U.S. patent application number 10/732669 was filed with the patent office on 2005-01-20 for mediators of hedgehog signaling pathways, compositions and uses related thereto.
This patent application is currently assigned to Curis, Inc.. Invention is credited to Baxter, Anthony David, Boyd, Edward Andrew, Guicherit, Oivin M., Porter, Jeffrey, Price, Stephen, Rubin, Lee.
Application Number | 20050014796 10/732669 |
Document ID | / |
Family ID | 26888847 |
Filed Date | 2005-01-20 |
United States Patent
Application |
20050014796 |
Kind Code |
A1 |
Baxter, Anthony David ; et
al. |
January 20, 2005 |
Mediators of hedgehog signaling pathways, compositions and uses
related thereto
Abstract
The present invention makes available methods and reagents for
inducing activation of a hedgehog pathway in a cell or tissue
comprising contacting the cell with a hedgehog agonist, such as a
small molecule, in a sufficient amount to achieve a therapeutic
result. In certain embodiments, the methods and reagents may be
employed to correct or inhibit an aberrant or unwanted growth
state, e.g., by antagonizing a normal ptc pathway or agonizing
smoothened or hedgehog activity.
Inventors: |
Baxter, Anthony David;
(Hertfordshire, GB) ; Boyd, Edward Andrew;
(Berkshire, GB) ; Guicherit, Oivin M.; (Belmont,
MA) ; Porter, Jeffrey; (Lexington, MA) ;
Price, Stephen; (Hertford, GB) ; Rubin, Lee;
(Wellesley, MA) |
Correspondence
Address: |
ROPES & GRAY LLP
ONE INTERNATIONAL PLACE
BOSTON
MA
02110-2624
US
|
Assignee: |
Curis, Inc.
Cambridge
MA
|
Family ID: |
26888847 |
Appl. No.: |
10/732669 |
Filed: |
December 9, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10732669 |
Dec 9, 2003 |
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09724492 |
Nov 28, 2000 |
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6683108 |
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60193279 |
Mar 30, 2000 |
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Current U.S.
Class: |
514/332 ;
514/340; 514/408; 514/422; 514/649; 514/651 |
Current CPC
Class: |
A61K 31/381 20130101;
C07D 333/70 20130101; C07D 409/14 20130101; A61K 31/422 20130101;
C07D 409/12 20130101; A61K 31/50 20130101; A61K 31/517 20130101;
A61K 31/44 20130101 |
Class at
Publication: |
514/332 ;
514/340; 514/408; 514/422; 514/649; 514/651 |
International
Class: |
A61K 031/444; A61K
031/44; A61K 031/40; A61K 031/137 |
Claims
1. A compound represented in general formula (III): 35wherein Ar
and Ar' independently represent substituted or unsubstituted aryl
or heteroaryl rings; Y, independently for each occurrence, is
absent or represents --NR--, --O--, --S--, or --Se--; X is selected
from --C(.dbd.O)--, --C(.dbd.S)--, --S(O.sub.2)--, --S(O)--,
--C(.dbd.NCN)--, --P(.dbd.O)(OR.sub.2)--, and a methylene group
optionally substituted with 1-2 groups selected from lower alkyl,
alkenyl, and alkynyl groups; M represents, independently for each
occurrence, a substituted or unsubstituted methylene group, or two
adjacent M taken together represent substituted or unsubstituted
ethene or ethyne: R represents, independently for each occurrence,
H or substituted or unsubstituted aryl, heterocyclyl, heteroaryl,
aralkyl, heteroaralkyl, alkynyl, alkenyl, or alkyl, or two R taken
together may form a 4- to 8-membered ring, Cy represents a
cyclohexyl ring, Cy' represents a substituted or unsubstituted
aryl, heterocyclyl, heteroaryl, or cycloalkyl; and i represents,
independently for each occurrence, an integer from 0 to 5; wherein
Y-M.sub.i adjacent to Cy is absent for both occurrences, and the
compound is enriched for the isomer in which the two nitrogen
substituents on Cy are disposed in a trans-relationship.
2. The compound of claim 1, wherein Y is absent from all
positions.
3. The compound of claim 1, wherein M represents, independently for
each occurrence, a substituted or unsubstituted methylene
group.
4. The compound of claim 1, wherein X is selected from
--C(.dbd.O)--, --C(.dbd.S)--, and --S(O.sub.2)--.
5. The compound of claim 1, wherein NR.sub.2 represents a primary
amine, or a secondary amine substituted with one lower alkyl
group.
6. The compound of claim 1, wherein Cy' is a substituted or
unsubstituted aryl or heteroaryl.
7. The compound of claim 1, wherein Cy' is bicyclic.
8. The compound of claim 7, wherein Cy' represents a substituted or
unsubstituted benzothiophene.
9. The compound of claim 8, wherein the benzothiophene ring is
substituted with one or more methyl, ethyl, fluorine, chlorine,
methoxy or cyano groups.
10. The compound of claim 9, wherein each R independently
represents H or a lower alkyl.
11. The compound of claim 9, wherein NR.sub.2 represents a
secondary amine.
12. The compound of claim 11, wherein each R independently
represents H or a lower alkyl.
13. The compound of claim 10, wherein i is zero except for M.sub.i
between Ar and N; and all Y are absent.
14. The compound of claim 12, wherein i is zero except for M.sub.i
between Ar and N; and all Y are absent.
15. The compound of claim 14, wherein X is selected from
--C(.dbd.O)--, --C(.dbd.S)--, and --S(O.sub.2)--.
16. The compound of claim 11, wherein X is selected from
--C(.dbd.O)--, --C(.dbd.S)--, and --S(O.sub.2)--.
17. The compound of claim 10, wherein X is selected from
--C(.dbd.O)--, --C(.dbd.S)--, and --S(O.sub.2)--.
18. The compound of claim 8, wherein X is selected from
--C(.dbd.O)--, --C(.dbd.S)--, and --S(O.sub.2)--.
19. The compound of claim 18, wherein NR.sub.2 represents a
secondary amine.
20. The compound of claim 19, wherein each R independently
represents H or a lower alkyl.
21. The compound of claim 8, wherein NR.sub.2 represents a
secondary amine.
22. The compound of claim 20, wherein i is zero except for M.sub.i
between Ar and N; and all Y are absent.
23. The compound of claim 21, wherein i is zero except for M,
between Ar and N, and all Y are absent.
24. The compound of claim 23, wherein each R independently
represents H or a lower alkyl.
25. The compound of claim 24, wherein NR.sub.2 represents a
secondary amine.
26. The compound of claim 25, wherein Cy' represents a substituted
or unsubstituted benzothiophene.
27. The compound of claim 26, wherein X is selected from
--C(.dbd.O)--, --C(.dbd.S)--, and --S(O.sub.2)--.
28. The compound of claim 27, wherein the benzothiophene ring is
substituted with one or more methyl, ethyl, fluorine, chlorine,
methoxy or cyano groups.
29. The compound of claim 27, wherein the benzothiophene ring is
substituted with one or more methyl, fluorine, or chlorine
groups.
30. A compound represented in general formula (Vet): 36wherein Cy
represents a cyclohexyl ring; Ar is a substituted or unsubstituted
aryl or heteroaryl ring; W is O or S, R represents, independently
for each occurrence, H or substituted or unsubstituted aryl,
heterocyclyl, heteroaryl, aralkyl, heteroaralkyl, alkynyl, alkenyl,
or alkyl, or two R taken together may form a 4- to 8-membered ring;
R.sub.1 and R.sub.2 represent, independently and as valency
permits, from 0-5 substituents on the ring to which each is
attached, selected from halogen, lower alkyl, lower alkenyl, aryl,
heteroaryl, carbonyl, thiocarbonyl, ketone, aldehyde, amino,
acylamino, amido, amidino, cyano, nitro, hydroxyl, azido, sulfonyl,
sulfoxido, sulfate, sulfonate, sulfamoyl, sulfonamido, phosphoryl,
phosphonate, phosphinate, --(CH.sub.2).sub.palkyl,
--(CH.sub.2).sub.palkenyl, --(CH.sub.2).sub.palkynyl,
--(CH.sub.2).sub.paryl, --(CH.sub.2).sub.paralkyl,
--(CH.sub.2).sub.pOH, --(CH.sub.2).sub.pO-lower alkyl,
--(CH.sub.2).sub.pO-lower alkenyl, --O(CH.sub.2).sub.nR,
--(CH.sub.2).sub.pSH, --(CH.sub.2).sub.pS-lower alkyl,
--(CH.sub.2).sub.pS-lower alkenyl, --S(CH.sub.2).sub.nR,
--(CH.sub.2).sub.pNR.sub.2, --(CH.sub.2).sub.pNR-lower alkyl,
--(CH.sub.2).sub.pNR-lower alkenyl, --NR(CH.sub.2).sub.nR, and
protected forms of the above; and n and p, individually for each
occurrence, represent integers from 0 to 10, wherein the compound
is enriched for the isomer in which the two nitrogen substituents
on Cy are disposed in a trans-relationship.
31. The compound of claim 30, wherein Ar is heteroaryl.
32. The compound of claim 30, wherein Ar is bicyclic.
33. The compound of claim 30, wherein NR.sub.2 represents NH).
34. The compound of claim 33, wherein Ar represents a substituted
or unsubstituted benzothiophene.
35. The compound of claim 34, wherein the benzothiophene ring is
substituted with one or more methyl, ethyl, fluorine, chlorine,
methoxy, or cyano groups.
36. The compound of claim 30, wherein each R independently
represents H or a lower alkyl group.
37. The compound of claim 36, wherein NR.sub.2 represents a
secondary amine.
38. The compound of claim 36, wherein Ar represents a substituted
or unsubstituted benzothiophene.
39. The compound of claim 38, wherein the benzothiophene ring is
substituted with one or more methyl, ethyl, fluorine, chlorine,
methoxy, or cyano groups.
40. The compound of claim 30, wherein Ar represents a substituted
or unsubstituted benzothiophene.
41. The compound of claim 40, wherein the benzothiophene ring is
substituted with one or more methyl, ethyl, fluorine, chlorine,
methoxy, or cyano groups.
42. The compound of claim 40, wherein each R independently
represents H or a lower alkyl group.
43. The compound of claim 42, wherein NR.sub.2 represents a
secondary amine.
44. The compound of claim 43, wherein the benzothiophene ring is
substituted with one or more methyl, ethyl, fluorine, chlorine,
methoxy, or cyano groups.
45. The compound of claim 43, wherein the benzothiophene ring is
substituted with one or more methyl, fluorine, or chlorine
groups.
46. A method for modulating proliferation or differentiation of a
cell, comprising contacting the cell with a compound of claim 1 or
claim 30.
47. The method of claim 46, wherein the compound is a hedgehog
agonist.
48. The method of claim 47, wherein the hedgehog agonist agonizes
hedgehog mediated signal transduction with an ED.sub.50 of 1 .mu.M
or less.
49. The method of claim 47, wherein the hedgehog agonist is
administered as part of a therapeutic or cosmetic application.
50. The method of claim 49, wherein the therapeutic or cosmetic
application is selected from regulation of neural tissues, bone and
cartilage formation and repair, regulation of spermatogenesis,
regulation of smooth muscle, regulation of lung, liver and other
organs arising from the primitive gut, regulation of hematopoietic
function, and regulation of skin and hair growth.
51. A method for treating neurological conditions deriving from
acute, subacute, or chronic injury to the nervous system: aging of
the nervous system; chronic neurodegenerative diseases of the
nervous system; or chronic immunological diseases of the nervous
system or affecting the nervous system, comprising administering to
a subject the preparation of claim 1 or 30.
52. The method of claim 51, wherein the injury is ischemic injury
resulting from stroke.
53. The method of claim 51, wherein the neurodegenerative disease
is Parkinson's disease.
54. The method of claim 51, wherein the neuromuscular disorders are
selected from chronic or progressive atrophies and chronic
peripheral neuropathy.
55. A method for treating or reducing the risk of developing
Parkinson's disease comprising administering to an animal a
compound of claim 1 or 30.
56. The method of claim 55, wherein the hedgehog agonist increases
the rate of survival of dopaminergic neurons.
57. A method for treating autonomic disorders of the peripheral
nervous system comprising administering to an animal a compound of
claim 1 or 30.
58. A method for treating neuronal damage due to ischemic events
comprising administering to an animal a compound of claim 1 or
30.
59. A method of modulating the growth, differentiation, or survival
of a cell, comprising contacting the cell with a compound of claim
1 or 30.
60. The method of claim 59, wherein the cell is contacted with the
compound in vitro.
61. The method of claim 59, wherein the cell is contacted with the
compound ex vivo.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This patent application is a continuation of U.S. patent
application Ser. No. 09/724,492, filed on Nov. 28, 2000, which
claims priority to U.S. Provisional Patent Application Ser. No.
60/193,279, filed on Mar. 30, 2000, the specifications of each of
which are incorporated by reference herein in their entirety.
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 fruitfly 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 Indian 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; Indian 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 hedghog 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. (11992) Cell 71:33-50; Tabata, T. et al.
(1992) Genes Dev. 2635-2645; Chang, D. E. et al. (11994)
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. (11994) 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. (11992) supra; Tabata et al.
(11992) supra; Chang et al. (1994) supra: Lee et al. (11994) 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. (11995) 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; Mart', 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. (11996) Cell 86, 21-34). Biochemical studies have shown that
the autoproteolytic cleavage of the HH precursor protein proceeds
through an internal thioester intermediate which subsequently is
cleaved in a nucleophilic substitution. It is likely that the
nucleophile is a small lipophilic molecule which 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; Mart', 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 which 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 embyros, 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 Doorplate 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; Mart'
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 (Mart' et al. (1995) supra). Thus, high
concentration 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 floorplate 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] BCNS also predisposes to multiple tumor types, the most
frequent being basal cell carcinomas (BCC) that occur in many
locations on the body and appear within the first two decades of
life. Most cases of BCC, however, are unrelated to the syndrome and
arise sporadically in small numbers on sun-exposed sites of
middle-aged or older people of northern European ancestry.
[0021] Recent studies in BCNS-related and sporadic BCC suggest that
a functional loss of both alleles of patched leads to development
of BCC. See Johnson, supra; Hahn, supra; and Gailani, M. R. et al.
(1996) Nature Genetics 14:78. Single allele deletions of chromosome
9q22.3 occur frequently in both sporadic and hereditary BCC.
Linkage analysis revealed that the defective inherited allele was
retained and the normal allele was lost in tumors from BCNS
patients.
[0022] 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.
[0023] 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.
[0024] 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
[0025] The present invention makes available methods and reagents
for activating the hedgehog signalling pathway of a cell, e.g.,
upregulating the hedgehog pathway in a normal cell or modulating
growth states resulting from deactivation of the hedgehog signaling
pathway, such as ptc gain-of-function, hedgehog loss-of-function,
or a smoothened loss-of-function, comprising contacting the cell
with an agent, such as a small molecule, in a sufficient amount to
modulate the growth state, for example, to reverse or control cell
death or growth inhibition, e.g., to antagonize a normal ptc
function, agonize a normal hedgehog function, or agonize smoothened
function. In certain embodiments of the present invention, a
hedgehog pathway agonist, according to the present invention,
increases hedgehog-dependent transcriptional activation, such as
expression of a ptc or gli gene (particularly Gli-1) but is
inhibited by the hedgehog antagonist jervine. In certain preferred
embodiments, the agonist upregulates expression of a gli gene by at
least 5%, at least 10%, at least 20%, or even at least about 50%
relative to a control in the absence of the agonist. The coding
sequences for exemplary human Gli genes include, for example, the
Gli-1 gene sequence of GenBank accession X07384 and the Gli-2 gene
sequence of GenBank accession AB007298. See also Kinzler et al.
Nature 1988, 332, 371. The level of gli expression can be
determined, for example, by measuring the level of mRNA
(transcription) or the level of protein (translation). While not
wishing to be bound by any particular theory, the agonist may exert
such activity by binding to smoothened or a complex including
smoothened, or otherwise disrupts a patched-smoothened complex.
[0026] In certain embodiments, a compound as set forth above may be
a small organic molecule, e.g., may have a molecular weight of less
than about 2000 amu, less than about 1500 amu, or even less than
about 1000 amu. In certain preferred embodiments, the antagonist is
a non-peptidyl organic molecule. In certain embodiments, a compound
useful in the present invention, such as described above, may have
an EC.sub.50 for inducing or augmenting one or more hedgehog
activities (such as upregulation of gli or ptc expression) of less
than about 1000 nm, less than about 100 nm, less than about 10 nm,
or even less than about 1 nm.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIGS. 1-31 depict reactions useful for synthesizing
compounds according to the present invention.
[0028] FIG. 32 illustrates representative compounds according to
the present invention.
[0029] FIGS. 34a and 34b depict results from hedgehog pathway
reporter assays using compounds of the present invention.
[0030] FIG. 35 presents assay results demonstrating upregulation of
Ptc and Gli by hedgehog agonists of the present invention.
[0031] FIGS. 36 and 37 portray the increased proliferation of
cerebellar neuron precursors in the presence of subject
agonists.
[0032] FIGS. 38A and B show the effect of subject agonists on
healing of a crushed sciatic nerve as measured in a grip assay.
[0033] FIGS. 39A-D demonstrate the effect of subject agonists on
healing of a crushed sciatic nerve as measured in a toe spread
assay.
[0034] FIG. 40 shows the effect of subject agonists in combination
with a low dose of hedgehog protein on lung, scapula, skin, and
skill tissue of developing mice.
[0035] FIG. 41 depicts the effect of subject agonists in with and
without added hedgehog protein on pancreas, kidney, skin, and heart
tissue of developing mice.
[0036] FIG. 42 exhibits the effect of a subject agonist on the
forelimb of a newborn mouse.
[0037] FIG. 43 presents the effect of a subject agonist on
forelimbs of a newborn mouse at different concentrations.
[0038] FIG. 44 portrays the effects of a subject agonist on
developing lung tissue.
[0039] FIG. 45 shows the effects of a subject agonist on developing
kidney tissue.
[0040] FIGS. 46A and B shows the effects of subject agonists on
mouse skin tissue in the presence and absence of hedgehog
protein.
[0041] FIGS. 47A and B compare the activity of subject agonists in
mouse and human reporter cells.
[0042] FIG. 48 depicts the upregulation of Gli in HEPM cells
treated with a modified N-terminal fragment of Sonic hedgehog or
with a subject agonist.
DETAILED DESCRIPTION OF THE INVENTION
[0043] I. Overview
[0044] The present invention relates to the discovery that signal
transduction pathways regulated by hedgehog, patched (ptc), gli
and/or smoothened can be modulated, at least in part, by small
molecules. While not wishing to be bound by any particular theory,
the activation of a receptor may be the mechanism by which these
agents act. For example, the ability of these agents to activate
the hedgehog pathway may be due to the ability of such molecules to
interact with or bind to smoothened, or at least to promote the
ability of those proteins to activate a hedgehog, ptc, and/or
smoothened-mediated signal transduction pathway.
[0045] Certain hedgehog agonists disclosed herein modulate hedgehog
activity in the absence of hedgehog protein itself, e.g., the
compounds mimic hedgehog, rather than merely promote hedgehog
binding to patched. Certain other compounds are useful for
enhancing the activity of hedgehog protein, and thus may be used in
therapeutic preparations or treatments which include hedgehog
protein, or may be used to increase the activity of hedgehog
protein naturally produced by the cells or tissue to be treated
with the agonist. The hedgehog agonists disclosed herein may bind
to smoothened, thereby activating the hedgehog pathway, or
otherwise induce dissociation of the patched-smoothened
complex.
[0046] In certain embodiments, hedgehog agonists useful in the
present induce hedgehog-dependent transcriptional regulation, such
as expression of the gli1 or ptc genes. Such agonists can thus
induce or increase the hedgehog-dependent pathway activation
resulting from, for example, increased levels of hedgehog
protein.
[0047] It is, therefore, specifically contemplated that these small
molecules which modulate aspects of hedgehog, ptc, or smoothened
signal transduction activity will likewise be capable of promoting
proliferation (or other biological consequences) in cells having a
ptc gain-of-function phenotype, hedgehog loss-of-function
phenotype, or a smoothened loss-of-function phenotype, as well as
cells wherein the hedgehog pathway functions normally. In preferred
embodiments, the subject agonists 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
inducing or augmenting at least some of the biological activities
of hedgehog proteins, preferably specifically in target cells.
Activation of the hedgehog pathway by a hedgehog agonist may be
quantified, for example, by detecting the increase in ptc or gli-1
transcription in the presence of the agonist relative to a control
in the absence of agonist. For example, an increase of at least 5%,
at least 10%, at least 20%, or even at least 50% may be indicative
of hedgehog pathway activation by a test compound. In certain
embodiments, the agonist activity of the subject compounds is not
inhibited by the hedgehog antibody 5E1, but is inhibited by jervine
or an antagonist having the formula: 1
[0048] This quality can be quantified, for example, by determining
whether the antibody or antagonist induces a decrease of more than
50%, more than 20%, more than 10%, or even more than 5% of the ptc
or gli-i upregulation induced by the agonist in the absence of the
hedgehog antagonist, etc. In certain embodiments, a compound useful
in the present invention, such as described above, may have an
EC.sub.50 for inducing or augmenting one or more hedgehog
activities (such as upregulation of gli expression) of less than
about 1000 nM, less than about 100 nM, less than about 10 nM, or
even less than about 1 nM. The coding sequences for exemplary human
Gli genes include, for example, the Gli-1 gene sequence of GenBank
accession X07384 and the Gli-2 gene sequence of GenBank accession
AB007298. See also Kinzler et al. Nature 1988, 332, 371. The level
of gli expression can be determined, for example, by measuring the
level of mRNA (transcription) or the level of protein
(translation).
[0049] Thus, the methods of the present invention include the use
of small molecules which antagonize ptc inhibition of hedgehog
signalling, such as by activating smoothened or downstream
components of the signal pathway, in the regulation of repair
and/or functional performance of a wide range of cells, tissues and
organs, including those having a ptc gain-of-function phenotype,
hedgehog loss-of-function phenotype, or a smoothened
loss-of-function phenotype. 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, urogenital organs (e.g., bladder), and 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 which 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).
[0050] In one embodiment, the subject method can be to treat
epithelial cells having a ptc gain-of-function phenotype, hedgehog
loss-of-function phenotype, or a smoothened loss-of-function
phenotype. In general, an epithelial cell may be contacted with an
amount of a hedgehog agonist to induce epithelial tissue formation.
The subject method can be carried out on epithelial cells which may
be either dispersed in culture or a part of an intact tissue or
organ. Moreover, the method can be performed on cells which are
provided in culture (in vitro), or on cells in a whole animal (in
vivo).
[0051] The hedgehog agonists of the present invention may be used
as part of regimens in the treatment of disorders of, or surgical
or cosmetic repair of, such epithelial tissues as skin and skin
organs; corneal, lens and other ocular tissue; mucosal membranes;
and periodontal epithelium. The methods and compositions disclosed
herein provide for the treatment or prevention of a variety of
damaged epithelial and mucosal tissues. For instance, the subject
method can be used to control wound healing processes, as for
example may be desirable in connection with any surgery involving
epithelial tissue, such as from dermatological or periodontal
surgeries. Exemplary surgical repair for which hedgehog agonists
may be useful include severe burn and skin regeneration, skin
grafts, pressure sores, dermal ulcers, fissures, post surgery scar
reduction, and ulcerative colitis.
[0052] In another aspect of the present invention, a hedgehog
agonist can be used to effect the growth of hair, as for example in
the treatment of alopecia whereby hair growth is potentiated.
[0053] In another preferred embodiment, the subject method can be
used as part of a treatment regimen for malignant medulloblastoma
and other primary CNS malignant neuroectodermal tumors.
