
red artificial color and superimposed with bright-field images.
Q-RT-PCR: Samples were collected in Trizol (GIBCO-BRL) and RNA
isolated according to the manufacturer's protocol. The RNA was then
transcribed into first strand cDNA according to standard protocols,
and amplified using an ABI Prism 7700 Sequence Detection System
(TaqMan) from Perkin Elmer and gene-specific primers. The
housekeeping gene GAPDH was used to normalize RNA concentration and
PCR efficiency, and GAPDH primers were added to the same reactions.
Since probes for both genes are labeled with different
fluorophores, the specific signal and that of GAPDH can be detected
in the same tube. Signal intensities were calculated using the
algorithms provided in Sequence Detector v1.7 , the software
provided by the manufacturer.
Example 8
Antagonism of Hedgehog Signaling in Colon Cancer
[0748] The growth of tumors is a complex process that requires
proliferation, angiogenesis, the inhibition of cell death, and many
other complex interactions between the cancer cells and the
surrounding tissue. An additional mechanism by which hedgehog
signaling may influence tumor growth and progression is through the
induction of factors that enhance proliferation, angiogenesis, and
the inhibition of cell death. For example, sonic hedgehog has been
shown to induce VEGF in fibroblasts. Thus, the use of hedgehog
antagonists may prevent hedgehog signaling from inducing factors
that promote tumor formation, and therefore inhibit tumor formation
or progression.
[0749] To help address this model, the ability of the antagonistic
hedgehog antibody 5E1 to inhibit tumor growth in mice injected with
a combination of hedgehog expressing colon cancer cells and
fibroblasts was investigated. Two experiments were performed to
assess the effects of 5E1 treatment on tumor size in mice injected
with hedgehog expressing colon cancer cells. In the first
experiment, treatment with 5E1, or PBS control, was initiated on
the same day as injection with the tumor cells. The results are
summarized in FIG. 36, and demonstrate that treatment with 5E1
significantly decreases tumor size, weight, and rate of growth in
comparison to that of mice treated with PBS (FIG. 36). The
experiment was performed using two separate colon cancer cell lines
with similar affects.
[0750] In the second experiment, treatment with 5E1 was delayed
until the eleventh day of tumor growth. The results are summarized
in FIG. 37, and demonstrate that treatment with 5E1 significantly
decreases the size and rate of growth of the tumor when compared to
control mice (FIG. 37). The experiment was performed using two
separate colon cancer cell lines with similar affects.
[0751] These results demonstrate the utility of hedgehog
antagonists in the inhibition of proliferation and growth of cancer
cells. Additionally, this model provides an in vivo method for
easily evaluating the efficacy of candidate hedgehog
antagonists.
[0752] METHODS: Experiment 1. Twenty nude mice were injected
subcutaneously with a combination of 10.sup.6 HT-29 cells (a Shh
expressing colon cancer cell line) and 10.sup.6 10T 1/2 cells (a
fibroblast cell line) in a volume of 100 .mu.l. The mice were
randomized into two groups. Group A was treated with PBS, and group
B was treated with 5E1. The treatments were initiated on the same
day as injection of the tumor cells. Treatment was administered IP,
3 times/week over a period of thirty days, and at a dose of 6
mg/kg. Additionally, this experiment was carried out under an
identical protocol using another Shh expressing colon cancer cell
line (Colo205) with similar results. Experiment 2--delayed
administration. Twenty nude mice were injected subcutaneously with
a combination of 10.sup.6 HT-29 cells (a Shh expressing colon
cancer cell line) and 10.sup.6 10T 1/2 cells (a fibroblast cell
line) in a volume of 100 .mu.l. The mice were randomized into two
groups. Group A was treated with PBS, and group B was treated with
5E1. Treatment was initiated after the tumor had grown to day 11.
Such tumors had a volume of approximately 90-210 mm.sup.3.
Treatment was administered IP, 3 times/week over a period of
twenty-nine days (until day 40 of total tumor growth), and at a
dose of 6 mg/kg. Additionally, this experiment was carried out
under an identical protocol using another Shh expressing colon
cancer cell line (Colo205) with similar results.
Example 9
Drug Screens
[0753] The foregoing examples present both in vitro and in vivo
models for examining the effects of hedgehog antagonist on cell
proliferation. The models provide assays for testing a range of
antagonistic agents for the ability to inhibit cell growth and
proliferation. Such screens can be used in initial assays to
identify lead compounds, and can also be used to evaluate the
relative efficacies of candidate compounds.
[0754] Antagonistic agents that can be analyzed in this way include
small molecules, blocking antibodies, antisense oligonucleotides,
and polypeptides. These agents may interfere with hedgehog
signaling at any point along the signal transduction pathway. For
example, preferred agents may interact with hedgehog, patched-1, or
smoothened. Additional preferred agents may interact with an
intracellular component of the hedgehog pathway including gli-1,
gli-2, or gli-3.
[0755] The in vitro and in vivo methods described above are not
specific for the cancer cell lines explicitly described herein. Any
cell type or cell line could be similarly tested, and these methods
could be easily used to assess the ability of hedgehog antagonists
to inhibit tumor growth and proliferation in other types of cancer
cells. Additionally, the in vitro assay could be employed to
analyze hedgehog signaling and the ability of hedgehog antagonists
to block hedgehog signaling in other non-cancerous
hyperproliferative cell types. For example, hyperproliferative
conditions include many other classes of disorders including skin
maladies such as psoriasis. The effects of candidate hedgehog
antagonists on these cell types can be easily assessed using the
methods described here.
[0756] All of the references cited above are hereby incorporated by
reference herein.
Equivalents
[0757] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
* * * * *