[0054] In another aspect, the present invention provides
pharmaceutical preparations comprising, as an active ingredient, a
hedgehog agonist, ptc antagonist, or smoothened agonist such as
described herein, formulated in an amount sufficient to promote, in
vivo, proliferation or other biological consequences of ptc
gain-of-function, hedgehog loss-of-function, or a smoothened
loss-of-function.
[0055] The subject treatments using hedgehog agonists, patched
antagonists, or smoothened agonists 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.
[0056] II. Definitions
[0057] For convenience, certain terms employed in the
specification, examples, and appended claims are collected
here.
[0058] 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, mis-expression 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.
[0059] "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.
[0060] 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.
[0061] "Dental tissue" refers to tissue in the mouth which is
similar to epithelial tissue, for example gum tissue. The method of
the present invention is useful for treating periodontal
disease.
[0062] "Dermal skin ulcers" refer to lesions on the skin caused by
superficial loss of tissue, usually with inflammation. Dermal skin
ulcers which 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.
[0063] The term "ED.sub.50" means the dose of a drug which produces
50% of its maximum response or effect.
[0064] An "effective amount" of, e.g., a hedgehog agonist, with
respect to the subject method of treatment, refers to an amount of
the agonist 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 the cosmetic
purpose.
[0065] 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 epithlelial
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.
[0066] The term "epithelialization" refers to healing by the growth
of epithelial tissue over a denuded surface.
[0067] 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.
[0068] 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.
[0069] "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.
[0070] 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.
[0071] 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 which 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.
[0072] The term "hedgehog agonist" refers to an agent which
potentiates or recapitulates the bioactivity of hedgehog, such as
to activate transcription of target genes. Preferred hedgehog
agonists can be used to overcome a ptc gain-of-function and/or a
smoothened loss-of-function, the latter also being refered to as
smoothened agonists. The term `hedgehog agonist` as used herein
refers not only to any agent that may act by directly activating
the normal function of the hedgehog protein, but also to any agent
that activates the hedgehog signalling pathway, and thus inhibits
the function of ptc.
[0073] The term "hedgehog loss-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 inhibitor, e.g., aberrant
inhibition of a hedgehog pathway. The loss-of-function may include
an increase in 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 loss-of-function` is also used herein to refer to
any similar cellular phenotype (e.g., exhibiting reduced
proliferation) which 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
cell with an abnormally low proliferation rate due to inactivation
of the hedgehog signalling pathway would have a `hedgehog
loss-of-function` phenotype, even if hedgehog is not mutated in
that cell.
[0074] As used herein, "immortalized cells" refers to cells which
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.
[0075] "Internal epithelial tissue" refers to tissue inside the
body which 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.
[0076] 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.
[0077] The term "LD.sub.50" means the dose of a drug which is
lethal in 50% of test subjects.
[0078] The term "nail" refers to the horny cutaneous plate on the
dorsal surface of the distal end of a finger or toe.
[0079] The term "patched gain-of-function" refers to an aberrant
modification or mutation of a ptc gene, or an increased level of
expression of the gene, which results in a phenotype which
resembles contacting a cell with a hedgehog inhibitor, e.g.,
aberrant deactivation of a hedgehog pathway. The gain-of-function
may include an increase of the ability of the ptc gene product to
regulate the level of expression of Ci genes, e.g., Gli1, Gli2 and
Gli3.
[0080] A "patient" or "subject" to be treated by the subject method
can mean either a human or non-human animal.
[0081] The term "prodrug" is intended to encompass compounds which,
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 which
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.
[0082] As used herein, "proliferating" and "proliferation" refer to
cells undergoing mitosis.
[0083] 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.
[0084] 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.
[0085] The term "small molecule" refers to a compound having a
molecular weight less than about 2500 amu, preferably less than
about 2000 amu, even more preferably less than about 1500 amu,
still more preferably less than about 1000 amu, or most preferably
less than about 750 amu.
[0086] The term "smoothened loss-of-function" refers to an aberrant
modification or mutation of a smo gene, or a decreased level of
expression of the gene, which results in a phenotype which
resembles contacting a cell with a hedgehog inhibitor, e.g.,
aberrant deactivation 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.
[0087] The term "therapeutic index" refers to the therapeutic index
of a drug defined as LD.sub.50/ED.sub.50.
[0088] The term "acylamino" is art-recognized and refers to a
moiety that can be represented by the general formula: 2
[0089] 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.
[0090] 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.
[0091] 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.
[0092] 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.
[0093] 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.
[0094] 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.
[0095] 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.
[0096] 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.
[0097] 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: 3
[0098] 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.
[0099] The term "amido" is art-recognized as an amino-substituted
carbonyl and includes a moiety that can be represented by the
general formula: 4
[0100] wherein R.sub.9, R.sub.10 are as defined above. Preferred
embodiments of the amide will not include imides which may be
unstable.
[0101] The term "aralkyl", as used herein, refers to an alkyl group
substituted with an aryl group (e.g., an aromatic or heteroaromatic
group).
[0102] 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.
[0103] The term "carbocycle", as used herein, refers to an aromatic
or non-aromatic ring in which each atom of the ring is carbon.
[0104] The term "carbonyl" is art-recognized and includes such
moieties as can be represented by the general formula: 5
[0105] 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
"thioformate." 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.
[0106] 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.
[0107] 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.
[0108] 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--.
[0109] A "phosphonamidite" can be represented in general formula:
6
[0110] wherein R.sub.9 and R.sub.10 are as defined above, Q.sub.2
represents O, S or N, and R.sub.48 represents a lower alkyl or an
aryl, Q.sub.2 represents O, S or N.
[0111] A "phosphoramidite" can be represented in general formula:
7
[0112] wherein R.sub.9 and R.sub.10 are as defined above, and
Q.sub.2 represents O, S or N.
[0113] A "phosphoryl" can in general be represented by the formula:
8
[0114] wherein Q.sub.1 represented S or O, and R.sub.46 represents
hydrogen, a lower alkyl or an aryl. When used to substitute, for
example, an alkyl, the phosphoryl group of the phosphorylalkyl can
be represented by the general formula: 9
[0115] wherein Q.sub.1 represented S or O, and each R.sub.46
independently represents hydrogen, a lower alkyl or an aryl,
Q.sub.2 represents O, S or N. When Q.sub.1 is an S, the phosphoryl
moiety is a "phosphorothioate".
[0116] 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.
[0117] The phrase "protecting group" as used herein means temporary
substituents which 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).
[0118] 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.
[0119] 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.
[0120] 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.
[0121] The term "sulfamoyl" is art-recognized and includes a moiety
that can be represented by the general formula: 10
[0122] in which R.sub.9 and R.sub.10 are as defined above.
[0123] The term "sulfate" is art recognized and includes a moiety
that can be represented by the general formula: 11
[0124] in which R.sub.41 is as defined above.
[0125] The term "sulfonamido" is art recognized and includes a
moiety that can be represented by the general formula: 12
[0126] in which R.sub.9 and R'.sub.11 are as defined above.
[0127] The term "sulfonate" is art-recognized and includes a moiety
that can be represented by the general formula: 13
[0128] in which R.sub.41 is an electron pair, hydrogen, alkyl,
cycloalkyl, or aryl.
[0129] The terms "sulfoxido" or "sulfinyl", as used herein, refers
to a moiety that can be represented by the general formula: 14
[0130] in which R.sub.44 is selected from the group consisting of
hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl,
aralkyl, or aryl.
[0131] 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.
[0132] 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.
[0133] 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
nonafilate 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.
[0134] 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.
[0135] 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.
[0136] 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.
[0137] 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
activate 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 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 which are in themselves known, but
are not mentioned here.
[0138] 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.
[0139] III. Exemplary Compounds of the Invention.
[0140] 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
represented by general formula (I): 15
[0141] wherein, as valence and stability permit,
[0142] Ar and Ar' independently represent substituted or
unsubstituted aryl or heteroaryl rings;
[0143] Y, independently for each occurrence, may be absent or
represent --N(R)--, --O--, --S--, or --Se--;
[0144] X 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)--,
and a methylene group optionally substituted with 1-2 groups such
as lower alkyl, alkenyl, or alkynyl groups;
[0145] M represents, independently for each occurrence, a
substituted or unsubstituted methylene group, such as --CH.sub.2--,
--CHF--, --CHOH--, --CH(Me)--, --C(.dbd.O)--, etc., or two M taken
together represent substituted or unsubstituted ethene or
ethyne;
[0146] R represents, independently for each occurrence, H or
substituted or unsubstituted aryl, heterocyclyl, heteroaryl,
aralkyl, heteroaralkyl, alkynyl, alkenyl, or alkyl, or two R taken
together may form a 4- to 8-membered ring, e.g., with N;
[0147] Cy and Cy' independenly represent substituted or
unsubstituted aryl, heterocyclyl, heteroaryl, or cycloalkyl,
including polycyclic groups;
[0148] i represents, independently for each occurrence, an integer
from 0 to 5, preferably from 0 to 2; and
[0149] n, individually for each occurence, represents an integer
from 0 to 10, preferably from 0 to 5.
[0150] In certain embodiments, M represents, independently for each
occurrence, a substituted or unsubstituted methylene group, such as
--CH.sub.2--, --CHF--, --CHOH--, --CH(Me)--, --C(.dbd.O)--,
etc.
[0151] In certain embodiments, Ar and Ar' represent phenyl rings,
e.g., unsubstituted or substituted with one or more groups
including heteroatoms such as O, N, and S. In certain embodiments,
at least one of Ar and Ar' represents a phenyl ring. In certain
embodiments, at least one of Ar and Ar' represents a heteroaryl
ring, e.g., a pyridyl, thiazolyl, thienyl, pyrimidyl, etc. In
certain embodiments, Y and Ar' are attached to Ar in a meta and/or
1,3-relationship.
[0152] In certain embodiments, Y is absent from all positions. In
embodiments wherein Y is present in a position, i preferably
represents an integer from 1-2 in an adjacent M.sub.i if i=0 would
result in two occurrences of Y being directly attached, or an
occurrence of Y being directly attached to N.
[0153] In certain embodiments, Cy' is a substituted or
unsubstituted aryl or heteroaryl. In certain embodiments, Cy' is
directly attached to X. In certain embodiments, Cy' is a
substituted or unsubstituted bicyclic or heteroaryl ring,
preferably both bicyclic and heteroaryl, such as benzothiophene,
benzofuran, benzopyrrole, benzopyridine, etc. In certain
embodiments, Cy' is a monocyclic aryl or heteroaryl ring
substituted at least with a substituted or unsubstituted aryl or
heteroaryl ring, e.g., forming a biaryl system. In certain
embodiments, Cy' includes two substituted or unsubstituted aryl or
heteroaryl rings, e.g., the same or different, directly connected
by one or more bonds, e.g., to form a biaryl or bicyclic ring
system.
[0154] In certain embodiments, X is selected from --C(.dbd.O)--,
--C(.dbd.S)--, and --S(O.sub.2)--.
[0155] In certain embodiments, Cy represents a substituted or
unsubstituted non-aromatic carbocyclic or heterocyclic ring, i.e.,
including at least one sp.sup.3 hybridized atom, and preferably a
plurality of sp.sup.3 hybridized atoms. In certain embodiments, Cy
includes an amine within the atoms of the ring or on a substitutent
of the ring, e.g., Cy is pyridyl, imidazolyl, pyrrolyl, piperidyl,
pyrrolidyl, piperazyl, etc., and/or bears an amino substituent. In
certain embodiments, Cy is a 5- to 7-membered ring. In certain
embodiments, Cy is directly attached to N. In embodiments wherein
Cy is a six-membered ring directly attached to N and bears an amino
substituent at the 4 position of the ring relative to N, the N and
amine substituents may be disposed trans on the ring.
[0156] In certain embodiments, substituents on Ar or Ar' are
selected from halogen, lower alkyl, lower alkenyl, aryl,
heteroaryl, carbonyl, thiocarbonyl, ketone, aldehyde, amino,
acylamino, cyano, nitro, hydroxyl, azido, sulfonyl, sulfoxido,
sulfate, sulfonate, sulfamoyl, sulfonamido, phosphoryl,
phosphonate, phosphinate, --(CH.sub.2).sub.palkyl,
--(CH.sub.2).sub.palkenyl, --(CH.sub.2).sub.palkynyl,
--(CH.sub.2).sub.paryl, --(CH.sub.2).sub.paralkyl,
--(CH.sub.2).sub.pOH, --(CH.sub.2).sub.pO-lower alkyl,
--(CH.sub.2).sub.pO-lower alkenyl, --O(CH.sub.2).sub.nR,
--(CH.sub.2).sub.pSH, --(CH.sub.2).sub.pS-lower alkyl,
--(CH.sub.2).sub.pS-lower alkenyl, --S(CH.sub.2).sub.nR,
--(CH.sub.2).sub.pN(R).sub.2, --(CH.sub.2).sub.pNR-lower alkyl,
--(CH.sub.2).sub.pNR-lower alkenyl, --NR(CH.sub.2).sub.nR, and
protected forms of the above, wherein p, individually for each
occurence, represents an integer from 0 to 10, preferably from 0 to
5.
[0157] In certain embodiments, compounds useful in the present
invention may be represented by general formula (II): 16
[0158] wherein, as valence and stability permit,
[0159] Ar and Ar' independently represent substituted or
unsubstituted aryl or heteroaryl rings;
[0160] Y, independently for each occurrence, may be absent or
represent --N(R)--, --O--, --S--, or --Se--;
[0161] X 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)--,
and a methylene group optionally substituted with 1-2 groups such
as lower alkyl, alkenyl, or alkynyl groups;
[0162] M represents, independently for each occurrence, a
substituted or unsubstituted methylene group, such as --CH.sub.2--,
--CHF--, --CHOH--, --CH(Me)--, --C(.dbd.O)--, etc., or two M taken
together represent substituted or unsubstituted ethene or ethyne,
wherein some or all occurrences of M in M.sub.j form all or part of
a cyclic structure;
[0163] R represents, independently for each occurrence, H or
substituted or unsubstituted aryl, heterocyclyl, heteroaryl,
aralkyl, heteroaralkyl, alkynyl, alkenyl, or alkyl, or two R taken
together may form a 4- to 8-membered ring, e.g., with N;
[0164] Cy' represents a substituted or unsubstituted aryl,
heterocyclyl, heteroaryl, or cycloalkyl, including polycyclic
groups;
[0165] j represents, independently for each occurrence, an integer
from 0 to 10, preferably from 2 to 7;
[0166] i represents, independently for each occurrence, an integer
from 0 to 5, preferably from 0 to 2; and
[0167] n, individually for each occurence, represents an integer
from 0 to 10, preferably from 0 to 5.
[0168] In certain embodiments, M represents, independently for each
occurrence, a substituted or unsubstituted methylene group, such as
--CH.sub.2--, --CHF--, --CHOH--, --CH(Me)--, --C(.dbd.O)--,
etc.
[0169] In certain embodiments, Ar and Ar' represent phenyl rings,
e.g., unsubstituted or substituted with one or more groups
including heteroatoms such as O, N, and S. In certain embodiments,
at least one of Ar and Ar' represents a phenyl ring. In certain
embodiments, at least one of Ar and Ar' represents a heteroaryl
ring, e.g., a pyridyl, thiazolyl, thienyl, pyrimidyl, etc. In
certain embodiments, Y and Ar' are attached to Ar in a meta and/or
1,3-relationship.
[0170] In certain embodiments, Y is absent from all positions. In
embodiments wherein Y is present in a position, i preferably
represents an integer from 1-2 in an adjacent M.sub.i if i=0 would
result in two occurrences of Y being directly attached, or an
occurrence of Y being directly attached to N or NR.sub.2.
[0171] In certain embodiments, Cy' is a substituted or
unsubstituted aryl or heteroaryl. In certain embodiments, Cy' is
directly attached to X. In certain embodiments, Cy' is a
substituted or unsubstituted bicyclic or heteroaryl ring,
preferably both bicyclic and heteroaryl, such as benzothiophene,
benzofuran, benzopyrrole, benzopyridine, etc. In certain
embodiments, Cy' is a monocyclic aryl or heteroaryl ring
substituted at least with a substituted or unsubstituted aryl or
heteroaryl ring, e.g., forming a biaryl system. In certain
embodiments, Cy' includes two substituted or unsubstituted aryl or
heteroaryl rings, e.g., the same or different, directly connected
by one or more bonds, e.g., to form a biaryl or bicyclic ring
system.
[0172] In certain embodiments, X is selected from --C(.dbd.O)--,
--C(.dbd.S)--, and --S(O.sub.2)--.
[0173] In certain embodiments, NR.sub.2 represents a primary amine
or a secondary or tertiary amine substituted with one or two lower
alkyl groups, aryl groups, or aralkyl groups, respectively,
preferably a primary amine.
[0174] In certain embodiments, substituents on Ar or Ar' are
selected from halogen, lower alkyl, lower alkenyl, aryl,
heteroaryl, carbonyl, thiocarbonyl, ketone, aldehyde, amino,
acylamino, cyano, nitro, hydroxyl, azido, sulfonyl, sulfoxido,
sulfate, sulfonate, sulfamoyl, sulfonamido, phosphoryl,
phosphonate, phosphinate, --(CH.sub.2).sub.palkyl,
--(CH.sub.2).sub.palkenyl, --(CH.sub.2).sub.palkynyl,
--(CH.sub.2).sub.paryl, --(CH.sub.2).sub.paralkyl,
--(CH.sub.2).sub.pOH, --(CH.sub.2).sub.pO-lower alkyl,
--(CH.sub.2).sub.pO-lower alkenyl, --O(CH.sub.2).sub.nR,
--(CH.sub.2).sub.pSH, --(CH.sub.2).sub.pS-lower alkyl,
--(CH.sub.2).sub.pS-lower alkenyl, --S(CH.sub.2).sub.nR,
--(CH.sub.2).sub.pN(R).sub.2, --(CH.sub.2).sub.pNR-lower alkyl,
--(CH.sub.2).sub.pNR-lower alkenyl, --NR(CH.sub.2).sub.nR, and
protected forms of the above, wherein p, individually for each
occurence, represents an integer from 0 to 10, preferably from 0 to
5.
[0175] In certain embodiments, compounds useful in the present
invention may be represented by general formula (III): 17
[0176] wherein, as valence and stability permit,
[0177] Ar and Ar' independently represent substituted or
unsubstituted aryl or heteroaryl rings;
[0178] Y, independently for each occurrence, may be absent or
represent --N(R)--, --O--, --S--, or --Se--;
[0179] X 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)--,
and a methylene group optionally substituted with 1-2 groups such
as lower alkyl, alkenyl, or alkynyl groups;
[0180] M represents, independently for each occurrence, a
substituted or unsubstituted methylene group, such as --CH.sub.2--,
--CHF--, --CHOH--, --CH(Me)--, --C(.dbd.O)--, etc., or two M taken
together represent substituted or unsubstituted ethene or
ethyne;
[0181] R represents, independently for each occurrence, H or
substituted or unsubstituted aryl, heterocyclyl, heteroaryl,
aralkyl, heteroaralkyl, alkynyl, alkenyl, or alkyl, or two R taken
together may form a 4- to 8-membered ring, e.g., with N;
[0182] Cy and Cy' independenly represent substituted or
unsubstituted aryl, heterocyclyl, heteroaryl, or cycloalkyl,
including polycyclic groups;
[0183] i represents, independently for each occurrence, an integer
from 0 to 5, preferably from 0 to 2; and
[0184] n, individually for each occurence, represents an integer
from 0 to 10, preferably from 0 to 5.
[0185] In certain embodiments, M represents, independently for each
occurrence, a substituted or unsubstituted methylene group, such as
--CH.sub.2--, --CHF--, --CHOH--, --CH(Me)--, --C(.dbd.O)--,
etc.
[0186] In certain embodiments, Ar and Ar' represent phenyl rings,
e.g., unsubstituted or substituted with one or more groups
including heteroatoms such as O, N, and S. In certain embodiments,
at least one of Ar and Ar' represents a phenyl ring. In certain
embodiments, at least one of Ar and Ar' represents a heteroaryl
ring, e.g., a pyridyl, thiazolyl, thienyl, pyrimidyl, etc. In
certain embodiments, Y and Ar' are attached to Ar in a meta and/or
1,3-relationship.
[0187] In certain embodiments, Y is absent from all positions. In
embodiments wherein Y is present in a position, i preferably
represents an integer from 1-2 in an adjacent M.sub.i if i=0 would
result in two occurrences of Y being directly attached, or an
occurrence of Y being directly attached to N or NR.sub.2.
[0188] In certain embodiments, Cy' is a substituted or
unsubstituted aryl or heteroaryl. In certain embodiments, Cy' is
directly attached to X. In certain embodiments, Cy' is a
substituted or unsubstituted bicyclic or heteroaryl ring,
preferably both bicyclic and heteroaryl, such as benzothiophene,
benzofuran, benzopyrrole, benzopyridine, etc. In certain
embodiments, Cy' is a monocyclic aryl or heteroaryl ring
substituted at least with a substituted or unsubstituted aryl or
heteroaryl ring, e.g., forming a biaryl system. In certain
embodiments, Cy' includes two substituted or unsubstituted aryl or
heteroaryl rings, e.g., the same or different, directly connected
by one or more bonds, e.g., to form a biaryl or bicyclic ring
system.
[0189] In certain embodiments, X is selected from --C(.dbd.O)--,
--C(.dbd.S)--, and --S(O.sub.2)--.
[0190] In certain embodiments, NR.sub.2 represents a primary amine
or a secondary or tertiary amine substituted with one or two lower
alkyl groups, aryl groups, or aralkyl groups, respectively,
preferably a primary amine.
[0191] In certain embodiments, Cy represents a substituted or
unsubstituted non-aromatic carbocyclic or heterocyclic ring, i.e.,
including at least one sp.sup.3 hybridized atom, and preferably a
plurality of sp.sup.3 hybridized atoms. In certain embodiments, Cy
is directly attached to N and/or to NR.sub.2. In certain
embodiments, Cy is a 5- to 7-membered ring. In embodiments wherein
Cy is a six-membered ring directly attached to N and bears an amino
substituent at the 4 position of the ring relative to N, the N and
amine substituents may be disposed trans on the ring.
[0192] In certain embodiments, substituents on Ar or Ar' are
selected from halogen, lower alkyl, lower alkenyl, aryl,
heteroaryl, carbonyl, thiocarbonyl, ketone, aldehyde, amino,
acylamino, cyano, nitro, hydroxyl, azido, sulfonyl, sulfoxido,
sulfate, sulfonate, sulfamoyl, sulfonamido, phosphoryl,
phosphonate, phosphinate, --(CH.sub.2).sub.palkyl,
--(CH.sub.2).sub.palkenyl, --(CH.sub.2).sub.palkynyl,
--(CH.sub.2).sub.paryl, --(CH.sub.2).sub.paralkyl,
--(CH.sub.2).sub.pOH, --(CH.sub.2).sub.pO-lower alkyl,
--(CH.sub.2).sub.pO-lower alkenyl, --O(CH.sub.2).sub.nR,
--(CH.sub.2).sub.pSH, --(CH.sub.2).sub.pS-lower alkyl,
--(CH.sub.2).sub.pS-lower alkenyl, --S(CH.sub.2).sub.nR,
--(CH.sub.2).sub.pN(R).sub.2, --(CH.sub.2).sub.pNR-lower alkyl,
--(CH.sub.2).sub.pNR-lower alkenyl, --NR(CH.sub.2).sub.nR, and
protected forms of the above, wherein p, individually for each
occurence, represents an integer from 0 to 10, preferably from 0 to
5.
[0193] In certain embodiments, compounds useful in the subject
methods include compounds represented by general forumla (IV):
18
[0194] wherein, as valence and stability permit,
[0195] Cy' represents a substituted or unsubstituted aryl or
heteroaryl ring, including polycyclics;
[0196] Y, independently for each occurrence, may be absent or
represent --N(R)--, --O--, --S--, or --Se--;
[0197] X 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)--,
and a methylene group optionally substituted with 1-2 groups such
as lower alkyl, alkenyl, or alkynyl groups;
[0198] M represents, independently for each occurrence, a
substituted or unsubstituted methylene group, such as --CH.sub.2--,
--CHF--, --CHOH--, --CH(Me)--, --C(.dbd.O)--, etc., or two M taken
together represent substituted or unsubstituted ethene or
ethyne;
[0199] R represents, independently for each occurrence, H or
substituted or unsubstituted aryl, heterocyclyl, heteroaryl,
aralkyl, heteroaralkyl, alkynyl, alkenyl, or alkyl, or two R taken
together may form a 4- to 8-membered ring, e.g., with N;
[0200] R.sub.1 and R.sub.2 represent, independently and as valency
permits, from 0-5 substituents on the ring to which it is attached,
selected from halogen, lower alkyl, lower alkenyl, aryl,
heteroaryl, carbonyl, thiocarbonyl, ketone, aldehyde, amino,
acylamino, amido, amidino, cyano, nitro, hydroxyl, azido, sulfonyl,
sulfoxido, sulfate, sulfonate, sulfamoyl, sulfonamido, phosphoryl,
phosphonate, phosphinate, --(CH.sub.2).sub.palkyl,
--(CH.sub.2).sub.palkenyl, --(CH.sub.2).sub.palkynyl,
--(CH.sub.2).sub.paryl, --(CH.sub.2).sub.paralkyl,
--(CH.sub.2).sub.pOH, --(CH.sub.2).sub.pO-lowe- r alkyl,
--(CH.sub.2).sub.pO-lower alkenyl, --O(CH.sub.2).sub.nR,
--(CH.sub.2).sub.pSH, --(CH.sub.2).sub.pS-lower alkyl,
--(CH.sub.2).sub.pS-lower alkenyl, --S(CH.sub.2).sub.nR,
--(CH.sub.2).sub.pN(R).sub.2, --(CH.sub.2).sub.pNR-lower alkyl,
--(CH.sub.2).sub.pNR-lower alkenyl, --NR(CH.sub.2).sub.nR, and
protected forms of the above;
[0201] Cy represents substituted or unsubstituted aryl,
heterocyclyl, heteroaryl, or cycloalkyl, including polycyclic
groups;
[0202] i represents, independently for each occurrence, an integer
from 0 to 5, preferably from 0 to 2; and
[0203] p and n, individually for each occurrence, represent
integers from 0 to 10, preferably from 0 to 5.
[0204] In certain embodiments, M represents, independently for each
occurrence, a substituted or unsubstituted methylene group, such as
--CH.sub.2--, --CHF--, --CHOH--, --CH(Me)--, --C(.dbd.O)--,
etc.
[0205] In certain embodiments, Cy' represents a substituted or
unsubstituted bicyclic or heterocyclic ring system, preferably both
bicyclic and heteroaryl, such as benzothiophene, benzofuran,
benzopyrrole, benzopyridine, etc. In certain embodiments, Cy' is
directly attached to X. In certain embodiments, Cy' is a monocyclic
aryl or heteroaryl ring substituted at least with a substituted or
unsubstituted aryl or heteroaryl ring, e.g., forming a biaryl
system. In certain embodiments, Cy' includes two substituted or
unsubstituted aryl or heteroaryl rings, e.g., the same or
different, directly connected by one or more bonds, e.g., to form a
biaryl or bicyclic ring system.
[0206] In certain embodiments, Y is absent from all positions. In
embodiments wherein Y is present in a position, i preferably
represents an integer from 1-2 in an adjacent M.sub.i if i=0 would
result in two occurrences of Y being directly attached, or an
occurrence of Y being directly attached to N.
[0207] In certain embodiments, X is selected from --C(.dbd.O)--,
--C(.dbd.S)--, and --S(O.sub.2)--.
[0208] In certain embodiments, Cy represents a substituted or
unsubstituted non-aromatic carbocyclic or heterocyclic ring, i.e.,
including at least one sp.sup.3 hybridized atom, and preferably a
plurality of sp.sup.3 hybridized atoms. In certain embodiments, Cy
includes an amine within the atoms of the ring or on a substitutent
of the ring, e.g., Cy is pyridyl, imidazolyl, pyrrolyl, piperidyl,
pyrrolidyl, piperazyl, etc., and/or bears an amino substituent. In
certain embodiments, Cy is directly attached to N. In certain
embodiments, Cy is a 5- to 7-membered ring. In embodiments wherein
Cy is a six-membered ring directly attached to N and bears an amino
substituent at the 4 position of the ring relative to N, the N and
amine substituents may be disposed trans on the ring.
[0209] In certain embodiments, R.sub.1 and R.sub.2 represent,
independently and as valency permits, from 0-5 substituents on the
ring to which it is attached, selected from halogen, lower alkyl,
lower alkenyl, carbonyl, thiocarbonyl, ketone, aldehyde, amino,
acylamino, cyano, nitro, hydroxyl, azido, sulfonyl, sulfoxido,
sulfate, sulfonate, sulfamoyl, sulfonamido, phosphoryl,
phosphonate, phosphinate, --(CH.sub.2).sub.palkyl,
--(CH.sub.2).sub.palkenyl, --(CH.sub.2).sub.palkynyl,
--(CH.sub.2).sub.paryl, --(CH.sub.2).sub.paralkyl,
--(CH.sub.2).sub.pOH, --(CH.sub.2).sub.pO-lowe- r alkyl,
--(CH.sub.2).sub.pO-lower alkenyl, --O(CH.sub.2).sub.nR,
--(CH.sub.2).sub.pSH, --(CH.sub.2).sub.pS-lower alkyl,
--(CH.sub.2).sub.pS-lower alkenyl, --S(CH.sub.2).sub.nR,
--(CH.sub.2).sub.pN(R).sub.2, --(CH.sub.2).sub.pNR-lower alkyl,
--(CH.sub.2).sub.pNR-lower alkenyl, --NR(CH.sub.2).sub.nR, and
protected forms of the above, wherein p, individually for each
occurence, represents an integer from 0 to 10, preferably from 0 to
5.
[0210] In certain embodiments, compounds useful in the present
invention may be represented by general formula (V): 19
[0211] wherein, as valence and stability permit,
[0212] Cy' represents a substituted or unsubstituted aryl or
heteroaryl ring, including polycyclics;
[0213] Y, independently for each occurrence, may be absent or
represent --N(R)--, --O--, --S--, or --Se--;
[0214] X 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)--,
and a methylene group optionally substituted with 1-2 groups such
as lower alkyl, alkenyl, or alkynyl groups;
[0215] M represents, independently for each occurrence, a
substituted or unsubstituted methylene group, such as --CH.sub.2--,
--CHF--, --CHOH--, --CH(Me)--, --C(.dbd.O)--, etc., or two M taken
together represent substituted or unsubstituted ethene or
ethyne;
[0216] R represents, independently for each occurrence, H or
substituted or unsubstituted aryl, heterocyclyl, heteroaryl,
aralkyl, heteroaralkyl, alkynyl, alkenyl, or alkyl, or two R taken
together may form a 4- to 8-membered ring, e.g., with N;
[0217] R.sub.1 and R.sub.2 represent, independently and as valency
permits, from 0-5 substituents on the ring to which it is attached,
selected from halogen, lower alkyl, lower alkenyl, aryl,
heteroaryl, carbonyl, thiocarbonyl, ketone, aldehyde, amino,
acylamino, amido, amidino, cyano, nitro, hydroxyl, azido, sulfonyl,
sulfoxido, sulfate, sulfonate, sulfamoyl, sulfonamido, phosphoryl,
phosphonate, phosphinate, --(CH.sub.2).sub.palkyl,
--(CH.sub.2).sub.palkenyl, --(CH.sub.2).sub.palkynyl,
--(CH.sub.2).sub.paryl, --(CH.sub.2).sub.paralkyl,
--(CH.sub.2).sub.pOH, --(CH.sub.2).sub.pO-lowe- r alkyl,
--(CH.sub.2).sub.pO-lower alkenyl, --O(CH.sub.2).sub.nR,
--(CH.sub.2).sub.pSH, --(CH.sub.2).sub.pS-lower alkyl,
--(CH.sub.2).sub.pS-lower alkenyl, --S(CH.sub.2).sub.nR,
--(CH.sub.2).sub.pN(R).sub.2, --(CH.sub.2).sub.pNR-lower alkyl,
--(CH.sub.2).sub.pNR-lower alkenyl, --NR(CH.sub.2).sub.nR, and
protected forms of the above;
[0218] Cy' represents a substituted or unsubstituted aryl,
heterocyclyl, heteroaryl, or cycloalkyl, including polycyclic
groups;
[0219] j represents, independently for each occurrence, an integer
from 0 to 10, preferably from 2 to 7;
[0220] i represents, independently for each occurrence, an integer
from 0 to 5, preferably from 0 to 2; and
[0221] p and n, individually for each occurrence, represent
integers from 0 to 10, preferably from 0 to 5.
[0222] In certain embodiments, M represents, independently for each
occurrence, a substituted or unsubstituted methylene group, such as
--CH.sub.2--, --CHF--, --CHOH--, --CH(Me)--, --C(.dbd.O)--,
etc.
[0223] In certain embodiments, Cy' represents a substituted or
unsubstituted bicyclic or heterocyclic ring system, preferably both
bicyclic and heteroaryl, such as benzothiophene, benzofuran,
benzopyrrole, benzopyridine, etc. In certain embodiments, Cy' is
directly attached to X. In certain embodiments, Cy' is a monocyclic
aryl or heteroaryl ring substituted at least with a substituted or
unsubstituted aryl or heteroaryl ring, e.g., forming a biaryl
system. In certain embodiments, Cy' includes two substituted or
unsubstituted aryl or heteroaryl rings, e.g., the same or
different, directly connected by one or more bonds, e.g., to form a
biaryl or bicyclic ring system.
[0224] In certain embodiments, Y is absent from all positions. In
embodiments wherein Y is present in a position, i preferably
represents an integer from 1-2 in an adjacent M.sub.i if i=0 would
result in two occurrences of Y being directly attached, or an
occurrence of Y being directly attached to N or NR.sub.2.
[0225] In certain embodiments, X is selected from --C(.dbd.O)--,
--C(.dbd.S)--, and --S(O.sub.2)--.
[0226] In certain embodiments, NR.sub.2 represents a primary amine
or a secondary or tertiary amine substituted with one or two lower
alkyl groups, aryl groups, or aralkyl groups, respectively,
preferably a primary amine.
[0227] In certain embodiments, R.sub.1 and R.sub.2 represent,
independently and as valency permits, from 0-5 substituents on the
ring to which it is attached, selected from halogen, lower alkyl,
lower alkenyl, carbonyl, thiocarbonyl, ketone, aldehyde, amino,
acylamino, cyano, nitro, hydroxyl, azido, sulfonyl, sulfoxido,
sulfate, sulfonate, sulfamoyl, sulfonamido, phosphoryl,
phosphonate, phosphinate, --(CH.sub.2).sub.palkyl,
--(CH.sub.2).sub.palkenyl, --(CH.sub.2).sub.palkynyl,
--(CH.sub.2).sub.paryl, --(CH.sub.2).sub.paralkyl,
--(CH.sub.2).sub.pOH, --(CH.sub.2).sub.pO-lowe- r alkyl,
--(CH.sub.2).sub.pO-lower alkenyl, --O(CH.sub.2).sub.nR,
--(CH.sub.2).sub.pSH, --(CH.sub.2).sub.pS-lower alkyl,
--(CH.sub.2).sub.pS-lower alkenyl, --S(CH.sub.2).sub.nR,
--(CH.sub.2).sub.pN(R).sub.2, --(CH.sub.2).sub.pNR-lower alkyl,
--(CH.sub.2).sub.pNR-lower alkenyl, --NR(CH.sub.2).sub.nR, and
protected forms of the above, wherein p, individually for each
occurence, represents an integer from 0 to 10, preferably from 0 to
5.
[0228] In certain embodiments, compounds useful in the present
invention may be represented by general formula (VI): 20
[0229] wherein, as valence and stability permit,
[0230] Cy' represents a substituted or unsubstituted aryl or
heteroaryl ring, including polycyclics;
[0231] Y, independently for each occurrence, may be absent or
represent --N(R)--, --O--, --S--, or --Se--;
[0232] X 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)--,
and a methylene group optionally substituted with 1-2 groups such
as lower alkyl, alkenyl, or alkynyl groups;
[0233] M represents, independently for each occurrence, a
substituted or unsubstituted methylene group, such as --CH.sub.2--,
--CHF--, --CHOH--, --CH(Me)--, --C(.dbd.O)--, etc., or two M taken
together represent substituted or unsubstituted ethene or
ethyne;
[0234] R represents, independently for each occurrence, H or
substituted or unsubstituted aryl, heterocyclyl, heteroaryl,
aralkyl, heteroaralkyl, alkynyl, alkenyl, or alkyl, or two R taken
together may form a 4- to 8-membered ring, e.g., with N;
[0235] Cy represents substituted or unsubstituted aryl,
heterocyclyl, heteroaryl, or cycloalkyl, including polycyclic
groups;
[0236] i represents, independently for each occurrence, an integer
from 0 to 5, preferably from 0 to 2; and
[0237] n and p, individually for each occurrence, represent
integers from 0 to 10, preferably from 0 to 5.
[0238] In certain embodiments, M represents, independently for each
occurrence, a substituted or unsubstituted methylene group, such as
--CH.sub.2--, --CHF--, --CHOH--, --CH(Me)--, --C(.dbd.O)--,
etc.
[0239] In certain embodiments, Cy' represents a substituted or
unsubstituted bicyclic or heteroaryl ring system, preferably both
bicyclic and heteroaryl, e.g., benzothiophene, benzofuran,
benzopyrrole, benzopyridyl, etc. In certain embodiments, Cy' is
directly attached to X. In certain embodiments, Cy' is a monocyclic
aryl or heteroaryl ring substituted at least with a substituted or
unsubstituted aryl or heteroaryl ring, e.g., forming a biaryl
system. In certain embodiments, Cy' includes two substituted or
unsubstituted aryl or heteroaryl rings, e.g., the same or
different, directly connected by one or more bonds, e.g., to form a
biaryl or bicyclic ring system.
[0240] In certain embodiments, Y is absent from all positions. In
embodiments wherein Y is present in a position, i preferably
represents an integer from 1-2 in an adjacent M.sub.i if i=0 would
result in two occurrences of Y being directly attached, or an
occurrence of Y being directly attached to N or NR.sub.2.
[0241] In certain embodiments, X is selected from --C(.dbd.O)--,
--C(.dbd.S)--, and --S(O.sub.2)--.
[0242] In certain embodiments, NR.sub.2 represents a primary amine
or a secondary or tertiary amine substituted with one or two lower
alkyl groups, aryl groups, or aralkyl groups, respectively,
preferably a primary amine.
[0243] In certain embodiments, Cy represents a substituted or
unsubstituted non-aromatic carbocyclic or heterocyclic ring, i.e.,
including at least one sp.sup.3 hybridized atom, and preferably a
plurality of sp.sup.3 hybridized atoms. In certain embodiments, Cy
is directly attached to N and/or to NR.sub.2. In certain
embodiments, Cy is a 5- to 7-membered ring. In embodiments wherein
Cy is a six-membered ring directly attached to N and bears an amino
substituent at the 4 position of the ring relative to N, the N and
amine substituents may be disposed trans on the ring.
[0244] In certain embodiments, R.sub.1 and R.sub.2 represent,
independently and as valency permits, from 0-5 substituents on the
ring to which it is attached, selected from halogen, lower alkyl,
lower alkenyl, carbonyl, thiocarbonyl, ketone, aldehyde, amino,
acylamino, cyano, nitro, hydroxyl, azido, sulfonyl, sulfoxido,
sulfate, sulfonate, sulfamoyl, sulfonamido, phosphoryl,
phosphonate, phosphinate, --(CH.sub.2).sub.palkyl,
--(CH.sub.2).sub.palkenyl, --(CH.sub.2).sub.palkynyl,
--(CH.sub.2).sub.paryl, --(CH.sub.2).sub.paralkyl,
--(CH.sub.2).sub.pOH, --(CH.sub.2).sub.pO-lowe- r alkyl,
--(CH.sub.2).sub.pO-lower alkenyl, --O(CH.sub.2).sub.nR,
--(CH.sub.2).sub.pSH, --(CH.sub.2).sub.pS-lower alkyl,
--(CH.sub.2).sub.pS-lower alkenyl, --S(CH.sub.2).sub.nR,
--(CH.sub.2).sub.pN(R).sub.2, --(CH.sub.2).sub.pNR-lower alkyl,
--(CH.sub.2).sub.pNR-lower alkenyl, --NR(CH.sub.2).sub.nR, and
protected forms of the above, wherein p, individually for each
occurence, represents an integer from 0 to 10, preferably from 0 to
5.
[0245] In certain embodiments, a subject compound has the structure
of Formula VII: 21
[0246] wherein, as valence and stability permit,
[0247] Cy represents a substituted or unsubstituted heterocyclyl or
cycloalkyl;
[0248] Cy' is a substituted or unsubstituted aryl or heteroaryl
ring;
[0249] W is O or S;
[0250] R represents, independently for each occurrence, H or
substituted or unsubstituted aryl, heterocyclyl, heteroaryl,
aralkyl, heteroaralkyl, alkynyl, alkenyl, or alkyl, or two R taken
together may form a 4- to 8-membered ring, e.g., with N;
[0251] R.sub.1 and R.sub.2 represent, independently and as valency
permits, from 0-5 substituents on the ring to which it is attached,
selected from halogen, lower alkyl, lower alkenyl, aryl,
heteroaryl, carbonyl, thiocarbonyl, ketone, aldehyde, amino,
acylamino, amido, amidino, cyano, nitro, hydroxyl, azido, sulfonyl,
sulfoxido, sulfate, sulfonate, sulfamoyl, sulfonamido, phosphoryl,
phosphonate, phosphinate, --(CH.sub.2).sub.palkyl,
--(CH.sub.2).sub.palkenyl, --(CH.sub.2).sub.palkynyl,
--(CH.sub.2).sub.paryl, --(CH.sub.2).sub.paralkyl,
--(CH.sub.2).sub.pOH, --(CH.sub.2).sub.pO-lowe- r alkyl,
--(CH.sub.2).sub.pO-lower alkenyl, --O(CH.sub.2).sub.nR,
--(CH.sub.2).sub.pSH, --(CH.sub.2).sub.pS-lower alkyl,
--(CH.sub.2).sub.pS-lower alkenyl, --S(CH.sub.2).sub.nR,
--(CH.sub.2).sub.pN(R).sub.2, --(CH.sub.2).sub.pNR-lower alkyl,
--(CH.sub.2).sub.pNR-lower alkenyl, --NR(CH.sub.2).sub.nR, and
protected forms of the above;
[0252] n and p, individually for each occurrence, represent
integers from 0 to 10.
[0253] In certain embodiments, Cy' represents a substituted or
unsubstituted bicyclic or heteroaryl ring system, preferably both
bicyclic and heteroaryl, e.g., benzothiophene, benzofuran,
benzopyrrole, benzopyridyl, etc. In certain embodiments, Cy' is
directly attached to X.
[0254] In certain embodiments, NR.sub.2 represents a primary amine
or a secondary or tertiary amine substituted with one or two lower
alkyl groups, aryl groups, or aralkyl groups, respectively,
preferably a primary amine.
[0255] In certain embodiments, Cy represents a substituted or
unsubstituted saturated carbocyclic or heterocyclic ring, i.e.,
composed of a plurality of sp.sup.3 hybridized atoms. In certain
embodiments, Cy is a 5- to 7-membered ring. In embodiments wherein
Cy is a six-membered ring directly attached to N and bears an amino
substituent at the 4 position of the ring relative to N, the N and
amine substituents may be disposed trans on the ring.
[0256] In certain embodiments, R.sub.1 and R.sub.2 represent,
independently and as valency permits, from 0-5 substituents on the
ring to which it is attached, selected from halogen, lower alkyl,
lower alkenyl, carbonyl, thiocarbonyl, ketone, aldehyde, amino,
acylamino, cyano, nitro, hydroxyl, sulfonyl, sulfoxido, sulfate,
sulfonate, sulfamoyl, sulfonamido, --(CH.sub.2).sub.palkyl,
--(CH.sub.2).sub.palkeny- l, --(CH.sub.2).sub.palkynyl,
--(CH.sub.2).sub.paryl, --(CH.sub.2).sub.paralkyl,
--(CH.sub.2).sub.pOH, --(CH.sub.2).sub.pO-lowe- r alkyl,
--(CH.sub.2).sub.pO-lower alkenyl, --O(CH.sub.2).sub.nR,
--(CH.sub.2).sub.pSH, --(CH.sub.2).sub.pS-lower alkyl,
--(CH.sub.2).sub.pS-lower alkenyl, --S(CH.sub.2).sub.nR,
--(CH.sub.2).sub.pN(R).sub.2, --(CH.sub.2).sub.pNR-lower alkyl,
--(CH.sub.2).sub.pNR-lower alkenyl, --NR(CH.sub.2).sub.nR, and
protected forms of the above.
[0257] In certain embodiments, a subject compound has a structure
of Formula VII: 22
[0258] wherein, as valence and stability permit,
[0259] U represents a substituted or unsubstituted aryl or
heteroaryl ring fused to the nitrogen-containing ring;
[0260] V represents a lower alkylene group, such as methylene,
1,2-ethylene, 1,1-ethylene, 1,1-propylene, 1,2-propylene,
1,3-propylene, etc.;
[0261] W represents S or O, preferably O;
[0262] X represents C.dbd.O, C.dbd.S, or SO.sub.2;
[0263] R.sub.3 represents substituted or unsubstituted aryl,
heteroaryl, lower alkyl, lower alkenyl, lower alkynyl, carbocyclyl,
carbocyclylalkyl, heterocyclyl, heterocyclylalkyl, aralkyl, or
heteroaralkyl;
[0264] R.sub.4 represents substituted or unsubstituted aralkyl or
lower alkyl, such as phenethyl, benzyl, or aminoalkyl, etc.;
[0265] R.sub.5 represents substituted or unsubstituted aryl,
heteroaryl, aralkyl, or heteroaralkyl, including polycyclic
aromatic or heteroaromatic groups.
[0266] In certain embodiments, U represents a phenyl ring fused to
the nitrogen-containing ring.
[0267] In certain embodiments, R.sub.3 is selected from substituted
or unsubstituted aryl, heteroaryl, lower alkyl, lower alkenyl,
aralkyl, and heteroaralkyl.
[0268] In certain embodiments, R.sub.4 is an unsubstituted lower
alkyl group, or is a lower alkyl group substituted with a secondary
or tertiary amine.
[0269] In certain embodiments, R.sub.5 is selected from substituted
or unsubstituted phenyl or naphthyl, or is a diarylalkyl group,
such as 2,2-diphenylethyl, diphenylmethyl, etc.
[0270] In certain embodiments, the subject agonists 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.,
ptc-1, ptc-2, etc.
[0271] In certain preferred embodiments, the subject agonists
inhibit ptc gain-of-function, hedgehog loss-of-function, or
smoothened loss-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 mM or less.
[0272] 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.
[0273] In certain embodiments, a compound which is an agonist of
the hedgehog pathway is chosen to selectively agonize hedgehog
activity over protein kinases other than PKA, such as PKC, e.g.,
the compound modulates the activity of the PKA/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
activator of the hedgehog pathway may activate hedgehog activity
with a K.sub.i at least an order of magnitude lower than its
K.sub.i for activation 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/hedgehog activation is less than 10 nM, preferably less than 1
nM, even more preferably less than 0.1 nM.
[0274] IV. Exemplary Applications of Method and Compositions
[0275] Another aspect of the present invention relates to a method
of modulating a differentiated state, survival, and/or
proliferation of a cell having a ptc gain-of-function, hedgehog
loss-of-function, or smoothened loss-of-function, by contacting the
cells with a hedgehog agonist according to the subject method and
as the circumstances may warrant.
[0276] 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 agonist, whether inductive or
anti-inductive with respect proliferation or differentiation of a
given tissue, can be, as appropriate, any of the preparations
described above.
[0277] For example, the present method is applicable to cell
culture techniques wherein, whether for genetic or biochemical
reasons, the cells have a ptc gain-of-function, hedgehog
loss-of-function, or smoothened loss-of-function phenotype. 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 agonist 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,
motorneurons. Such neuronal cultures can be used as convenient
assay systems as well as sources of implantable cells for
therapeutic treatments.
[0278] According to the present invention, large numbers of
non-tumorigenic neural progenitor cells can be perpetuated in vitro
and their rate of proliferation and/or differentiation can be
affected by contact with hedgehog agonists of the present
invention. Generally, a method is provided comprising the steps of
isolating neural progenitor cells from an animal, perpetuating
these cells in vitro or in vivo, preferably in the presence of
growth factors, and regulating the differentiation of these cells
into particular neural phenotypes, e.g., neurons and glia, by
contacting the cells with a hedgehog agonist.
[0279] Progenitor cells are thought to be under a tonic inhibitory
influence which maintains the progenitors in a suppressed state
until their differentiation is required. However, recent techniques
have been provided which permit these cells to proliferate, and
unlike neurons which are terminally differentiated and therefore
non-dividing, they can be produced in unlimited number and are
highly suitable for transplantation into heterologous and
autologous hosts with neurodegenerative diseases.
[0280] By "progenitor" it is meant an oligopotent or multipotent
stem cell which is able to divide without limit and, under specific
conditions, can produce daughter cells which terminally
differentiate such as into neurons and glia. These cells can be
used for transplantation into a heterologous or autologous host. By
heterologous is meant a host other than the animal from which the
progenitor cells were originally derived. By autologous is meant
the identical host from which the cells were originally
derived.
[0281] Cells can be obtained from embryonic, post-natal, juvenile
or adult neural tissue from any animal. By any animal is meant any
multicellular animal which contains nervous tissue. More
particularly, is meant any fish, reptile, bird, amphibian or mammal
and the like. The most preferable donors are mammals, especially
mice and humans.
[0282] In the case of a heterologous donor animal, the animal may
be euthanized, and the brain and specific area of interest removed
using a sterile procedure. Brain areas of particular interest
include any area from which progenitor cells can be obtained which
will serve to restore function to a degenerated area of the host's
brain. These regions include areas of the central nervous system
(CNS) including the cerebral cortex, cerebellum, midbrain,
brainstem, spinal cord and ventricular tissue, and areas of the
peripheral nervous system (PNS) including the carotid body and the
adrenal medulla. More particularly, these areas include regions in
the basal ganglia, preferably the striatum which consists of the
caudate and putamen, or various cell groups such as the globus
pallidus, the subthalamic nucleus, the nucleus basalis which is
found to be degenerated in Alzheimer's disease patients, or the
substantia nigra pars compacta which is found to be degenerated in
Parkinson's disease patients.
[0283] Human heterologous neural progenitor cells may be derived
from fetal tissue obtained from elective abortion, or from a
post-natal, juvenile or adult organ donor. Autologous neural tissue
can be obtained by biopsy, or from patients undergoing neurosurgery
in which neural tissue is removed, in particular during epilepsy
surgery, and more particularly during temporal lobectomies and
hippocampalectomies.
[0284] Cells can be obtained from donor tissue by dissociation of
individual cells from the connecting extracellular matrix of the
tissue. Dissociation can be obtained using any known procedure,
including treatment with enzymes such as trypsin, collagenase and
the like, or by using physical methods of dissociation such as with
a blunt instrument or by mincing with a scalpel to a allow
outgrowth of specific cell types from a tissue. Dissociation of
fetal cells can be carried out in tissue culture medium, while a
preferable medium for dissociation of juvenile and adult cells is
artificial cerebral spinal fluid (aCSF). Regular aCSF contains 124
mM NaCl, 5 mM KCl, 1.3 mM MgCl.sub.2, 2 mM CaCl.sub.2, 26 mM
NaHCO.sub.3, and 10 mM D-glucose. Low Ca.sup.2+ aCSF contains the
same ingredients except for MgCl.sub.2 at a concentration of 3.2 mM
and CaCl.sub.2 at a concentration of 0.1 mM.
[0285] Dissociated cells can be placed into any known culture
medium capable of supporting cell growth, including MEM, DMEM,
RPMI, F-12, and the like, containing supplements which are required
for cellular metabolism such as glutamine and other amino acids,
vitamins, minerals and useful proteins such as transferrin and the
like. Medium may also contain antibiotics to prevent contamination
with yeast, bacteria and fungi such as penicillin, streptomycin,
gentamicin and the like. In some cases, the medium may contain
serum derived from bovine, equine, chicken and the like. A
particularly preferable medium for cells is a mixture of DMEM and
F-12.
[0286] Conditions for culturing should be close to physiological
conditions. The pH of the culture media should be close to
physiological pH, preferably between pH 6-8, more preferably close
to pH 7, even more particularly about pH 7.4. Cells should be
cultured at a temperature close to physiological temperature,
preferably between 30.degree. C.-40.degree. C., more preferably
between 32.degree. C.-38.degree. C., and most preferably between
35.degree. C.-37.degree. C.
[0287] Cells can be grown in suspension or on a fixed substrate,
but proliferation of the progenitors is preferably done in
suspension to generate large numbers of cells by formation of
"neurospheres" (see, for example, Reynolds et al. (1992) Science
255:1070-1709; and PCT Publications WO93/01275, WO94/09119,
WO94/10292, and WO94/16718). In the case of propagating (or
splitting) suspension cells, flasks are shaken well and the
neurospheres allowed to settle on the bottom corner of the flask.
The spheres are then transferred to a 50 ml centrifuge tube and
centrifuged at low speed. The medium is aspirated, the cells
resuspended in a small amount of medium with growth factor, and the
cells mechanically dissociated and resuspended in separate aliquots
of media.
[0288] Cell suspensions in culture medium are supplemented with any
growth factor which allows for the proliferation of progenitor
cells and seeded in any receptacle capable of sustaining cells,
though as set out above, preferably in culture flasks or roller
bottles. Cells typically proliferate within 3-4 days in a
37.degree. C. incubator, and proliferation can be reinitiated at
any time after that by dissociation of the cells and resuspension
in fresh medium containing growth factors.
[0289] In the absence of substrate, cells lift off the floor of the
flask and continue to proliferate in suspension forming a hollow
sphere of undifferentiated cells. After approximately 3-10 days in
vitro, the proliferating clusters (neurospheres) are fed every 2-7
days, and more particularly every 2-4 days by gentle centrifugation
and resuspension in medium containing growth factor.
[0290] After 6-7 days in vitro, individual cells in the
neurospheres can be separated by physical dissociation of the
neurospheres with a blunt instrument, more particularly by
triturating the neurospheres with a pipette. Single cells from the
dissociated neurospheres are suspended in culture medium containing
growth factors, and differentiation of the cells can be control in
culture by plating (or resuspending) the cells in the presence of a
hedgehog agonist.
[0291] To further illustrate other uses of the subject hedgehog
agonists, 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.
[0292] 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 agonists
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 agonists can be
used alone, or can be used in combination with other neurotrophic
factors which act to more particularly enhance a particular
differentiation fate of the neuronal progenitor cell.
[0293] In addition to the implantation of cells cultured in the
presence of the subject hedgehog agonists, yet another aspect of
the present invention concerns the therapeutic application of a
hedgehog agonist 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 agonists
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, amylotrophic 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. For
example, in the specific case of Parkinson's disease, intervention
by increasing the activity of hedgehog by a subject agonist can
improve the in vivo survival of fetal and adult dopaminergic
neurons, and thus can provide a more effective treatment of this
disease. Thus, in one embodiment, the subject method comprises
administering to an animal afflected with Parkinson's disease, or
at risk of developing Parkinson's disease, an amount of a hedgehog
agonist effective for increasing the rate of survival of
dopaminergic neurons in the animal.
[0294] The present method is applicable to cell culture techniques.
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). Once a neuronal cell has become
terminally differentiated it typically will not change to another
terminally differentiated cell-type. However, neuronal cells can
nevertheless readily lose their differentiated state. This is
commonly observed when they are grown in culture from adult tissue,
and when they form a blastema during regeneration. The present
method provides a means for ensuring an adequately restrictive
environment in order to maintain dopaminergic and GABAergic cells
in differentiated states, and can be employed, for instance, in
cell cultures designed to test the specific activities of other
trophic factors.
[0295] In such embodiments of the subject method, a culture of
differentiated cells inlcuding dopaminergic and/or GABAergic cells
can be contacted with a hedgehog agonist in order to maintain the
integrity of a culture of terminally differentiated neuronal cells
by preventing loss of differentiation. The subject method can be
used in conjunction with agents which induce the differentiation of
neuronal precursors, e.g., progenitor or stem cells, into
dopaminergic or GABAergic neurons.
[0296] Many neurological disorders are associated with degeneration
of discrete populations of neuronal elements and may be treatable
with a therapeutic regimen which includes a hedgehog agonist. For
example, Alzheimer's disease is associated with deficits in several
neurotransmitter systems, both those that project to the neocortex
and those that reside with the cortex. For instance, the nucleus
basalis in patients with Alzheimer's disease have been observed to
have a profound (75%) loss of neurons compared to age-matched
controls. Although Alzheimer's disease is by far the most common
form of dementia, several other disorders can produce dementia.
Several of these are degenerative diseases characterized by the
death of neurons in various parts of the central nervous system,
especially the cerebral cortex. However, some forms of dementia are
associated with degeneration of the thalamus or the white matter
underlying the cerebral cortex. Here, the cognitive dysfunction
results from the isolation of cortical areas by the degeneration of
efferents and afferents. Huntington's disease involves the
degeneration of intrastraital and cortical cholinergic neurons and
GABAergic neurons. Pick's disease is a severe neuronal degeneration
in the neocortex of the frontal and anterior temporal lobes,
sometimes accompanied by death of neurons in the striatum.
Treatment of patients suffering from such degenerative conditions
can include the application of hedgehog agonists in order to
control, for example, differentiation and apoptotic events which
give rise to loss of neurons (e.g., to enhance survival of existing
neurons) as well as promote differentiation and repopulation by
progenitor cells in the area affected.
[0297] In addition to degenerative-induced dementias, a
pharmaceutical preparation of one or more of the subject hedgehog
agonists can be applied opportunely in the treatment of
neurodegenerative disorders which have manifestations of tremors
and involuntary movements. Parkinson's disease, for example,
primarily affects subcortical structures and is characterized by
degeneration of the nigrostriatal pathway, raphe nuclei, locus
cereleus, and the motor nucleus of vagus. Ballism is typically
associated with damage to the subthalmic nucleus, often due to
acute vascular accident. Also included are neurogenic and myopathic
diseases which ultimately affect the somatic division of the
peripheral nervous system and are manifest as neuromuscular
disorders. Examples include chronic atrophies such as amyotrophic
lateral sclerosis, Guillain-Barre syndrome and chronic peripheral
neuropathy, as well as other diseases which can be manifest as
progressive bulbar palsies or spinal muscular atrophies. The
present method is amenable to the treatment of disorders of the
cerebellum which result in hypotonia or ataxia, such as those
lesions in the cerebellum which produce disorders in the limbs
ipsilateral to the lesion. For instance, a preparation of a
hedgehog agonist can used to treat a restricted form of cerebellar
cortical degeneration involving the anterior lobes (vermis and leg
areas) such as is common in alcoholic patients.
[0298] In an illustrative embodiment, the subject method is used to
treat amyotrophic lateral sclerosis. ALS is a name given to a
complex of disorders that comprise upper and lower motor neurons.
Patients may present with progressive spinal muscular atrophy,
progressive bulbar palsy, primary lateral sclerosis, or a
combination of these conditions. The major pathological abnormality
is characterized by a selective and progressive degeneration of the
lower motor neurons in the spinal cord and the upper motor neurons
in the cerebral cortex. The therapeutic application of a hedgehog
agonist can be used alone, or in conjunction with other
neurotrophic factors such as CNTF, BDNF or NGF to prevent and/or
reverse motor neuron degeneration in ALS patients.
[0299] Hedgehog agonists of the present invention can also be used
in the treatment of autonomic disorders of the peripheral nervous
system, which include disorders affecting the innervation of smooth
muscle and endocrine tissue (such as glandular tissue). For
instance, the subject method can be used to treat tachycardia or
atrial cardiac arrythmias which may arise from a degenerative
condition of the nerves innervating the striated muscle of the
heart.
[0300] Furthermore, a potential role for certain of the hedgehog
agonists derives from the role of hedgehog proteins in development
and maintenance of dendritic processes of axonal neurons. Potential
roles for hedgehog agonists consequently include guidance for
axonal projections and the ability to promote differentiation
and/or maintenance of the innervating cells to their axonal
processes. Accordingly, compositions comprising hedgehog agonists
may be employed to support the survival and reprojection of several
types of ganglionic neurons sympathetic and sensory neurons as well
as motor neurons. In particular, such therapeutic compositions may
be useful in treatments designed to rescue, for example, various
neurons from lesion-induced death as well as guiding reprojection
of these neurons after such damage. Such diseases include, but are
not limited to, CNS trauma infarction, infection (such as viral
infection with varicella-zoster), metabolic disease, nutritional
deficiency, toxic agents (such as cisplatin treatment).
[0301] 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
agonists can be added to the prosthetic device to regulate the rate
of growth and regeneration of the dendridic processes. Exemplary
nerve guidance channels are described in U.S. Pat. Nos. 5,092,871
and 4,955,892.
[0302] 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
agonists 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.
[0303] The subject method has wide applicability to the treatment
or prophylaxis of disorders affecting the regulation of peripheral
nerves, including peripheral ganglionic neurons, sympathetic,
sensory neurons, and motor neurons. In general, the method can be
characterized as including a step of administering to an animal an
amount of a hedgehog agonist effective to alter the proliferative
and/or differentiation state of treated peripheral nerve cells.
Such therapeutic compositions may be useful in treatments designed
to rescue, for example, retinal ganglia, inner ear and accoustical
nerves, and motorneurons, from lesion-induced death as well as
guiding reprojection of these neurons after such damage. Such
diseases and conditions include, but are not limited to, chemical
or mechanical trauma, infection (such as viral infection with
varicella-zoster), metabolic disease such as diabetes, nutritional
deficiency, toxic agents (such as cisplatin treatment). The goals
of treatment in each case can be twofold: (1) to eliminate the
cause of the disease and (2) to relieve its symptoms.
[0304] Peripheral neuropathy is a condition involving nerve-ending
damage in the hands and feet. Peripheral neuropathy generally
refers to a disorder that affects the peripheral nerves, most often
manifested as one or a combination of motor, sensory, sensorimotor,
or autonomic neural dysfunction. The wide variety of morphologies
exhibited by peripheral neuropathies can each be uniquely
attributed to an equally wide variety of causes. For instance,
peripheral neuropathies can be genetically acquired, can result
from a systemic disease, or can be induced by a toxic agent. Some
toxic agents that cause neurotoxicities are therapeutic drugs,
antineoplastic agents, contaminants in foods or medicinals, and
environmental and industrial pollutants.
[0305] In particular, chemotherapeutic agents known to cause
sensory and/or motor neuropathies include vincristine, an
antineoplastic drug used to treat haematological malignancies and
sarcomas. The neurotoxicity is dose-related, and exhibits as
reduced intestinal motility and peripheral neuropathy, especially
in the distal muscles of the hands and feet, postural hypotension,
and atony of the urinary bladder. Similar problems have been
documented with taxol and cisplatin (Mollman, J. E., 1990, New Eng
Jour Med. 322:126-127), although cisplatin-related neurotoxicity
can be alleviated with nerve growth factor (NGF) (Apfel, S. C. et
al, 1992, Annals of Neurology 31:76-80). Although the neurotoxicity
is sometimes reversible after removal of the neurotoxic agent,
recovery can be a very slow process (Legha, S., 1986, Medical
Toxicology 1:421-427; Olesen, et al., 1991, Drug Safety
6:302-314).
[0306] There are a number of inherited peripheral neuropathies,
including: Refsum's disease, abetalipoproteinemia, Tangier disease,
Krabbe's disease, metachromatic leukodystrophy, Fabry's disease,
Dejerine-Sottas syndrome, and others. Of all the inherited
neuropathies, the most common by far is Charcot-Marie-Tooth
disease.
[0307] Charcot-Marie-Tooth (CMT) Disease (also known as peroneal
muscular atrophy, or hereditary motor sensory neuropathy (HMSN)) is
the most common hereditary neurological disorder. It is
characterized by weakness and atrophy, primarily of the peroneal
muscles, due to segmental demyelination of peripheral nerves and
associated degeneration of axons and anterior horn cells. Autosomal
dominant inheritance is usual, and associated degenerative CNS
disorders, such as Friedreich's ataxia, are common.
[0308] In one aspect, the method of the present invention can be
used in the treatment and maintenance of hereditary neuropathies.
This group of neuropathies is now becoming increasingly recognized
due to the dramatic advances in molecular genetics. The symptoms of
the various hereditary neuropathies are wide-ranging. A common
denominator is usually the early onset of mild numbness and
tingling in the feet that slowly progresses to involve the legs and
the hands and later the rest of the upper extremities. Most of the
hereditary neuropathies do have a motor component consisting of
distal weakness in the lower and upper extremities. A majority of
patients with hereditary neuropathies have high arches in their
feet or other bony deformities. The symptoms are very slowly
progressive and the majority of the patients are still walking two
decades after the onset of their symptoms.
[0309] The diagnosis of a hereditary neuropathy is usually
suggested with the early onset of neuropathic symptoms, especially
when a positive family history is also present. Prior to the recent
genetic advances, the diagnosis was supported by typical findings
of marked slowing of the nerve conduction studies on
electromyography and a nerve biopsy. Typical findings on a nerve
biopsy include the presence of so-called onion-bulbs, indicating a
recurring demyelinating and remyelinating of the nerve fibers. With
the most recent genetic advances, two major hereditary neuropathies
known as Charcot-Marie-Tooth disease and hereditary neuropathy with
liability to pressure palsies can be diagnosed with a simple blood
test that identifies the different mutations responsible for these
two entities.
[0310] Hereditary neuropathies are caused by genetic abnormalities
which are transmitted from generation to generation. For several of
these, the genetic defect is known, and tests are available for
diagnosis and prenatal counseling.
[0311] As set foth above, the subject method can be used as part of
a therapeutic regimen in the treatment of Charcot-Marie Tooth
Disease (CMT). This is a general term given to the hereditary
sensorimotor neuropathies. CMT type 1 (CMT 1) is associated with
demyelination or breakdown of the myelin sheaths. Several different
abnormalities have been identified. CMT Type 1A is most commonly
caused by duplication of a gene encoding a myelin protein called
PMP-22, and CMT type 1B is caused by a mutation in a myelin protein
called the Po glycoprotein. CMTX is a hereditary sensorimotor
neuropathy which primarily affects men. It is caused by a mutation
in a gene encoding a protein called Connexin 32 on the
X-chromosome.
[0312] In another embodiment, the subject method can be used in the
treatment of familial amyloidotic neuropathy and other related
hereditary neuropathies. Amyloidotic neuropathy usually presents
with pain, sensory loss and autonomic dysfunction. It is caused by
a mutation in a protein called Transthyretin, resulting in
deposition of the protein as amyloid in the peripheral nerves.
[0313] The subject method can be used in the treatment of
hereditary porphyria, which can have components of peripheral
neuropathy. Still another hereditary neuropathy for which the
subject methods can be used for treatment is hereditary sensory
neuropathy Type II (HSN II). The methods and compositions of the
present invention can also be used in the treatment and maintenance
of acquired neuropathies.
[0314] For example, hedgehog agonists can be used to prevent
diabetic neuropathies. Diabetes is the most common known cause of
neuropathy. It produces symptoms in approximately 10% of people
with diabetes. In most cases, the neuropathy is predominantly
sensory, with pain and sensory loss in the hands and feet. But some
diabetics have mononeuritis or mononeuritis multiplex which causes
weakness in one or more nerves, or lumbosacral plexopathy or
amyotrophy which causes weakness in the legs.
[0315] The instant method can also be used in the treatment of
immune-mediated neuropathies. The main function of the immune
system is to protect the body against infectious organisms which
enter from outside. In some cases, however the immune system turns
against the body and causes autoimmune disease. The immune system
consists of several types of white blood cells, including
T-lymphocytes, which also regulate the immune response; and
B-lymphocytes or plasma cells, which secrete specialized proteins
called "antibodies" Sometimes, for unknown reasons, the immune
system mistakenly attacks parts of the body such as the peripheral
nenes. This is "autoimmune" Peripheral Neuropathy. There are
several different types, depending on the part of the peripheral
nerve which is attacked and the type of the immune reaction. The
following are brief descriptions of the neuropathies which are
mediated by the immune system.
[0316] For instance, a hedgehog agonist can be used to treat
Guillain-Barre syndrome (GBS). An acute neuropathy because it comes
on suddenly or rapidly. Guillain-Barre syndrome can progress to
paralysis and respiratory failure within days or weeks after onset.
The neuropathy is caused when the immune system destroys the myelin
sheaths of the motor and sensory nerves. It is often preceded by
infection, vaccination or trauma, and that is thought to be what
triggers the autoimmune reaction. The disease is self-limiting,
with spontaneous recovery within six to eight weeks. But the
recovery is often incomplete.
[0317] Other neuropathies which begin acutely, and which can be
treated by the method of the present invention, include acute motor
neuropathy, acute sensory neuropathy, and acute autonomic
neuropathy, in which there is an immune attack against the motor,
sensory or autonomic nerves, respectively. The Miller-Fisher
syndrome is another variant in which there is paralysis of eye
gaze, incoordination, and unsteady gait.
[0318] Still another acquired neuropathy which is may be treated by
the subject method is chronic inflammatory demyelinating
polyneuropathy (CIDP). CIDP is thought to be a chronic and more
indolent form of the Guillain-Barre syndrome. The disease
progresses either with repeated attacks, called relapses, or in a
stepwise or steady fashion. As in GBS, there appears to be
destruction of the myelin sheath by antibodies and T-lymphocytes.
But since there is no specific test for CIDP, the diagnosis is
based on the clinical and laboratory characteristics.
[0319] Chronic polyneuropathies with antibodies to peripheral
nerves is still another peripheral neuropathy for which the subject
methods can be employed to treat or prevent. In some types of
chronic neuropathies, antibodies to specific components of nerve
have been identified. These include demyelinating neuropathy
associated with antibodies to the myelin associated glycoprotein
(MAG), motor neuropathy associated with antibodies to the
gangliosides GM1 or GD1a, and sensory neuropathy associated with
anti-sulfatide or GD1b ganglioside antibodies. The antibodies in
these cases bind to oligosaccharide or sugar like molecules, which
are linked to proteins (glycoproteins) or lipids (glycolipids or
gangliosides) in the nerves. It is suspected that these antibodies
may be responsible for the neuropathies.
[0320] The subject method can also be used as part of a therapeutic
plan for treating neuropathies associated with vasculitis or
inflammation of the blood vessels in peripheral nerves. Neuropathy
can also be caused by vasculitis--an inflammation of the blood
vessels in peripheral nerve. It produces small "strokes" along the
course of the peripheral nerves, and may be restricted to the
nerves or it may be generalized, include a skin rash, or involve
other organs. Several rheumatological diseases like rheumatoid
arthritis, lupus, periarteritis nodosa, or Sjogren's syndrome, are
associated with generalized vasculitis, which can also involve the
peripheral nerves. Vasculitis can cause polyneuritis, mononeuritis,
or mononeuritis multiplex, depending on the distribution and
severity of the lesions.
[0321] In still another embodiment, the method of the present
invention can be used for treatment of brachial or lumbosacral
plexitis. The brachial plexus, which lies under the armpit,
contains the nerves to the arm and hand. Brachial plexitis is the
result of inflamation of that nerve bundle, and produces weakness
and pain in one or both arms. Umbosacral plexitis, which occurs in
the pelvis, causes weakness and pain in the legs.
[0322] Hedgehog agonists may also be suitable for use in the
treatment of neuropathies associated with monoclonal gammopathies.
In monoclonal gammopathy, single clones of B-cells or plasma cells
in the bone marrow or lymphoid organs expand to form benign or
malignant tumors and secrete antibodies. "Monoclonal" is because
there are single clones of antibodies, and "gammopathy" stands for
gammaglobulins, which is another name for antibodies. In some
cases, the antibodies react with nerve components; in others,
fragments of the antibodies form amyloid deposits.
[0323] Yet another aspect of the present invention relates to the
use of the subject method in the treatment of neuropathies
associated with tumors or neoplasms. Neuropathy can be due to
direct infiltration of nerves by tumor cells or to indirect effect
of the tumor. The latter is called paraneoplastic neuropathy.
Several types have been described. For instance, the subject
methods can be used to manage sensory neuropathy associated with
lung cancer. This neuropathy is associated with antibodies to a
protein called Hu, which is present in the sensory neurons of the
peripheral nerves. Likewise, the subject method can be used to
treat neuropathies associated with multiple myeloma. Multiple
myeloma is a bony tumor which is caused by antibody-secreting
plasma cells in the bone marrow. The tumor is made up of a single
clone of plasma cells, and the antibodies they produce are
identical or monoclonal. Some people with multiple myeloma develop
sensorimotor polyneuropathy with degeneration of axons in the
peripheral nerves. In other embodiments, the subject method can be
used to treat neuropathies associated with Waldenstrom's
macroglobulemia, chronic lymphocytic leukemia, or B-cell lymphoma.
These are tumors caused by antibody-secreting B-lymphocytes in the
spleen, bone marrow or lymph nodes. These antibodies are monoclonal
and frequently react with peripheral nerve components such as MAG,
GM1, or sulfatide. In still other embodiments, the hedgehog
agonists of the present invention can be used as part of
therapeutic protocol for the treatment of patients with cancers
where neuropathy is a consequence of local irradiation or be caused
by medications such as vincristine and cisplatin.
[0324] The present invention also contemplates the use of hedgehog
agonists for the treatment of neuropathies associated with
amyloidosis. Amyloid is a substance deposited in the peripheral
nerves and interferes with their operation: the disorder is
amyloidosis. There are two main types: primary amyloidosis, in
which the deposits contain fragments of monoclonal antibodies (see
monoclonal gammopathy above); and hereditary amyloidosis in which
the deposits contain a mutated protein called Transthyretin.
Primary amyloidosis is usually associated with monoclonal
gammopathies or myeloma.
[0325] Still another aspect of the present invention provides the
subject method as a means for treating neuropathies caused by
infections. Peripheral neuropathies can be caused by infection of
the peripheral nerves. Viruses that cause peripheral neuropathies
include the AIDS virus, HIV-I, which causes slowly progressive
sensory neuropathy, Cytomegalovirus which causes a rapidly
progressive paralytic neuropathy, Herpes zoster which cause
shingles, and poliovirus which causes a motor neuropathy. Hepatitis
B or C infections are sometimes associated with vasculitic
neuropathy.
[0326] Bacterial infections that cause neuropathy include leprosy,
which causes a patchy sensory neuropathy, and diphtheria which can
cause a rapidly progressive paralytic neuropathy. Other infectious
diseases that cause neuropathy include Lyme disease, which is
caused by a spirochete, and trypanosomiasis which is caused by a
parasite. Both commonly present with a multifocal neuropathy
[0327] Neuropathies caused by nutritional imbalance are also
candidate disorders for treatment by the subject method.
Deficiencies of vitamins B12, B1 (thiamine), B6 (pyridoxine), or E,
for example, can produce polyneuropathies with degeneration of
peripheral nerve axons. This can be due to poor diet, or inability
to absorb the nutrients from the stomach or gut. Moreover,
megadoses of vitamin B6 can also cause a peripheral neuropathy, and
the subject method can be used as part of a detoxification program
in such cases.
[0328] Yet another use of the subject method is in the treatment of
neuropathies arising in kidney diseases. Chronic renal failure can
cause a predominantly sensory peripheral neuropathy with
degeneration of peripheral nerve axons.
[0329] Another aspect of the present invention provides a method
for treating hypothyroid neuropathies. Hypothyroidism is sometimes
associated with a painful sensory polyneuropathy with axonal
degeneration. Mononeuropathy or mononeuropathy multiplex can also
occur due to compression of the peripheral nerves by swollen
tissues.
[0330] The subject method can also be used in the treatment of
neuropathies caused by alcohol and toxins. Certain toxins can cause
peripheral neuropathy. Lead toxicity is associated with a motor
neuropathy; arsenic or mercury cause a sensory neuropathy, and
thallium can cause a sensory and autonomic neuropathy. Several
organic solvents and insecticides can also cause polyneuropathy.
Alcohol is directly toxic to nerves and alcohol abuse is a major
cause of neuropathy. The subject method can be used, in certain
embodiments, as part of a broader detoxification program.
[0331] In still another embodiment, the methods and compositions of
the present invention can be used for the treatment of neuropathies
caused by drugs. Several drugs are known to cause neuropathy. They
include, among others, vincristine and cisplatin in cancer,
nitrofurantoin, which is used in pyelonephritis, amiodarone in
cardiac arrhythmias, disulfiram in alcoholism, ddC and ddI in AIDS,
and dapsone which is used to treat leprosy. As above, the subject
method can be used, in certain embodiments, as part of a broader
detoxification program.
[0332] The method of the present invention can also be used in the
treatment of neuropathies caused by trauma or compression.
Localized neuropathies can result from compression of nerves by
external pressure or overlying tendons and other tissues. The best
known of these are the carpal tunnel syndrome which results from
compression at the wrist, and cervical or lumbar radiculopathies
(sciatica) which result from compression of nerve roots as they
exit the spine. Other common areas of nerve compression include the
elbows, armpits, and the back of the knees.
[0333] The subject method is also useful in variety of idiopathic
neuropathies. The term "idiopathic" is used whenever the cause of
the neuropathy cannot be found. In these cases, the neuropathy is
classified according to its manifestations, i.e., sensory, motor,
or sensorimotor idiopathic polyneuropathy.
[0334] The subject method has wide applicability to the treatment
or prophylaxis of disorders afflicting muscle tissue. In general,
the method can be characterized as including a step of
administering to an animal an amount of a hedgehog agonist
effective to alter the proliferative state of a treated muscle
tissue. The mode of administration and dosage regimens will vary
depending on the muscle tissue(s) which is to be treated.
[0335] In one aspect, the invention is directed to a muscle-trophic
factor, and its use in stimulating muscle growth or differentiation
in mammals. Such stimulation of muscle growth is useful for
treating atrophy, or wasting, in particular, skeletal muscle
atrophy and cardiac muscle atrophy. In addition, certain diseases
wherein the muscle tissue is damaged, is abnormal or has atrophied,
are treatable using the invention, such as, for example, normal
aging, disuse atrophy, wasting or cachexia, and various secondary
disorders associated with age and the loss of muscle mass, such as
hypertension, glucose intolerance and diabetes, dyslipidemia and
atherosclerotic cardiovascular disease. The treatment of muscular
myopathies such as muscular dystrophies is also embodied in the
invention.
[0336] With denervation or disuse, skeletal muscles undergo rapid
atrophy which leads to a profound decrease in size, protein content
and contractile strength. This atrophy is an important component of
many neuromuscular diseases in humans. In a clinical setting,
compositions comprising the subject hedgehog agonists can be used
for inhibiting muscle degeration, e.g., for decreasing the loss of
muscle mass, such as part of a treatment for such muscle wasting
disorders.
[0337] In preferred embodiments pharmaceutical compositions
according to the invention are administered to patients suffering
from a disorder, i.e., an abnormal physical condition, a disease or
pathophysiological condition associated with abnormal and/or
aberrant regulation of muscle tissue. The disorders for which the
compositions of the invention are administered are preferably those
which directly or indirectly produce a wasting (i.e., loss) of
muscle mass, that is, a muscle wasting disorder. These include
muscular dystrophies, cardiac cachexia, emphysema, leprosy,
malnutrition, osteomalacia, child acute leukemia, AIDS cachexia and
cancer cachexia.
[0338] The muscular dystrophies are genetic diseases which are
characterized by progressive weakness and degeneration of muscle
fibers without evidence of neural degeneration. In Duchenne
muscular dystrophy (DMD) patients display an average of a 67%
reduction in muscle mass, and in myotonic dystrophy, fractional
muscle protein synthesis has been shown to be decreased by an
average of 28%, without any corresponding decrease in non-muscle
protein synthesis (possibly due to impaired end-organ response to
anabolic hormones or substrates). Accelerated protein degradation
has been demonstrated in the muscles of DMD patients. The subject
method can be used as part of a therapeutic strategy for
preventing, and in some instance reversing, the muscle wasting
conditions associated with such dystrophies.
[0339] Severe congestive heart failure (CHF) is characterized by a
"cardiac cachexia," i.e., a muscle protein wasting of both the
cardiac and skeletal muscles, with an average 19% body weight
decrease. The cardiac cachexia is caused by an increased rate of
myofibrillar protein breakdown. The subject method can be used as
part of a treatment for cardiac cachexia.
[0340] Emphysema is a chronic obstructive pulmonary disease,
defined by an enlargement of the air spaces distal to the terminal
non-respiratory bronchioles, accompanied by destructive changes of
the alveolar walls. Clinical manifestations of reduced pulmonary
functioning include coughing, wheezing, recurrent respiratory
infections, edema, and functional impairment and shortened
life-span. The efflux of tyrosine is increased by 47% in
emphysematous patients. Also, whole body leucine flux remains
normal, whole-body leucine oxidation is increased, and whole-body
protein synthesis is decreased. The result is a decrease in muscle
protein synthesis, accompanied by a decrease in whole body protein
turnover and skeletal muscle mass. This decrease becomes
increasingly evident with disease progression and long term
deterioration. The subject hedgehog agonists may be used to prevent
and/or reverse, the muscle wasting conditions associated with such
diseases.
[0341] In diabetes mellitus, there is a generalized wasting of
small muscle of the hands, which is due to chronic partial
denervation (neuropathy). This is most evident and worsens with
long term disease progression and severity. The subject method can
be used as part of a therapeutic strategy for treatement of
diabetes mellitus.
[0342] Leprosy is associated with a muscular wasting which occurs
between the metacarpals of the thumb and index finger. Severe
malnutrition is characterized by, inter alia, severe muscle
wasting. The subject method can be used to treat muscle wasting
effects of leprosy.
[0343] Osteomalacia is a nutritional disorder caused by a
deficiency of vitamin D and calcium. It is referred to as "rickets"
in children, and "osteomalacia" in adults. It is marked by a
softening of the bones (due to impaired mineralization, with excess
accumulation of osteoid), pain, tenderness, muscle wasting and
weakness, anorexia, and overall weight loss. It can result from
malnutrition, repeated pregnancies and lactation (exhausting or
depleting vitamin D and calcium stores), and vitamin D resistance.
The subject method can be used as part of a therapeutic strategy
for treatment of osteomalacia.
[0344] In childhood acute leukemia there is protein energy
malnutrition which results in skeletal muscle wasting. Studies have
shown that some children exhibit the muscle wasting even before
diagnosis of the leukemia, with an average 27% decrease in muscle
mass. There is also a simultaneous 33%-37% increase in adipose
tissue, resulting in no net change in relative body weight and limb
circumference. Such patients may be amenable to treatment with a
hedgehog agonist according to the method of the present
invention.
[0345] Cancer cachexia is a complex syndrome which occurs with
variable incidence in patients with solid tumors and hematological
malignancies. Clinically, cancer cachexia is manifested as weight
loss with massive depletion of both adipose tissue and lean muscle
mass, and is one cause of death which results from cancer. Cancer
cachexia patients have shorter survival times, and decreased
response to chemotherapy. In addition to disorders which produce
muscle wasting, other circumstances and conditions appear to be
linked in some fashion with a decrease in muscle mass. Such
afflictions include muscle wasting due to chronic back pain,
advanced age, long-term hospitalization due to illness or injury,
alcoholism and corticosteroid therapy. The subject method can be
used as part of a therapeutic strategy for preventing, and in some
instance reversing, the muscle wasting conditions associated with
such cancers.
[0346] Studies have shown that in severe cases of chronic lower
back pain, there is paraspinal muscle wasting. Decreasing
paraspinal muscle wasting alleviates pain and improves function. A
course of treatment for disorder can include administration of a
therapeutic amount of a hedgehog agonist.
[0347] It is also believed that general weakness in old age is due
to muscle wasting. As the body ages, an increasing proportion of
skeletal muscle is replaced by fibrous tissue. The result is a
significant reduction in muscle power, but only a marginal
reduction in fat-free mass. The subject method can be used as part
of a treatment and preventive strategies for preventing/reversing
muscle wasting in elderly patients.
[0348] Studies have also shown that in patients suffering injuries
or chronic illnesses, and hospitalized for long periods of time,
there is long-lasting unilateral muscle wasting, with an average
31% decrease in muscle mass. Studies have also shown that this can
be corrected with intensive physiotherapy. However, it may be more
effective for many patients to at least augment such therapies with
treatment by the subject method
[0349] In alcoholics there is wasting of the anterior tibial
muscle. This proximal muscle damage is caused by neurogenic damage,
namely, impaired glycolytic and phosphorylase enzyme activity. The
damage becomes apparent and worsens the longer the duration of the
alcohol abuse. Patients treated with corticosteroids experience
loss of muscle mass. Such patients may also be amenable to
treatment by the subject method.
[0350] The compounds of the invention can be used to alleviate the
muscle mass loss resulting from the foregoing conditions, as well
as others. Additionally, the hedgehog agonists of the present
invention are useful in veterinary and animal husbandry
applications to counter weight loss in animals, or to promote
growth. For instance, the invention may also find use for
increasing the efficiency of animal meat production. Specifically,
animals may be fed or injected with a hedgehog agonist in order to
increase overall skeletal muscle mass, e.g., to increase the weight
of such farm animals as cows, pigs, sheep, chickens and salmon.
[0351] The maintenance of tissues and organs ex vivo is also highly
desirable. Tissue replacement therapy is well established in the
treatment of human disease. There are many situations where one may
wish to transplant muscle cells, especially muscle stem cells, into
a recipient host where the recipient's cells are missing, damaged
or dysfunctional muscle cells in muscle wasting disease. For
example, transplantation of normal myoblasts may be useful to treat
Duchenne muscular dystrophy and other muscle degeneration and
wasting diseases. See, for example, Partridge (1991) Muscle &
Nerve 14:197-212. In the case of myoblasts, they may be injected at
various sites to treat muscle wasting diseases.
[0352] The subject method can be used to regulate the growth of
muscle cells and tissue in vitro, as well as to accelerate the
grafting of impanted muscle tissue to an animal host. In this
regard, the present invention also concerns myoblast cultures which
have been expanded by treatment with a hedgehog agonist. In an
illustrative embodiment, such a method comprises obtaining a muscle
sample, preferably one including myoblasts; optionally treating the
cell sample enzymically to separate the cells; culturing, in the
presence of a hedgehog agonist.
[0353] 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 agonists can also
be utilized for both cell culture and therapeutic methods involving
generation and maintenance of non-neuronal tissue.
[0354] 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
agonists of the instant method can be employed for regulating the
development and maintenance of an artificial liver which 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.
[0355] In another embodiment, therapeutic compositions of hedgehog
agonists 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.
[0356] 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 agonists can be utilized in liver
repair subsequent to a partial hepatectomy.
[0357] 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.
[0358] In the context of the present invention, it is contemplated
therefore that the subject hedgehog agonists can be used to control
or regulate the proliferation and/or differentiation of pancreatic
tissue both in vivo and in vitro.
[0359] There are a wide variety of pathological cell proliferative
and differentiative conditions for which the agonists 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 agonists. 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 .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, and other organs
which derive from the primitive gut.
[0360] 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. To the
extent that aberrant ptc, hedgehog, and smoothened signaling may be
indicated in disease progression, the subject agonists, can be used
to enhance regeneration of the tissue after anti-tumor therapy.
[0361] 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 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 reappearence of embryonal traits in mature
.beta.-cells can be observed. By utilizing the subject hedgehog
agonists, the differentiation path or proliferative index of the
cells can be regulated.
[0362] Furthermore, manipulation of the differentiative state of
pancreatic tissue can be utilized in conjunction with
transplantation of artificial pancreas so as to promote
implantation, vascularization, and in vivo differentiation and
maintenance of the engrafted tissue. For instance, manipulation of
hedgehog function to affect tissue differentiation can be utilized
as a means of maintaining graft viability.
[0363] 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.
[0364] In still another embodiment of the present invention,
compositions comprising hedgehog agonists 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 agonists 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.
[0365] 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.
[0366] In one embodiment of the present invention, the subject
method comprises treating the afflicted connective tissue with a
therapeutically sufficient amount of a hedgehog agonist,
particularly an agonist selective for Indian 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.
[0367] 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 agonists 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.
[0368] 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.
[0369] 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
which 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.
[0370] In one embodiment of the subject method, the implants are
contacted with a hedgehog agonist 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.
[0371] In another embodiment, the implanted device is treated with
a hedgehog agonist in order to actively remodel the implanted
matrix and to make it more suitable for its intended function. As
set forth 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.
[0372] 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.
[0373] 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. Indian hedgehog is particularly associated with the
hypertrophic chondrocytes that are ultimately replaced by
osteoblasts. For instance, administration of a hedgehog agonist 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 agonists can be employed, for
example, to control endochondral ossification in the formation of a
"model" for ossification.
[0374] In yet another embodiment of the present invention, a
hedgehog agonist 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, a hedgehog agonist can be used as a fertility
agent. In similar fashion, hedgehog agonists of the subject method
are potentially useful for modulating normal ovarian function.
[0375] 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 agonist 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) which is
to be treated. For example, topical formulations will be preferred
where the treated tissue is epidermal tissue, such as dermal or
mucosal tissues.
[0376] A method which "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.
[0377] Complications are a constant risk with wounds that have not
fully healed and remain open. Although most wounds heal quickly
without treatment, some types of wounds resist healing. Wounds
which cover large surface areas also remain open for extended
periods of time. In one embodiment of the present invention, the
subject method can be used to accelerate the healing of wounds
involving epithelial tissues, such as resulting from surgery,
burns, inflammation or irritation. Certain of the hedgehog agonists
of the present invention can also be applied prophylactically, such
as in the form of a cosmetic preparation, to enhance tissue
regeneration processes, e.g., of the skin, hair and/or
fingernails.
[0378] 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.
[0379] Full and partial thickness burns are an example of a wound
type which often covers large surface areas and therefore requires
prolonged periods of time to heal. As a result, life-threatening
complications such as infection and loss of bodily fluids often
arise. In addition, healing in burns is often disorderly, resulting
in scarring and disfigurement. In some cases wound contraction due
to excessive collagen deposition results in reduced mobility of
muscles in the vicinity of the wound. The compositions and method
of the present invention can be used to accelerate the rate of
healing of burns and to promote healing processes that result in
more desirable cosmetic outcomes and less wound contraction and
scarring.
[0380] Severe burns which cover large areas are often treated by
skin autografts taken from undamaged areas of the patient's body.
The subject method can also be used in conjunction with skin grafts
to impove "take" rates of the graft by accelerating growth of both
the grafted skin and the patient's skin that is proximal to the
graft.
[0381] Dermal ulcers are yet another example of wounds that are
amenable to treatment by the subject method, e.g., to cause healing
of the ulcer and/or to prevent the ulcer from becoming a chronic
wound. For example, one in seven individuals with diabetes develop
dermal ulcers on their extremities, which are susceptible to
infection. Individuals with infected diabetic ulcers often require
hospitalization, intensive services, expensive antibiotics, and, in
some cases, amputation. Dermal ulcers, such as those resulting from
venous disease (venous stasis ulcers), excessive pressure
(decubitus ulcers) and arterial ulcers also resist healing. The
prior art treatments are generally limited to keeping the wound
protected, free of infection and, in some cases, to restore blood
flow by vascular surgery. According to the present method, the
afflicted area of skin can be treated by a therapy which includes a
hedgehog agonist which promotes epithelization of the wound, e.g.,
accelerates the rate of the healing of the skin ulcers.
[0382] 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,
lend 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 which includes application
of an hedgehog agonist can reduce the abnormal proliferation and
differentiation of the affected epithelium, helping to reduce the
severity of subsequent inflammatory events.
[0383] In another exemplary embodiment, the subject method is
provided for treating or preventing gastrointestinal diseases.
Briefly, a wide variety of diseases are associated with disruption
of the gastrointestinal epithelium or villi, including
chemotherapy- and radiation-therapy-induce- d enteritis (i.e., gut
toxicity) and mucositis, peptic ulcer disease, gastroenteritis and
colitis, villus atrophic disorders, and the like. For example,
chemotherapeutic agents and radiation therapy used in bone marrow
transplantation and cancer therapy affect rapidly proliferating
cells in both the hematopoietic tissues and small intestine,
leading to severe and often dose-limiting toxicities. Damage to the
small intestine mucosal barrier results in serious complications of
bleeding and sepsis. The subject method can be used to promote
proliferation of gastrointenstinal epithelium and thereby increase
the tolerated doses for radiation and chemotherapy agents.
Effective treatment of gastrointestinal diseases may be determined
by several criteria, including an enteritis score, other tests well
known in the art.
[0384] 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 bromodeoxuridine, 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 degenerative diseases of retinal cells and
regulate photoreceptor differentiation.
[0385] With age, the epidermis thins and the skin appendages
atrophy. Hair becomes sparse and sebaceous secretions decrease,
with consequent susceptibility to dryness, chapping, and fissuring.
The dermis diminishes with loss of elastic and collagen fibers.
Moreover, keratinocyte proliferation (which is indicative of skin
thickness and skin proliferative capacity) decreases with age. An
increase in keratinocyte proliferation is believed to conteract
skin aging, i.e., wrinkles, thickness, elasticity and repair.
According to the present invention, a proliferative form of a
hedgehog agonist can be used either therapeutically or cosmetically
to counteract, at least for a time, the effects of aging on
skin.
[0386] Yet another aspect of the present invention relates to the
use of the subject method to promote hair growth. Hair is basically
composed of keratin, a tough and insoluble protein; its chief
strength lies in its disulphide 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.
[0387] 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.
[0388] Thus, in certain embodiments, the subject method can be
employed as a way of promoting the growth of human hair, e.g., to
correct baldness, alopecia, or other diseases characterized by hair
loss.
[0389] The subject method can also be used in treatment of a wound
to eye tissue. Generally, damage to corneal tissue, whether by
disease, surgery or injury, may affect epithelial and/or
endothelial cells, depending on the nature of the wound. Corneal
epithelial cells are the non-keratinized epithelial cells lining
the external surface of the cornea and provide a protective barrier
against the external environment. Corneal wound healing has been of
concern to both clinicians and researchers. Opthomologists are
frequently confronted with corneal dystrophies and problematic
injuries that result in persistent and recurrent epithelial
erosion, often leading to permanent endothelial loss. The use of
proliferative forms of the subject hedgehog agonists can be used in
these instances to promote epithelialization of the affected
corneal tissue.
[0390] To further illustrate, specific disorders typically
associated with epithelial cell damage in the eye, and for which
the subject method can provide beneficial treatment, include
persistent corneal epithelial defects, recurrent erosions,
neurotrophic corneal ulcers, keratoconjunctivitis sicca, microbial
corneal ulcers, viral cornea ulcers, and the like. Surgical
procedures typically causing injury to the epithelial cell layers
include laser procedures performed on the ocular surface, any
refractive surgical procedures such as radial keratotomy and
astigmatic keratotomy, conjunctival flaps, conjunctival
transplants, epikeratoplasty, and corneal scraping. Moreover,
superficial wounds such as scrapes, surface erosion, inflammation,
etc. can cause lose of epithelial cells. According to the present
invention, the corneal epithelium is contacted with an amount of a
hedgehog agonist effective to cause proliferation of the corneal
epithelial cells to appropriately heal the wound.
[0391] In another aspect of the invention, the subject method can
be used to induce differentiation and/or promote 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.
[0392] The present method can be used for improving the "take rate"
of a skin graft. Grafts of epidermal tissue can, if the take rate
of the graft is to long, blister and shear, decreasing the
likelihood that the autograft will "take", i.e. adhere to the wound
and form a basement membrane with the underlying granulation
tissue. Take rates can be increased by the subject method by
inducing proliferation of the keratinocytes. The method of
increasing take rates comprises contacting the skin autograft with
an effective wound healing amount of a hedgehog agonist described
in the method of promoting wound healing and in the method of
promoting the growth and proliferation of keratinocytes, as
described above.
[0393] Skin equivalents have many uses not only as a replacement
for human or animal skin for skin grafting, but also as test skin
for determining the effects of pharmaceutical substances and
cosmetics on skin. A major difficulty in pharmacological, chemical
and cosmetic testing is the difficulties in determining the
efficacy and safety of the products on skin. One advantage of the
skin equivalents of the invention is their use as an indicator of
the effects produced by such substances through in vitro testing on
test skin.
[0394] Thus, in one embodiment of the subject method can be used as
part of a protocol for skin grafting of, e.g., denuded areas,
granulating wounds and burns. The use of hedgehog agonists can
enhance such grafting techniques as split thickness autografts and
epidermal autografts (cultured autogenic keratinocytes) and
epidermal allografts (cultured allogenic keratinocytes). In the
instance of the allograft, the use of the subject method to enhance
the formation of skin equivalents in culture helps to
provide/maintain a ready supply of such grafts (e.g., in tissue
banks) so that the patients might be covered in a single procedure
with a material which allows permanent healing to occur.
[0395] In this regard, the present invention also concerns
composite living skin equivalents comprising an epidermal layer of
cultured keratinocyte cells which have been expanded by treatment
with a hedgehog agonist. The subject method can be used as part of
a process for the preparation of composite living skin equivalents.
In an illustrative embodiment, such a method comprises obtaining a
skin sample, treating the skin sample enzymically to separate the
epidermis from the dermis, treating the epidermis enzymically to
release the keratinocyte cells, culturing, in the presence of a
hedgehog agonist, the epidermal keratinocytes until confluence, in
parallel, or separately, treating the dermis enzymatically to
release the fibroblast cells, culturing the fibroblasts cells until
sub-confluence, inoculating a porous, cross-linked collagen sponge
membrane with the cultured fibroblast cells, incubating the
inoculated collagen sponge on its surface to allow the growth of
the fibroblast cells throughout the collagen sponge, and then
inoculating it with cultured keratinocyte cells, and further
incubating the composite skin equivalent complex in the presence of
a hedgehog agonist to promote the growth of the cells.
[0396] In other embodiments, skin sheets containing both epithelial
and mesenchymal layers can be isolated in culture and expanded with
culture media supplemented with a proliferative form of a hedgehog
agonist. Any skin sample amenable to cell culture techniques can be
used in accordance with the present invention. The skin samples may
be autogenic or allogenic.
[0397] In another aspect of the invention, the subject method can
be used in conjunction with various periodontal procedures in which
control of epithelial cell proliferation in and around periodontal
tissue is desired. In one embodiment, hedgehog agonists can be used
to enhance reepithelialization around natural and prosthetic teeth,
e.g., to promote formation of gum tissue.
[0398] Hedgehog gene products are able to regulate maturation of T
lymphocytes. Certain aspects of the invention are directed to
hedgehog agonists and their uses as immunomodulatory agents against
both acquired and hereditary immunological disorders.
[0399] For instance, such compositions can be used to increase the
population of T-helper cells to optimum levels in the host, e.g, to
stimulate the immune system of the animal. Such uses of the subject
compoistions can be used in the treatment of bacterial or viral
infections, as well as to help the body fight against cancer cells.
Alternatively, these substances also enable the host to adjust to
diseases arising from disarrangement of self-recognition processes
in which there is excessive attack by host T-cells against
endogenous tissues. In such instances, the subject compositions can
be used to reduce T-cell population so that the signs and symptoms
of self-directed inflammatory (autoimmune) diseases such rheumatoid
arthritis and multiple sclerosis are ameliorated.
[0400] As described herein, hedgehog proteins inhibit maturation of
T lymphocytes. Based upon its inhibitory effect, the administration
of hedgehog agonists is suggested herein as a treatment for several
types of immunological disorders involving unwanted activation of
cellular immunity, e.g., graft rejection, autoimmune disorders, and
the like.
[0401] In general, the method of the present invention comprises
administering to animal, or to cultured lymphocytes in vitro, an
amount of a hedgehog agonist which produces a non-toxic response by
the cell of inhibition of maturation. The subject method can be
carried out on cells which may be either dispersed in culture or a
part of an intact tissue or organ. Moreover, the method can be
performed on cells which are provided in culture (in vitro), or on
cells in a whole animal (in vivo). The invention also relates to
methods of controlling the functional performance of T cells by use
of the pharmaceutical preparations of the invention.
[0402] Without wishing to be bound by any particular theory, the
inhibitory effect of hedgehog on T cell maturation may be due at
least in part to the ability of hedgehog proteins to antagonize
(directly or indirectly) patched-mediated regulation of gene
expression and other physiological effects mediated by that
protein. The patched gene product, a cell surface protein, is
understood to signal through a pathway which causes transcriptional
repression of members of the Wnt and Dpp/BMP families of
morphogens, proteins which impart positional information. In other
tissue, the introduction of hedgehog relieves (derepresses) this
inhibition conferred by patched, allowing expression of particular
gene programs.
[0403] In another aspect, the present invention provides
pharmaceutical preparations comprising hedgehog agonists. The
hedgehog agonists 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 agonist, 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".
[0404] Pharmaceutical formulations of the present invention can
also include veterinary compositions, e.g., pharmaceutical
preparations of the hedgehog agonists suitable for veterinary uses,
e.g., for the treatment of live stock or domestic animals, e.g.,
dogs.
[0405] 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 proteinacious
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 agonist at a particular target site.
[0406] 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.
[0407] 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, intraarticulare, subcapsular,
subarachnoid, intraspinal and intrasternal injection and
infusion.
[0408] 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.
[0409] 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, intracistemally and topically, as by
powders, ointments or drops, including buccally and
sublingually.
[0410] 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.
[0411] 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 which is effective to
achieve the desired therapeutic response for a particular patient,
composition, and mode of administration, without being toxic to the
patient.
[0412] 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 agonist 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.
[0413] 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.
[0414] In general, a suitable daily dose of a compound of the
invention will be that amount of the compound which 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, preferably from about 0.001 to about 10 mg per kilogram, even
more preferably from about 0.01 to about 1 mg per kilogram.
[0415] 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.
[0416] The term "treatment" is intended to encompass also
prophylaxis, therapy and cure.
[0417] 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.
[0418] 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 therapeutical effects of the
first administered one is not entirely disappeared when the
subsequent is administered.
[0419] V. Pharmaceutical Compositions
[0420] 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 agonists 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.
[0421] 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.
[0422] 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 ptc
gain-of-function, hedgehog loss-of-function, or smoothened
loss-of-function 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.
[0423] 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.
[0424] 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 agonists 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.
[0425] As set out above, certain embodiments of the present
hedgehog agonists 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)
[0426] 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.
[0427] 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)
[0428] 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.
[0429] 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.
[0430] 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 which can be combined with a carrier material to
produce a single dosage form will generally be that amount of the
compound which produces a therapeutic effect. Generally, out of one
hundred percent, this amount will range from about 1 percent to
about ninety-nine percent of active ingredient, preferably from
about 5 percent to about 70 percent, most preferably from about 10
percent to about 30 percent.
[0431] 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.
[0432] 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.
[0433] 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.
[0434] 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.
[0435] 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 which
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 which 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.
[0436] 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.
[0437] 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.
[0438] 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.
[0439] 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 agonist.
[0440] 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.
[0441] 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 which may be required.
[0442] 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,
tragacanth, cellulose derivatives, polyethylene glycols, silicones,
bentonites, silicic acid, talc and zinc oxide, or mixtures
thereof.
[0443] 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.
[0444] 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 agonists in the proper medium. Absorption enhancers can
also be used to increase the flux of the hedgehog agonists 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.
[0445] Ophthalmic formulations, eye ointments, powders, solutions
and the like, are also contemplated as being within the scope of
this invention.
[0446] 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.
[0447] Examples of suitable aqueous and nonaqueous carriers which
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.
[0448] 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 which delay
absorption such as aluminum monostearate and gelatin.
[0449] 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.
[0450] 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 which are
compatible with body tissue.
[0451] 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.
[0452] 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.
[0453] 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, Ore., U.S.A., 1977).
[0454] VI. Synthetic Schemes and Identification of Active
Agonists
[0455] The subject inhibitors, and congeners thereof, can be
prepared readily by employing the cross-coupling technologies of
Suzuki, Stille, and the like. These coupling reactions are carried
out under relatively mild conditions and tolerate a wide range of
"spectator" functionality.
[0456] a. Combinatorial Libraries
[0457] The compounds of the present invention, particularly
libraries of variants having various representative classes of
substituents, are amenable to combinatorial chemistry and other
parallel synthesis schemes (see, for example, PCT WO 94/08051). The
result is that large libraries of related compounds, e.g., a
variegated library of compounds represented above, can be screened
rapidly in high throughput assays in order to identify potential
hedgehog agonist lead compounds, as well as to refine the
specificity, toxicity, and/or cytotoxic-kinetic profile of a lead
compound. For instance, ptc, hedgehog, or smoothened bioactivity
assays, such as may be developed using cells with either a ptc
gain-of-function, hedgehog loss-of-function, or smoothened
loss-of-function, can be used to screen a library of the subject
compounds for those having antagonist activity toward ptc or
agonist activity towards hedgehog or smoothened.
[0458] Simply for illustration, a combinatorial library for the
purposes of the present invention is a mixture of chemically
related compounds which may be screened together for a desired
property. The preparation of many related compounds in a single
reaction greatly reduces and simplifies the number of screening
processes which need to be carried out. Screening for the
appropriate physical properties can be done by conventional
methods.
[0459] Diversity in the library can be created at a variety of
different levels. For instance, the substrate aryl groups used in
the combinatorial reactions can be diverse in terms of the core
aryl moiety, e.g., a variegation in terms of the ring structure,
and/or can be varied with respect to the other substituents.
[0460] A variety of techniques are available in the art for
generating combinatorial libraries of small organic molecules such
as the subject hedgehog agonists. See, for example, Blondelle et
al. (1995) Trends Anal. Chem. 14:83; the Affymax U.S. Pat. Nos.
5,359,115 and 5,362,899: the Ellman U.S. Pat. No. 5,288,514: the
Still et al. PCT publication WO 94/08051; the ArQule U.S. Pat. Nos.
5,736,412 and 5,712,171; Chen et al. (1994) JACS 116:2661: Kerr et
al. (1993) JACS 115:252; PCT publications WO92/10092, WO93/09668
and WO91/07087; and the Lerner et al. PCT publication WO93/20242).
Accordingly, a variety of libraries on the order of about 100 to
1,000,000 or more diversomers of the subject hedgehog agonists can
be synthesized and screened for particular activity or
property.
[0461] In an exemplary embodiment, a library of candidate hedgehog
agonists diversomers can be synthesized utilizing a scheme adapted
to the techniques described in the Still et al. PCT publication WO
94/08051, e.g., being linked to a polymer bead by a hydrolyzable or
photolyzable group, optionally located at one of the positions of
the candidate agonists or a substituent of a synthetic
intermediate. According to the Still et al. technique, the library
is synthesized on a set of beads, each bead including a set of tags
identifying the particular diversomer on that bead. The bead
library can then be "plated" with ptc gain-of-function, hedgehog
loss-of-function, or smoothened loss-of-function cells for which an
hedgehog agonist is sought. The diversomers can be released from
the bead, e.g., by hydrolysis.
[0462] The structures of the compounds useful in the present
invention lend themselves readily to efficient synthesis. The
nature of the structures of the subject compounds, as generally set
forth above, allows the rapid combinatorial assembly of such
compounds. For example, as in the scheme set forth below, an
activated aryl group, such as an aryl triflate or bromide, attached
to a bead or other solid support can be linked to another aryl
group by performing a Stille or Suzuki coupling with an aryl
stannane or an aryl boronic acid. If the second aryl group is
functionalized with an aldehyde, an amine substituent can be added
through a reductive amination. Alternatively, the second aryl group
could be functionalized with a leaving group, such as a triflate,
tosylate, or halide, capable of being displaced by an amine. Or,
the second aryl group may be functionalized with an amine group
capable of undergoing reductive amination with an amine, e.g.,
CyKNH.sub.2. Other possible coupling techniques include transition
metal-mediated amine arylation reactions. The resultant secondary
amine can then be further functionalized by an acylation,
alkylation, or arylation to generate a tertiary amine or amide
which can then be cleaved from the resin or support. These
reactions generally are quite mild and have been successfully
applied in combinatorial solid-phase synthesis schemes.
Furthermore, the wide range of substrates and coupling partners
suitable and available for these reactions permits the rapid
assembly of large, diverse libraries of compounds for testing in
assays as set forth herein. For certain schemes, and for certain
substitutions on the various substituents of the subject compounds,
one of skill in the art will recognize the need for masking certain
functional groups with a suitable protecting group. Such techniques
are well known in the art and are easily applied to combinatorial
synthesis schemes. 23
[0463] Many variations on the above and related pathways permit the
synthesis of widely diverse libraries of compounds which may be
tested as agonists of hedgehog function.
[0464] b. Screening Assays
[0465] There are a variety of assays available for determining the
ability of a compound to antagonize ptc function or agonize
smoothened or hedgehog function, many of which can be disposed in
high-throughput formats. In many drug screening programs which test
libraries of compounds and natural extracts, high throughput assays
are desirable in order to maximize the number of compounds surveyed
in a given period of time. Thus, libraries of synthetic and natural
products can be sampled for other compounds which are hedgehog
agonists.
[0466] In addition to cell-free assays, test compounds can also be
tested in cell-based assays. In one embodiment, cell which have a
ptc gain-of-function, hedgehog loss-of-function, or smoothened
loss-of-function phenotype can be contacted with a test agent of
interest, with the assay scoring for, e.g., promotion of
proliferation of the cell in the presence of the test agent.
[0467] A number of gene products have been implicated in
patched-mediated signal transduction, including patched,
transcription factors of the cubitus interruptus (ci) family, the
serine/threonine kinase fused (fu) and the gene products of
costal-2, smoothened and suppressor of fused.
[0468] The induction of cells by hedgehog proteins sets in motion a
cascade involving the activation and inhibition of downstream
effectors, the ultimate consequence of which is, in some instances,
a detectable change in the transcription or translation of a gene.
Potential transcriptional targets of hedgehog-mediated signaling
are the patched gene (Hidalgo and Ingham, 1990 Development 110,
291-301; Marigo et al., 1996) and the vertebrate homologs of the
drosophila cubitus interruptus gene, the Gli genes (Hui et al.
(1994) Dev Biol 162:402-413). Patched gene expression has been
shown to be induced in cells of the limb bud and the neural plate
that are responsive to Shh. (Marigo et al. (1996) PNAS 93:9346-51;
Marigo et al. (1996) Development 122:1225-1233). The Gli genes
encode putative transcription factors having zinc finger DNA
binding domains (Orenic et al. (1990) Genes & Dev 4:1053-1067;
Kinzler et al. (1990) Mol Cell Biol 10:634-642). Transcription of
the Gli gene has been reported to be upregulated in response to
hedgehog in limb buds, while transcription of the Gli3 gene is
downregulated in response to hedgehog induction (Marigo et al.
(1996) Development 122:1225-1233). By selecting transcriptional
regulatory sequences from such target genes, e.g., from patched or
Gli genes, that are responsible for the up- or down-regulation of
these genes in response to hedgehog signalling, and operatively
linking such promoters to a reporter gene, one can derive a
transcription based assay which is sensitive to the ability of a
specific test compound to modify hedgehog-mediated signalling
pathways. Expression of the reporter gene, thus, provides a
valuable screening tool for the development of compounds that act
as agonists of hedgehog.
[0469] Reporter gene based assays of this invention measure the end
stage of the above-described cascade of events, e.g.,
transcriptional modulation. Accordingly, in practicing one
embodiment of the assay, a reporter gene construct is inserted into
the reagent cell in order to generate a detection signal dependent
on ptc loss-of-function, hedgehog gain-of-function, smoothened
gain-of-function, or stimulation by Shh itself. The amount of
transcription from the reporter gene may be measured using any
method known to those of skill in the art to be suitable. For
example, mRNA expression from the reporter gene may be detected
using RNAse protection or RNA-based PCR, or the protein product of
the reporter gene may be identified by a characteristic stain or an
intrinsic biological activity. The amount of expression from the
reporter gene is then compared to the amount of expression in
either the same cell in the absence of the test compound or it may
be compared with the amount of transcription in a substantially
identical cell that lacks the target receptor protein. Any
statistically or otherwise significant increase in the amount of
transcription indicates that the test compound has in some manner
antagonized the normal ptc signal (or agonized the hedgehog or
smoothened signal), e.g., the test compound is a potential hedgehog
agonist.
[0470] Exemplification
[0471] 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.
[0472] In the experimental section below, the term `Hh protein` is
used to designate octyl-Shh-N, a lipophilic form of a bacterially
derived fragment of human sonic hedgehog protein (amino acids
24-198, Shh-N). Specifically, Shh-N has been covalently linked in
vitro via its amino terminal cysteine to an octyl maleimide group.
This modified form, like others described recently (Pepinsky et
al., J. Biol. Chem. 1998, 273, 14037-45) exhibits higher specific
potency than the corresponding unmodified fragment in several
cell-based assays of hedgehog signalling.
[0473] Compound Screening
[0474] To measure the hedgehog agonist activity of compounds, we
used [10T1/2(s12)] cells containing the hedgehog-responsive Gli-Luc
reporter-construct. In each MTP (MicroTiter Plate; 96-well plate),
10,000-20,000 cells were plated per well, in full media (10% FBS).
After about 24-48 hr, plates were switched to low-serum media
(=0.5% FBS). Subsequently, a test compound was added at 1-5 .mu.M
in the presence or absence of octyl-hedgehog (see below). After
another 24 hr, the media from the MTPs was discarded and replaced
with luciferase assay-mix, containing lysis-buffer with luciferase
substrate. The plates were incubated at RT for about 15-30 min and
read in a luminometer.
[0475] Screen A
[0476] Plates were incubated for 48 hr before switching to
low-serum. Compounds were screened at 1-2 .mu.M in the presence of
Hh protein (0.01 .mu.g/ml; EC.sub.30=about 30% of max-induced
activity).
[0477] Screen B
[0478] Plates were incubated for 24 hr before switching to
low-serum. Compounds were screened at 5 .mu.M without adding Hh
protein.
[0479] Compound Counter-Screen (SV-Luc)
[0480] For the counter-screen we used the [10T1/2(SV-Luc)] cells
containing a SV40-luciferase expression cassette that allows for a
constitutive level of luciferase-activity in the cells. This assay
allows one to assess the specificity of the compounds selected in
the Gli-Luc assay, i.e., whether the compounds specifically
stimulate the hedgehog signalling pathway only, or
reporter-constructs in general. Cell plating and cultivation as
well as compound handling were performed as in the Gli-Luc assay.
In this assay, no Hh protein was added because the
reporter-construct is constitutively active already.
[0481] From Screen A, we identified hits identified in FIGS. 32 and
33. The 1,4-diaminocyclohexane subunit of the compounds of FIG. 32
which include this moiety have the two amino substituents disposed
in a trans-relationship, e.g., both substituents equatorial on the
cyclohexane subunit. These hits were confirmed in Screen B. The
data shown in FIGS. 34a and 34b essentially involved dosing the two
compounds in both assays (Gli-Luc & SV-Luc).
[0482] The data from the Gli-Luc assay is converted to percent
activity where full activity with Hh protein is set at 100% (FIG.
35; Table 1). The data shows that the two compounds can
significantly stimulate activity from the Gli-luc reporter
construct (i.e., activate hedgehog signalling) even in the absence
of Hh protein; concentration range 0.1-15 .mu.M, EC50=2 .mu.M,
ECmax=50-70% of max-induced activity with Hh protein). Furthermore,
the compounds show a more striking induction in the presence of
hedgehog (EC50=0.2-0.3 uM, ECmax=70-90% of max induced activity
with Hh protein), indicating a synergy between the compounds and Hh
protein, consistent with the compounds serving as agonists for the
hedgehog pathway.
1 TABLE 1 Compound EC50 (.mu.M) A B <10 C <1 D <0.1 E
<10 F <10 G <10 H <1 I >10 J <1 K <1 L
<0.05 M <1 N <1 O <1 P <1 Q <1 R <1 S <1 T
<10 U <10 V <0 W <10 X <10 Y <10 Z <10 A'
<10 B' <10 C' <10 D' <10 E' <10 F' <10 G' <10
H' >10 I' <1 J' <1 K' <10 L' <.05 M' <10 N' <1
O' <1 P' <1 Q' <1
[0483] The data from the SV-Luc assay (FIG. 35) indicate that the
two compounds do not affect the luciferase activity over the range
of concentrations (up to 15 .mu.M) tested, suggesting that the
compounds do not stimulate activity from a reporter-construct per
se, but are probably specific to the hedgehog pathway. Also, the
unaffected activity suggests that the compounds are not
intrinsically toxic to the cells.
[0484] Hh Agonist: Quantitative RT-PCR Measurement of Gli
Upregulation
[0485] The compounds of FIGS. 32 and 33 were tested for their
ability to activate transcription of two well-studied targets of
the Hedgehog pathway: the transcription factor Gli-1, and the
putative Hedgehog receptor component Ptc-1. As depicted in FIG. 35,
we found that, at a compound concentration of 8 .mu.M, induction of
both targets was approximately 60% for the p-cyanophenyl compound
and 40% for the m-nitrophenyl compound of that obtained with the
optimal concentration of Hh protein.
[0486] Assays were performed as follows:
[0487] Murine C3H 10T1/2 cells were seeded at approximately 200,000
cells per well in a 24-well plate in complete medium (10% FBS).
After 24 hr, medium was removed and replaced with "starvation"
medium (0.5% FBS) containing dilutions of compound or Hh protein.
After 16-18 hr, total RNA was prepared using TriZol (Life
Technologies, inc.).
[0488] One microgram aliquots of total RNA were used to prepare
random hexamer-primed cDNA with M-MTV Reverse transcriptase (Life
Technologies, Inc.).
[0489] To measure the relative levels of Gli-1 and Ptc-1
transcripts, TaqMan assays (PE Biosystems) were performed with an
ABI Prism 7700 Sequence Detection System. As an internal control,
GAPDH transcript levels were simultaneously measured in each
reaction. Data were analyzed using Sequence Detector v1.6.3 (Perkin
Elmer).
[0490] Induction of Cerebellar Neuron Precursor Proliferation by
Hedgehog Signaling Agonists.
[0491] In order to test the efficacy of Hedgehog signaling
agonists, the ability of the agonists to stimulate the
proliferation of cerebellar neuron precursors was determined.
Cerebellar granule neuron precursors are known to respond to Hh
protein by proliferating. To test the agonists, cerebellar neurons
were dissected out of postnatal (one week) rat brains, and placed
into primary cell culture. Treatment agents were added once, on the
first day of culture (0 DIV). Cells were left in culture until 2
DIV, when 3H-thymidine was added for 5 hours; the amount of
incorporation by the cells of this 3H-thymidine provides a measure
of the level of proliferation of the cells. Cells were then lysed,
and the incorporation of 3H-thymidine was determined. The positive
control was Hh protein, at 1.0 .mu.g/ml final concentration. Hh
protein alone caused approximately a 20-fold increase in
3H-thymidine incorporation, consistent with the known ability of
Shh to stimulate proliferation of these cells.
Non-Hh-protein-stimulated cells, including cells treated with
control vehicle (DMSO), had very low levels of proliferation.
However, the two of the Hh agonists in FIG. 32 that were tested, A
and B, were found to significantly stimulate 3H-thymidine
incorporation in the absence of Hh protein treatment, as depicted
in FIG. 36. At 5.0 .mu.M, the p-cyanophenyl compound B caused
10.2-fold induction of 3H-thymidine incorporation. FIG. 37 shows
that agonist D also strongly stimulated 3H-thymidine incorporation
in the absence of Shh. At 1.0, 0.3, or 0.1 .mu.M, D caused 16-,
17-, or 13-fold induction, respectively, of 3H thymidine
incorporation into the cells. These observations demonstrate that
the hedgehog agonists can stimulate known hedgehog biological
responses in target cells.
[0492] Nerve Crush Assay
[0493] CD-1 male mice were anesthetized with Avertin, 240 mg/kg IP.
The area on the ventral side of the mouse leg around the knee was
shaved and cleaned with 70% ethanol to remove the hair followed by
swabbing with Betadine. The skin over the thigh was tented up with
forceps and small scissors were used to make a 1/4-inch cut in the
skin. Fat was moved away to expose the femoral nerve, artery, and
vein. With the tips of the curved #7 forceps pointing down, the
muscle was spread apart just below the femoral artery/vein to
reveal the sciatic nerve deep in the muscle. While using one pair
of forceps to hold the muscle apart, a second pair was used to
carefully lift up the nerve. Care was taken to not lift up the
muscle fibers. The forceps were opened and closed 3 times to
separate the nerve from the muscle. The nerve was held elevated
from the muscle with the curved forceps and the hemostats were
clamped down on the nerve to keep the nerve in the middle of the
hemostats. The hemostats were held in place for 10 seconds. In this
manner, both sciatic nerves were crushed approximately 1+ cm above
the knee (and branches). The hemostats were unclipped and the nerve
fell back into the muscle. A small pipet tip (P2) was used to apply
a small amount of histological tissue marking dye to the crushed
area. A surgical clip was used to close the incision. Care was
taken not to clip the skin to the muscle. The clips were left in
place throughout the entire experiment.
[0494] Control surgery was done to a group of mice. This involved
lifting the nerve up with the forceps and letting it fall back into
place without any crush. This site can also be marked with the
histological dye.
[0495] Drug treatment began on the day of surgery. For Shh protein,
a fusion protein of Shh and immunoglobin (as described in U.S.
Provisional Application No. 60/164,025, incorporated herein by
reference) administered at a dose of 1 mg/kg in a solution of PBS,
a subcutaneous injection was given in the middle of the back of the
animal with a 28 gauge, 1/2 cc insulin syringe and repeated every
other day until day 12-13. (Recovery was complete by then.) For
agonist administration, a solution of the agonist in 43% DMSO/PBS
(FIGS. 38A and 39B), or in 10% DMSO/water (FIGS. 38B and 39D), was
delivered by a minipump (1 .mu.L/hour). Behavioral tests were
initiated on day 4 post-surgery and continued until day 12 or
13.
[0496] Behavioral Testing--Grip Assay
[0497] A mouse was placed on an 8".times.8" metal wire grid (like a
test-tube rack) with 1 cm openings, and the grid was slowly
inverted 10 times with a constant steady motion. The number of
times the mouse failed to grip with its left and right hind limbs
was recorded. The mouse was kept away from the edges of the grid by
repositioning the mouse on the grid as necessary. If the mouse just
hooked its leg around or through the grid it was considered a
failure. An inversion was repeated if the mouse was walking, and
failed to grip the grid.
[0498] Failures of the left and right leg were pooled with other
animals from that experimental group (6 animals/group.times.2 foot
grip scores/animal=12 grip scores/group on any given time point).
Results for agonist D are depicted in FIG. 38A. Animals treated
with vehicle alone generally began to recover on their own by day 9
post-surgery. FIG. 38B depicts results obtained using agonist D
dissolved in a vehicle of 10% DMSO/water in the treatment
protocol.
[0499] Behavioral Testing--Toespread Measurements
[0500] Toespread measuring began at Day 4 post-surgery and was
measured every other day. The mouse was held by the proximal part
of the tail and permitted to hold onto the wire top of the cage
with its front limbs. A small paint brush or cotton applicator was
used to paint the hind toes and footpads of the mouse. The mouse
was allowed to walk across a clean sheet of paper, to leave at
least two clean prints for each hind limb. Using a ruler, a line
was drawn through the widest toe prints on each foot and the
distance between them was measured. As the animals recovered, the
distance increased to normal measurements.
[0501] The left and right leg scores were averaged and pooled with
the other animals from that experimental group (6
animals/group.times.2 foot grip scores/animal=12 toe spread
scores/group on any given time point). Results using agonist D are
depicted in FIGS. 39A and B. FIG. 39D depicts the effects of
agonist D dissolved in a vehicle of 10% DMSO/water in this
protocol.
[0502] Reporter Mice Assays
[0503] Mice with a .beta.-galactosidase transgene (ptc-lacZ mice)
under the regulatory control of the patched locus expresss the
.beta.-galactosidase protein in the same cells where the endogenous
mouse patched gene is expressed. The .beta.-galactosidase protein
can thus be used as a faithful reporter of the expression of the
endogenous patched gene. The patched gene is a known component of
the hedgehog signalling pathway and is upregulated when the
hedgehog pathway is activated. Hence, in the ptc-lacZ mice
.beta.-galactosidase protein is overexpressed in mice in which the
hedgehog pathway has been activated, resulting in more intense blue
staining (due to higher lexels of the .beta.-galactosidase enzyme)
when tissues are stained for enzymatic activity with the X-gal
substrate.
[0504] Ptc-lacZ mice were divided into four treatment groups and
were treated with the following compounds or combinations of
compounds for four days, beginning on the first day after birth
(dipal-Shh=dipalmitoyla- ted Sonic hedgehog):
2 1) dipal-Shh 10 mg/kg/injection 2X/day 2) dipal-Shh 1
mg/kg/injection 2X/day vehicle 10-15 .mu.l/injection 4Xday 3)
dipal-Shh 1 mg/kg/injection 2X/day agonist B 15 mg/kg/injection
4X/day 4) no treatment
[0505] The vehicle for the agonist was 10% DMSO in PBS (pH 7.2).
The agonist was injected from a 4.67 mM stock dissolved in the
vehicle solution.
[0506] 18 hours following the last injections, the mice were
sacrificed and the following tissues were collected: skull, kidney,
lung, scapula, skin and heart. All tissues were fixed in 0.2%
glutaraldehyde, 5 mM EDTA (pH 8.0), 20 mM MgCl.sub.2, 100 mM
Na.sub.2HPO.sub.4 for 30 minutes before staining in 1 mg/ml X-gal,
12.5 mM potassium ferrocyanide, 12.5 mM potassium ferricyanide, 2
mM MgCl.sub.2, 0.01% deoxycholate, 0.02% NP-40, and 100 mM
Na.sub.2HPO.sub.4 for 30 hours. Tissues were judged for relative
intensity and photographed.
[0507] All tissues from treatment group 3 (agonist+low dose
dipal-Shh) showed significant upregulation of .beta.-galactosidase
(as visualized by more intense blue staining) compared to those
from treatment group 2 (vehicle+low dose dipal-Shh), as seen in
FIG. 40, wherein tissues from group 3 are depicted on the right
side of each frame. The images in FIG. 41 show an example of tissue
from each of the four treatment groups. The upregulation observed
in group 3 was similar to that seen in treatment group 1 (high dose
dipal-Shh). The level of .beta.-galactosidase staining in treatment
group 2 was similar to that seen in treatment group 4 (no
treatment).
[0508] Agonist B was clearly capable of upregulating the hedgehog
pathway in vivo in the presence of a low dose of dipal-shh which by
itself is insufficient to produce detectable upregulation.
[0509] FIG. 42: Ptc-lacZ mice were divided into two treatment
groups and were treated with the following compounds for three
days, beginning on the first day after birth:
3 1) vehicle 8-15 .mu.l/injection 4X/day 2) agonist D 4
mg/kg/injection 4X/day
[0510] The vehicle for the agonist was 10% DMSO in PBS (pH 7.2).
The agonist was injected from a 1.0 mM stock dissolved in the
vehicle solution.
[0511] Eighteen hours following the last injections, the mice were
sacrificed and the forelimbs were collected and processed as
described above. The forelimbs from the agonist-treated mice showed
strong upregulation in the nerves, blood vessels, cartilage, and
connective tissue.
[0512] Agonist D was clearly capable of upregulating the hedgehog
pathway in vivo in the absence of dipal-Shh. The upregulation seen
at this dose of agonist D is greater than that seen with injections
of dipal-Shh at 10 mg/kg/injection, 2.times./day.
[0513] In a third experiment, Ptc-lacZ mice were divided into five
treatment groups and were treated with the following compounds for
four days, beginning on the first day after birth:
4 1) vehicle 9-20 .mu.l/injection 4X/day 2) agonist D 0.9
mg/kg/injection 4X/day 3) agonist D 0.3 mg/kg/injection 4X/day 4)
agonist D 0.1 mg/kg/injection 4X/day 5) octyl-shh 10
mg/kg/injection 2X/day
[0514] The vehicle for the agonist was 10% DMSO in PBS (pH 7.2).
The agonist was injected from stocks of 0.3, 0.1 and 0.03 mM
dissolved in the vehicle solution.
[0515] Eighteen hours following the last injections, the mice were
sacrificed and the forelimbs were collected and processed as
described above. Results of this experiment are depicted in FIG.
43, in which the vehicle control is not shown. The forelimbs from
the agonist-treated mice again showed strong upregulation in the
nerves, blood vessels, cartilage, and connective tissue (compared
to the vehicle group). The 0.9 mg/kg group showed the highest
levels of upregulation in this experiment. Both the 0.9 mg/kg and
the 0.3 mg/kg dose groups showed greater upregulation than the 10
mg/kg Hh protein group. The 0.1 mg/kg group showed very weak
upregulation that was clearly less than that seen in the Hh protein
group.
[0516] Agonist D was clearly capable of upregulating the hedgehog
pathway in vivo in a dose-responsive manner. The upregulation seen
at the 0.9 and 0.3 mg/kg doses of agonist D is greater than that
seen with injections of Hh protein at 10 mg/kg/injection,
2.times./day.
[0517] Lung Branching Assay
[0518] E12.5 old ptc-1 (d11) lacZ lungs were harvested and
transgenic embryos identified by lacZ detection using tails.
Explants were assembled on 1 .mu.m polycarbonate filters (Costar)
placed on top of plastic grids (histology embedding chamber) and
placed in standard 12-well tissue culture plates filled with lung
explant culture 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.
[0519] Results are depicted in FIG. 44. In the control panel on the
left, LacZ expression can be observed in the mesenchyme immediately
adjacent to distal branching tips, a pattern reflective of
endogenous patched expression. Treatment with 5 .mu.M of agonist B
leads to significantly increased reporter gene expression and
expansion of the expression domain of the transgene, indicative of
hedgehog pathway upregulation. The rightmost panel shows the
results of treatment with 5 .mu.g/mL of Hh protein.
[0520] Kidney Branching Assay
[0521] E13.5 old ptc-1 (d11) lacZ lungs were harvested and
transgenic embryos identified by lacZ detection using tails.
Explants were assembled on 1 .mu.m polycarbonate filters (Costar)
placed on top of plastic grids (histology embedding chamber) and
placed in standard 12-well tissue culture plates filled with kidney
explant culture 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.
[0522] Results are depicted in FIG. 45. In the control panel on the
left, LacZ expression can be observed in the mesenchyme immediately
adjacent to proximalmost ureteric epitheilium, a pattern reflective
of endogenous patched expression. Treatment with 5 .mu.M of agonist
B leads to significantly increased reporter gene expression and
expansion of the expression domain of the transgene, indicative of
hedgehog pathway upregulation. Note that the signal remains
localized to the mesenchyme and does not expand into the more
distally located ureteric and tubular epithelia, indicating that
only the mesenchymal cell type(s) responding to hedgehog signaling
in the endogenous situation respond to the agonist, while cell
types which usually do not activate this pathway are unaffected by
agonist treatment. The rightmost panel shows the results of
treatment with 5 .mu.g/mL of Hh protein.
[0523] Skin Explants
[0524] Skin from ptc-lacZ E17.5 pups was excised with a 2 mm skin
punch. Those skin punches were then cultured for 6 days in control
media, or media including either agonist B or D or Hh protein, or
media including both an agonist and Hh protein. The explants were
then stained with X-Gal stain. FIG. 46A shows results for agonist
B. Treatment with the agonist alone shows greater staining than
culturing with a low dose of Hh protein alone, and treatment with
agonist and a low dose of Hh protein shows staining similar to that
of a substantially higher dose of Hh protein. Analogous results for
agonist D are presented in FIG. 46B.
[0525] Activity in Human Cells
[0526] FIGS. 47A and B compare the activity of subject compounds 0
and R in mouse reporter cells (TM3 cells with a Gli-Luc reporter
construct, as described above) and in human reporter cells (human
embryonic palatal mesenchyme (HEPM) cells with a Gli-Luc
construct). FIG. 48 shows a quantitative PCR analysis of RNA
expressed from the hedgehog target gene Gli-1 in human cells
treated with the vehicle, Hh protein, and agonist O. The activation
of the reporter cell line and the elevated Gli-1 message in
response to the compounds demonstrate that this agonist functions
in human cells. quisel
[0527] Preparation of Compounds of the Present Invention
[0528] a. Illustrative Synthetic Schemes
[0529] Exemplary synthesis schemes for generating hedgehog agonists
useful in the methods and compositions of the present invention are
shown in FIGS. 1-31.
[0530] The reaction conditions in the illustrated schemes of FIGS.
1-31 are as follows:
[0531] 1) R.sub.1CH.sub.2CN, NaNH.sub.2, toluene
[0532] (Arzneim-Forsch, 1990, 40, 11, 1242)
[0533] 2) H.sub.2SO.sub.4, H.sub.2O, reflux
[0534] (Arzneim-Forsch, 1990, 40, 11, 1242)
[0535] 3) H.sub.2SO.sub.4, EtOH, reflux
[0536] (Arzneim-Forsch, 1990, 40, 11, 1242)
[0537] 4) NaOH, EtOH, reflux
[0538] 5) (Boc).sub.2O, 2M NaOH, THF
[0539] 6) LiHDMS, R.sub.1X, THF
[0540] (Merck Patent Applic # WO 96/06609)
[0541] 7) Pd--C, H.sub.2, MeOH
[0542] 8) t-BuONO, CuBr, HBr, H.sub.2O
[0543] (J. Org. Chem. 1977, 42, 2426)
[0544] 9) ArB(OH).sub.2, Pd(PPh.sub.3).sub.4, Dioxane
[0545] (J. Med. Chem. 1996, 39, 217-223)
[0546] 10) R.sub.12(H)C.dbd.CR.sub.13R.sub.14, Pd(OAc).sub.2,
Et.sub.3N, DMF
[0547] (Org. React. 1982, 27, 345)
[0548] 11) Tf.sub.2O, THF
[0549] (J. Am. Chem. Soc. 1987, 109, 5478-5486)
[0550] 12) ArSnBu.sub.3, Pd(PPh.sub.3).sub.4, Dioxane
[0551] (J. Am. Chem. Soc. 1987, 109, 5478-5486)
[0552] 13) KMnO.sub.4, Py, H.sub.2O
[0553] (J. Med. Chem. 1996, 39, 217-223)
[0554] 14) NaOR.sub.1, THF
[0555] 15) NaSR.sub.1, THF
[0556] 16) HNR.sub.1R.sub.13, THF
[0557] 17) HONO, NaBF.sub.4
[0558] (Adv. Fluorine Chem. 1965, 4, 1-30)
[0559] 18) Pd(OAc).sub.2, NaH, DPPF, PhCH.sub.3, R.sub.1OH
[0560] (J. Org. Chem. 1997, 62, 5413-5418)
[0561] 19) i. R.sub.1X, Et.sub.3N, CH.sub.2Cl.sub.2, ii.
R.sub.13X
[0562] 20) SOCl.sub.3, cat DMF
[0563] 21) CH.sub.2N.sub.2, Et.sub.2O
[0564] 22) Ag.sub.2O, Na.sub.2CO.sub.3, Na.sub.2S.sub.2O.sub.3,
H.sub.2O
[0565] (Tetrahedron Lett. 1979, 2667)
[0566] 23) AgO.sub.2CPh, Et.sub.3N, MeOH
[0567] (Org. Syn., 1970, 50, 77; J. Am. Chem. Soc. 1987, 109,
5432)
[0568] 24) LiOH, THF-MeOH
[0569] 25) (EtO).sub.2P(O)CH.sub.2CO.sub.2R, BuLi, THF
[0570] 26) MeO.sub.2CCH(Br).dbd.P(Ph).sub.3, benzene
[0571] 27) KOH or KOtBu
[0572] 28) Base, X(CH.sub.2).sub.nCO.sub.2R
[0573] 29) DPPA, Et.sub.3N, toluene
[0574] (Synthesis 1985, 220)
[0575] 30) HONO, H.sub.2O
[0576] 31) SO.sub.2, CuCl, HCl, H.sub.2O
[0577] (Synthesis 1969, 1-10, 6)
[0578] 32) Lawesson's reagent, toluene
[0579] (Tetrahedron Asym. 1996, 7, 12, 3553)
[0580] 33) R.sub.2M, solvent
[0581] 34) 30% H.sub.2O.sub.2, glacial CH.sub.3CO.sub.2H
[0582] (Helv. Chim. Acta. 1968, 349, 323)
[0583] 35) triphosgene, CH.sub.2Cl.sub.2
[0584] (Tetrahedron Lett., 1996, 37, 8589)
[0585] 36) i. (EtO).sub.2P(O)CHLiSO.sub.2Oi-Pr, THF, ii. NaI
[0586] 37) Ph.sub.3PCH.sub.3I, NaCH.sub.2S(O)CH.sub.3, DMSO
[0587] (Synthesis 1987, 498)
[0588] 38) Br.sub.2, CHCl.sub.3 or other solvent
[0589] (Synthesis 1987, 498)
[0590] 39) BuLi, Bu.sub.3SnCl
[0591] 40) ClSO.sub.2OTMS, CCl.sub.4
[0592] (Chem. Ber. 1995, 128, 575-580)
[0593] 41) MeOH--HCl, reflux
[0594] 42) LAH, Et.sub.2O or LiBH.sub.4, EtOH or BH.sub.3--THF
[0595] (Tetrahedron Lett., 1996, 37, 8589)
[0596] 43) MsCl, Et.sub.3N, CH.sub.2Cl.sub.2
[0597] (Tetrahedron Lett., 1996, 37, 8589)
[0598] 44) Na.sub.2SO.sub.3, H.sub.2O
[0599] (Tetrahedron Lett., 1996, 37, 8589)
[0600] 45) R.sub.2R.sub.4NH, Et.sub.3N, CH.sub.2Cl.sub.2
[0601] 46) R.sub.2M, solvent
[0602] 47) CH.sub.3NH(OCH.sub.3), EDC, HOBt, DIEA, CH.sub.2Cl.sub.2
or DMF
[0603] (Tetrahedron Lett, 1981, 22, 3815)
[0604] 48) MeLi, THF
[0605] 49) mCPBA, CH.sub.2Cl.sub.2
[0606] 50) HONO, Cu.sub.2O, Cu(NO.sub.3).sub.2, H.sub.2O
[0607] (J. Org. Chem. 1977, 42, 2053)
[0608] 51) R.sub.1M, solvent
[0609] 52) HONO, NaS(S)COEt, H.sub.2O
[0610] (Org. Synth. 1947, 27, 81)
[0611] 53) HSR.sub.2 or HSR.sub.4, CH.sub.2Cl.sub.2
[0612] 54) i-BuOC(O)Cl, Et.sub.3N, NH.sub.3, THF
[0613] 55) R.sub.2R.sub.4NH, CH.sub.2Cl.sub.2, NaBH(OAc).sub.3
[0614] 56) R.sub.2R.sub.4NH, MeOH/CH.sub.3CO.sub.2H,
NaBH.sub.3CN
[0615] 57) R.sub.2OH, EDC, HOBt, DIEA, CH.sub.2Cl.sub.2 or DMF
[0616] 58) R.sub.2OH, HBTU, HOBt, DIEA, CH.sub.2Cl.sub.2 or DMF
[0617] 59) R.sub.2R.sub.4NH, EDC, HOBt, DIEA, CH.sub.2Cl.sub.2 or
DMF
[0618] 60) R.sub.2R.sub.4NH, HBTU, HOBt, DIEA, CH.sub.2Cl.sub.2 or
DMF
[0619] 61) POCl.sub.3, Py, CH.sub.2Cl.sub.2
[0620] 62) R.sub.2R.sub.4NCO, solvent
[0621] 63) R.sub.2OC(O)CI, Et.sub.3N, solvent
[0622] 64) R.sub.2CO.sub.2H, EDC or HBTU, HOBt, DIEA,
CH.sub.2Cl.sub.2 or DMF
[0623] 65) R.sub.2X, Et.sub.3N, solvent
[0624] 66) (CH.sub.3S).sub.2C.dbd.N(CN), DMF, EtOH
[0625] (J. Med. Chem. 1994, 37, 57-66)
[0626] 67) R.sub.2SO.sub.2Cl, Et.sub.3N, CH.sub.2Cl.sub.2
[0627] 68) R.sub.2-- or R.sub.3-- or R.sub.4CHO,
MeOH/CH.sub.3CO.sub.2H, NaBH.sub.3CN
[0628] (Synthesis 1975, 135-146)
[0629] 69) Boc(Tr)-D or L-CysOH, HBTU, HOBt, DIEA, CH.sub.2Cl.sub.2
or DMF
[0630] 70) Boc(Tr)-D or L-CysH, NaBH.sub.3CN,
MeOH/CH.sub.3CO.sub.2H
[0631] (Synthesis 1975, 135-146)
[0632] 71) S-Tr-N-Boc cysteinal, ClCH.sub.2CH.sub.2Cl or THF,
NaBH(OAc).sub.3
[0633] (J. Org. Chem. 1996, 61, 3849-3862)
[0634] 72) TFA, CH.sub.2Cl.sub.2, Et.sub.3SiH or (3:1:1)
thioanisole/ethanedithiol/DMS
[0635] 73) TFA, CH.sub.2Cl.sub.2
[0636] 74) DPPA, Et.sub.3N, toluene,
HOCH.sub.2CH.sub.2SiCH.sub.3
[0637] (Tetrahedron Lett. 1984, 25, 3515)
[0638] 75) TBAF, THF
[0639] 76) Base, TrSH or BnSH
[0640] 77) Base, R.sub.2X or R.sub.4X
[0641] 78) R.sub.3NH.sub.2, MeOH/CH.sub.3CO.sub.2H,
NaBH.sub.3CN
[0642] 79) N.sub.2H.sub.4, KOH
[0643] 80) Pd.sub.2(dba).sub.3, P(o-tol).sub.3, RNH.sub.2, NaOtBu,
Dioxane, R.sub.1NH.sub.2
[0644] (Tetrahedron Lett. 1996, 37, 7181-7184).
[0645] 81) Cyanamide.
[0646] 82) Fmoc-Cl, sodium bicarbonate.
[0647] 83) BnCOCl, sodium carbonate.
[0648] 84) AllylOCOCl, pyridine.
[0649] 85) Benzyl bromide, base.
[0650] 86) Oxalyl chloride, DMSO.
[0651] 87) RCONH.sub.2.
[0652] 88) Carbonyldiimidazole, neutral solvents (e.g., DCM, DMF,
THF, toluene).
[0653] 89) Thiocarbonyldiimidazole, neutral solvents (e.g., DCM,
DMF, THF, toluene).
[0654] 90) Cyanogen bromide, neutral solvents (e.g., DCM, DMF, THF,
toluene).
[0655] 91) RCOCl, Triethylamine
[0656] 92) RNHNH.sub.2, EDC.
[0657] 93) RO.sub.2CCOCl, Et.sub.3N, DCM.
[0658] 94) MsOH, Pyridine (J. Het. Chem., 1980, 607.)
[0659] 95) Base, neutral solvents (e.g., DCM, toluene, THF).
[0660] 96) H.sub.2NOR, EDC.
[0661] 97) RCSNH.sub.2.
[0662] 98) RCOCHBrR, neutral solvents (e.g., DCM, DMF, THF,
toluene), (Org. Proc. Prep. Intl., 1992, 24, 127).
[0663] 99) CH.sub.2N.sub.2, HCl. (Synthesis, 1993, 197).
[0664] 100) NH2NHR, neutral solvents (e.g., DCM, DMF, THF,
toluene).
[0665] 101) RSO.sub.2Cl, DMAP. (Tetrahedron Lett., 1993, 34,
2749).
[0666] 102) Et.sub.3N, RX. (J. Org. Chem., 1990, 55, 6037).
[0667] 103) NOCl or Cl.sub.2 (J. Org. Chem., 1990, 55, 3916).
[0668] 104) H.sub.2NOH, neutral solvents (e.g., DCM, DMF, THF,
toluene).
[0669] 105) RCCR, neutral solvents (DCM, THF, Toluene).
[0670] 106) RCHCHR, neutral solvents (DCM, THF, Toluene).
[0671] 107) H.sub.2NOH, HCl.
[0672] 108) Thiocarbonyldiimidazole, SiO.sub.2 or
BF.sub.3OEt.sub.2. (J. Med. Chem., 1996, 39, 5228).
[0673] 109) Thiocarbonyldiimidazole, DBU or DBN. (J. Med. Chem.,
1996, 39, 5228).
[0674] 110) HNO.sub.2, HCl.
[0675] 111) ClCH.sub.2CO.sub.2Et (Org. Reactions, 1959,
10,143).
[0676] 112) Morpholine enamine (Eur. J. Med. Chem., 1982, 17,
27).
[0677] 113) RCOCHR'CN
[0678] 114) RCOCHR'CO.sub.2Et
[0679] 115) Na.sub.2SO.sub.3
[0680] 116) H.sub.2NCHRCO.sub.2Et
[0681] 117) EtO.sub.2CCHRNCO
[0682] 118) RCNHNH.sub.2.
[0683] 119) RCOCO.sub.2H, (J. Med. Chem., 1995, 38, 3741).
[0684] 120) RCHO, KOAc.
[0685] 121) 2-Fluoronitrobenzene.
[0686] 122) SnCl.sub.2, EtOH, DMF.
[0687] 123) RCHO, NaBH.sub.3CN, HOAc.
[0688] 124) NH.sub.3, MeOH.
[0689] 125) 2,4,6-Me.sub.3PhSO.sub.2NH.sub.2.
[0690] 126) Et.sub.2NH, CH.sub.2Cl.sub.2
[0691] 127) MeOC(O)CI, Et.sub.3N, CH.sub.2Cl.sub.2
[0692] 128) R.sub.2NH2, EDC, HOBT, Et.sub.3N, CH.sub.2Cl.sub.2
[0693] 129) DBU, PhCH.sub.3
[0694] 130) BocNHCH(CH.sub.2STr)CH.sub.2NH.sub.2, EDC, HOBT,
Et.sub.3N, CH.sub.2Cl.sub.2
[0695] 131) R.sub.2NHCH.sub.2CO.sub.2Me, HBTU, HOBT, Et.sub.3N,
CH.sub.2Cl.sub.2
[0696] 132) BocNHCH(CH.sub.2STr)CH.sub.2OMs, LiHMDS, THF
[0697] 133) R.sub.2NHCH.sub.2CO.sub.2Me, NaBH(OAc).sub.3,
ClCH.sub.2CH.sub.2Cl or THF
[0698] 134) R.sub.2NHCH.sub.2CH(OEt).sub.2, HBTU, HOBT, Et.sub.3N,
CH.sub.2Cl.sub.2
[0699] 135) NaBH(OAc).sub.3, ClCH.sub.2CH.sub.2Cl or THF, AcOH.
[0700] 136) Piperidine, DMF.
[0701] 137) Pd(Ph.sub.3P).sub.4, Bu.sub.3SnH.
[0702] 138) RCO.sub.2H, EDC, HOBT, Et.sub.3N, DCM.
[0703] 139) RNH.sub.2, neutral solvents.
[0704] 140) RCHO, NaBH.sub.3CN, HOAc.
[0705] 141) RNCO, solvent.
[0706] 142) RCO.sub.2H, EDC or HBTU, HOBt, DIEA, CH.sub.2Cl.sub.2
or DMF.
[0707] 143) RCOCl, Triethylamine
[0708] 144) RSO.sub.2Cl, Et.sub.3N, CH.sub.2Cl.sub.2.
[0709] 145) SnCl.sub.2, EtOH, DMF.
[0710] 146) RNH.sub.2, EDC, HOBt, DIEA, CH.sub.2Cl.sub.2 or
DMF.
[0711] 147) Dibromoethane, Et.sub.3N, CH.sub.2Cl.sub.2
[0712] 148) Oxalyl chloride, neutral solvents.
[0713] 149) LiOH, THF-MeOH.
[0714] 150) Carbonyldiimidazole, neutral solvents (e.g., DCM, DMF,
THF, toluene).
[0715] 151) RNH.sub.2, Et.sub.3N, CH.sub.2Cl.sub.2.
[0716] 152) Base, RX.
[0717] 153) DBU, PhCH.sub.3
[0718] 154) DPPA, Et.sub.3N, toluene (Synthesis 1985, 220)
[0719] 155) SOCl.sub.2, cat DMF.
[0720] 156) ArH, Lewis Acid (AlCl.sub.3, SnCl.sub.4, TiCl.sub.4),
CH.sub.2Cl.sub.2.
[0721] 157) H.sub.2NCHRCO.sub.2Et, neutral solvents.
[0722] 158) BocHNCHRCO.sub.2H, EDC OR HBTU, HOBt, DIEA,
CH.sub.2Cl.sub.2 or DMF.
[0723] 159) TFA, CH.sub.2Cl.sub.2.
[0724] b. Illustrative Preparation of Aryl Subunits
[0725] Ary subunits may be functionalized using a wide variety of
reactions known to those in the art. The chemistry of aromatic and
heteroaromatic rings is rich, and only a sampling of useful
reactions can be presented here. A number of illustrative examples,
particularly useful for generating the biaryl portion of the
subject compounds, are shown below.
[0726] Suzuki Coupling No. 1: 24
[0727] Suzuki Coupling No. 2: 25
[0728] Stille Coupling No. 1: 26
[0729] Stille Coupling No. 2 27
[0730] Stille Coupling No. 3 28
[0731] c. Illustrative Preparation of Coupling Substrates
[0732] Members of the general classes of coupling substrates
outlined above--arylstannanes, arylboronic acids, aryl triflates
and aryl halides--are available from the parent heterocycles. In
general, the transformations required to prepare a coupling
substrate are reliable and amenable to scale-up. Illustrative
examples are shown below.
[0733] Preparation of an Aryl Iodide 29
[0734] Preparation of an Aryl Stannane 30
[0735] Preparation of an Aryl Triflate 31
[0736] Preparation of Aryl Boronic Acid 32
[0737] Solid Phase Synthesis of Subject Compounds
[0738] General:
[0739] Washing Protocols
[0740] Method 1: water (3.times.), acetone (2.times.),
N,N-dimethylformamide (3.times.), water (2.times.), acetone
(1.times.), N,N-dimethylformamide (3.times.), water (2.times.),
acetone (3.times.), methanol (3.times.), acetone (3.times.) and
methanol (3.times.);
[0741] Method 2: dichloromethane, hexane, N,N-dimethylformamide,
dichloromethane, hexane, dichloromethane and hexane;
[0742] Method 3: water, N,N-dimethylformamide, water, 1.0 M aqueous
sodium hydroxide solution, water, N-N dimethylformamide, water, 1.0
aqueous sodium hydroxide solution, water, N,N-dimethylformamide,
dichloromethane, methanol, dichloromethane, and methanol.
[0743] Method 4: N,N-dimethylformamide, dichloromethane,
N,N-dimethylformamide, dichloromethane, methanol, dichloromethane,
methanol (2.times.) and ether (2.times.). 33
[0744] Step A--Preparation of
(Nitrophen-4'-yloxycarboxy)benz-4-yloxymethy- l Polystyrene-(Wang
PNP Carbonate Polystyrene)
[0745] Hydroxybenz-4-yloxymethyl polystyrene (Wang Resin)
[0746] Sodium methoxide (233 g, 4.31 mol) was added slowly to a
stirred mixture of chloromethyl polystyrene (2.4 kg, 3.6 mol
functionalised loading) and 4-hydroxybenzyl alcohol (581 g, 4.68
mol) in N,N-dimethylacetamide (10 L) at room temperature under
nitrogen. After dilution with N,N-dimethylacetamide (13 L), the
mixture was heated at 50.degree. C. for 5 h and then filtered via
cannula through a P-ETFE mesh (70 .mu.m). The crude product was
washed extensively using the sequence of Method 1, then dried under
vacuum at 60.degree. C. to give 2630 g of the title resin.
[0747] (Nitrophen-4'-yloxycarboxy)benz-4-yloxymethyl
polystyrene-(Wang PNP Carbonate Polystyrene)
[0748] 4-Methylmorpholine (660 mL, 6.0 mol) was added dropwise over
2 h to a stirred mixture of hydroxybenz-4-yloxymethyl polystyrene
(2000 g, 2.5 mol functionalised loading) and 4-nitrophenol
chloroformate (1209 g, 6.0 mol) in dichloromethane (22 L) at
(0.degree. C. under nitrogen. The mixture was warmed gradually to
room temperature, stirred overnight and filtered via cannula
through a P-ETFE mesh (70 .mu.m). The crude resin was washed
extensively using the sequence of Method 2, then dried under vacuum
at room temperature to give 2728 g of a mixture of the title resin
and 4-methylmorpholine hydrochloride.
[0749] Step B--The Preparation of Wang Resin-Bound Diamines
[0750] General Method (For Piperazine, Homopiperazine and
trans-1,4-diaminocyclohexane):
[0751] Crude (nitrophen-4'-yloxycarboxy)benz-4-yloxymethyl
polystryrene (1002.5 g, .about.0.9 mol functionalised loading) was
swollen over 15 min in a 50% v/v mixture of anhydrous
dichloromethane and N,N-dimethylformamide (9 L) under nitrogen.
N,N-diisopropylamine (626 mL, 5 mol equivalents) and the
appropriate diamine (5 mol equivalents) were added and the mixture
was stirred vigorously overnight at room temperature. The mixture
was filtered through a P-ETFE mesh (70 .mu.m), washed extensively
using the sequence of Method 3 and dried under vacuum at 60.degree.
C. to give the resin-bound diamine.
[0752] Ethylenediamine Bound to Wang Resin
[0753] Crude (nitrophen-4'-yloxycarboxy)benz-4-yloxymethyl
polystyrene (1002.5 g, .about.0.9 mol functionalised loading) was
swollen over 15 min in dichloromethane (7 L) under nitrogen and
treated with ethylenediamine (181 mL, 2.7 mol). The resulting
thick, yellow suspension was diluted with dichloromethane (2 L) and
vigorously stirred overnight at room temperature. The mixture was
filtered through a P-PETFE mesh (70 .mu.m), washed extensively
using the sequence of Method 3 and dried under vacuum at 60.degree.
C. to give the title resin-bound diamine.
[0754] m-Xylylenediamine Bound to Wang Resin
[0755] Crude (nitrophen-4'-yloxycarboxy)benz-4-yloxymethyl
polystyrene (1002.5 g, .about.0.9 mol functionalised loading) was
swollen in tetrahydrofuran (7 L) over 15 min under nitrogen and
treated with a solution of m-xylylenediamine (828 mL, 6.27 mol) in
tetrahydrofuran (1 L). The resulting thick yellow suspension was
diluted with dichloromethane (2 L) and vigorously stirred overnight
at room temperature. The mixture was filtered through a P-ETFE mesh
(70 .mu.m), washed extensively using the sequence of Method 3 and
dried under vacuum at 60.degree. C. to give the resin-bound
diamine.
[0756] Step C: Preparation of the Building Block Using a Suzuki
Coupling Procedure
[0757] A suspension of the appropriate aryl bromide (1 equivalent)
and potassium carbonate (2.2 equivalents) in toluene (13 volumes)
was stirred and degassed at room temperature.
Tetrakis(triphenylphosphine)palladium(0- ) (0.01 equivalent) was
added and the reaction vessel evacuated and purged with nitrogen
(three times). After 15 min, a degassed solution of
2-methoxy-5-formylphenylboronic acid (1.2 equivalents) in ethanol
(6.3 volumes) was added via cannula, then the mixture was heated
under reflux and stirred overnight under nitrogen. After cooling,
the solid was filtered from solution and washed thoroughly with
toluene. The filtrate was evaporated to dryness under reduced
pressure to give the crude product. This was triturated with
diethyl ether (5 volumes) and the resulting slurry was filtered,
washed with diethyl ether and dried under vacuum. The biaryl
aldehyde was obtained as a yellow powder.
[0758] Step D: Building Block Loading onto Wang Diamine:
[0759] Reductive Alkylation
[0760] The appropriate resin (1 equivalent, .about.0.75 mmol
functionalised loading) was swollen in a mixture of
tetrahydrofuran, trimethylorthoformate and dichloromethane (1:1:1,
v/v/v, 10 mL) over 15 min, then gently agitated and treated with
the appropriate aldehyde (2 equivalents). After gentle agitation
overnight at room temperature, the resin was filtered, washed
thoroughly with tetrahydrofuran and dried under vacuum at
40.degree. C. The dried resin was then swollen in tetrahydrofuran
over 15 min and treated with acetic acid (0.12 equivalent) and
sodium triacetoxyborohydride (5 equivalents). The resin suspension
was gently agitated overnight at room temperature, then filtered,
washed extensively using the sequence of Method 4 and dried under
vacuum at 60.degree. C.
[0761] Acid Chloride Capping
[0762] The appropriate resin (1 equivalent) was swollen in
dichloromethane (10 volumes) over 10 min and treated with the
appropriate acid chloride (3 equivalents) and
N.N-diisopropylethylamine (3 equivalents). The resin suspension was
gently agitated overnight at room temperature, filtered, and washed
extensively using the sequence of Method 4 and dried under vacuum
at 40.degree. C.
[0763] Solution Phase Synthesis of Subject Compounds
[0764] The following exemplary scheme illustrates one route through
which hedgehog agonists of the present invention may be prepared.
Variations on this exemplary pathway will be readily comprehended
and executed by those of ordinary skill in the art, permitting the
preparation of a wide range of compounds that fall within the
disclosed general formulae. The compound numbers used in this
scheme are consistent with the procedures below, and are
independent of the compound numbers used elsewhere in the
application, such as the figures.
[0765] Synthesis of 3-Chloro-benzo[b]thiophene-2-carboxylic acid
(4'-cyano-6-methoxy-biphenyl-3-ylmethyl)-(4-methylaminocyclohexyl)-amide
hydrochloride (7) 34
[0766] (4-Amino-cyclohexyl)-carbamic acid t-butyl ester (1)
[0767] A solution of di-t-butyl-dicarbonate (12.0 g, 54.7 mmol) and
tetrahydrofuran (250 mL) was added slowly under nitrogen over 3 h
to a suspension of 1,4-diaminocyclohexane (50.0 g, 0.44 mol) in
tetrahydrofuran (250 mL) while maintaining the temperature below
10.degree. C. The mixture was allowed to warm to room temperature
and subsequently stirred for 16 hours, then filtered. The filtrate
was concentrated in vacuo to give a residue. Water (500 mL) was
added to the residue, followed by stirring for approximately 15
min, after which the mixture was filtered and the aqueous layer was
extracted with dichloromethane (3.times.200 mL). The organic
extracts were combined and concentrated to give a residue which was
dissolved in t-butylmethyl ether (350 mL) and washed with water
(3.times.50 mL). The t-butylmethyl ether was removed in vacuo to
give the title compound 1 (8.1 g, 69%) as a solid: .delta..sub.H
(360 MHz: CDCl.sub.3) 1.06-1.24 (m, 4H), 1.43 (s, 9H), 1.83 (d,
2H), 1.98 (d, 2H), 2.56-2.66 (m, 1H), 3.30-3.35 (m, 1H) and
4.31-4.38 (m, 1H).
[0768] N-Methyl-cyclohexane-1,4-diamine (2)
[0769] Amine 1 (15.0 g, 0.7 mol) was added slowly over 45 min to a
1N solution of lithium aluminium hydride in THF (450 mL, 0.36 mol)
under nitrogen. The mixture was stirred for 30 min at room
temperature, then heated at reflux for 5-6 hours under nitrogen.
Water (13.2 mL) was added to the mixture followed by 15% aqueous
sodium hydroxide (13.2 mL), and water (39.7 mL). The mixture was
then stirred for 15-30 min. The solid was filtered off and washed
with t-butylmethyl ether (200 mL), dichloromethane (200 mL), and
t-butylmethyl ether (200 mL). The organic extracts were collected,
dried (MgSO.sub.4), and filtered. The drying agent was then washed
with dichloromethane and the organic extracts combined and
concentrated in vacuo to give the title compound 2 (7.52 g, 84%) as
a pale yellow solid: .delta..sub.H (360 MHz: CDCl.sub.3) 1.04-1.20
(q, 4H), 1.51 (br s, 3H), 1.80-1.96 (m, 4H), 2.25-2.35 (m, 1H),
2.41 (s, 3H), 2.61-2.72 (m, 1H).
[0770] (4-Amino-cyclohexyl)-methyl-carbamic acid t-butyl ester
(3)
[0771] Benzaldehyde (12.8 mL, 0.13 mol) was added in a single
portion to a solution of N-methylamine 2 (16.2 g, 0.13 mol) and
toluene (150 mL) under nitrogen The resulting mixture was heated to
reflux using Dean-Stark apparatus for 4 h. After allowing the
mixture to cool to room temperature, di-t-butyl dicarbonate (27.5
g, 0.13 mol) was added in portions and the mixture stirred for 16
h. The mixture was concentrated in vacuo to leave a yellow oil, to
which 1N aqueous potassium hydrogen sulfate (90 mL) was added
followed by vigorously stirring until TLC indicated the reaction
was complete (.about.2.5 h). The mixture was extracted into ether
(3.times.100 mL) and the aqueous layer made alkaline (pH .about.12)
with aqueous sodium hydroxide. The aqueous layer was then saturated
with sodium chloride and the product extracted into chloroform
(3.times.40 mL). The combined extracts were concentrated in vacuo
to give the title compound 3 (16.3 g, 59%) as a yellow oil:
.delta..sub.H (360 MHz: CDCl.sub.3) 1.11-1.34 (m, 5H), 1.45 (s,
9H), 1.66 (br d, 2H), 1.90 (br d, 2H), 2.56-2.66 (m, 1H), 2.71 (s,
3H) and 3.98 (br s, 2H).
[0772]
{4-[(4'-Cyano-6-methoxy-biphenyl-3-ylmethyl)-amino]-cyclohexyl}-met-
hyl-carbamic acid t-butyl ester (5)
[0773] A solution of amine 3 (5.0 g, 21.92 mmol), aldehyde 4 (5.19
g, 21.92 mmol) and trimethyl orthoformate (50 ml) were stirred at
room temperature under nitrogen for 16 h. Sodium
triacetoxyborohydride (6.5 g, 30.7 mmol) was then added portion
wise and the mixture stirred at room temperature until the reaction
was complete, as determined by LC-MS analysis. Water was added
carefully and mixture stirred for a 5 min, followed by separation
of the layers. The trimethyl orthoformate layer was poured onto 1N
aqueous potassium hydrogen sulfate (100 mL) and stirrred for 15
min. The precipitated solid was filtered, washed with water (50
mL), cold tributyl methyl ether (3.times.30 mL). The washed
precipitate was then suspended in dichloromethane (150 mL) to which
saturated aqueous sodium hydrogen carbonate (50 mL) was added and
the pH made alkaline (pH .about.10) whilst maintaining vigorous
stirring. The dichloromethane layer was washed with water, and
brine, and the organic extract was dried (MgSO.sub.4) and
concentrated in vacuo to give the title compound 5 (6.9 g, 69%) as
an off white solid: .delta..sub.H (360 MHz: CDCl.sub.3) 1.18-1.31
(m, 4H), 1.43 (s, 9H), 1.68 (d, 2H), 2.01 (d, 2H), 2.44-2.56 (m,
1H), 2.69 (s, 3H), 3.76 (s, 2H), 3.80 (s, 3H), 6.92 (d, 1H), 7.24
(s, 1H), 7.29 (d, 1H), 7.61 (d, 2H) and 7.66 (d, 2H).
[0774]
{4-[(3-Chloro-benzo[b]thiophene-2-carbonyl)-(4'-cyano-6-methoxy-bip-
henyl-3-ylmethyl)-amino]-cyclohexyl}-methyl-carbamic acid t-butyl
ester (6)
[0775] N,N-Diisopropylethylamine (2.1 mL, 12.1 mmol) was added to a
solution of amine 5 (2.2 g, 4.9 mmol),
3-chlorobenzo[b]thiophene-2-carbon- yl chloride (1.3 g, 5.86 mmol)
and anhydrous dichloromethane (22 mL), with stirring under argon.
Once all of the starting material had been consumed as monitored by
TLC (2.5 hours), the mixture was washed with water, saturated
aqueous sodium hydrogen carbonate, and brine. The organic layer was
dried (MgSO.sub.4), and concentrated in vacuo. The yellow residue
was then purified by silica-gel chromatography using hexane/ethyl
acetate 3:1 to give the title compound 6 (2.93 g, 93%) as a pale
yellow solid:
[0776] 3-Chloro-benzo[b]thiophene-2-carboxylic acid
(4'-cyano-6-methoxy-biphenyl-3-ylmethyl)-(4-methylamino-cyclohexyl)-amide
hydrochloride (7)
[0777] Concentrated hydrochloric acid (27.5 mL) was added to a
solution of compound 6 (11.0 g, 17.1 mmol) and ethanol (82.5 mL).
The mixture was stirred until reaction was complete as monitored by
TLC. The mixture was concentrated in vacuo and dichloromethane
added and concentrated again, this was repeated until a solid was
obtained. The solid was then slurried with t-butylmethyl ether (30
mL), filtered, and the organic layer dried (MgSO.sub.4) and
concentrated in vacuo to give the title compound 7 (10.0 g, 99%):
.delta..sub.H (360 MHz: DMSO, 70.degree. C.) 1.30-1.50 (m, 2H),
1.85 (br s, 4H), 2.12 (br d, 2H), 2.48 (s, 3H), 2.91 (br t, 1H),
3.81 (s, 3H), 3.87 (br s, 1H), 4.71 (s, 2H), 7.14 (d, 1H), 7.29 (s,
1H), 7.40 (d, 1H), 7.57-7.68 (m, 4H), 7.80-7.90 (m, 3H), 8.09 (d,
1H), 8.82 (br s, 2H).
[0778] All publications and patents cited herein are hereby
incorporated by reference in their entirety.
[0779] 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.
* * * * *