U.S. patent application number 09/090622 was filed with the patent office on 2002-01-17 for inhibitors of hedgehog signaling pathways, compositions and uses related thereto.
Invention is credited to BEACHY, PHILIP A., COOPER, MICHAEL K., PORTER, JEFFREY A..
Application Number | 20020006931 09/090622 |
Document ID | / |
Family ID | 26765298 |
Filed Date | 2002-01-17 |
United States Patent
Application |
20020006931 |
Kind Code |
A1 |
BEACHY, PHILIP A. ; et
al. |
January 17, 2002 |
INHIBITORS OF HEDGEHOG SIGNALING PATHWAYS, COMPOSITIONS AND USES
RELATED THERETO
Abstract
The present invention makes availables assays and reagents
inhibiting paracrine and/or autocrine signals produced by a
hedgehog protein comprising contacting a cell sensitive to the
hedgehog protein with a steroidal alkaloid, or other small
molecule, in a sufficient amount to reduce the sensitivity of the
cell to the hedgehog protein.
Inventors: |
BEACHY, PHILIP A.;
(BALTIMORE, MD) ; COOPER, MICHAEL K.; (BALTIMORE,
MD) ; PORTER, JEFFREY A.; (CAMBRIDGE, MA) |
Correspondence
Address: |
ROPES & GRAY
ONE INTERNATIONAL PLACE
BOSTON
MA
02110-2624
US
|
Family ID: |
26765298 |
Appl. No.: |
09/090622 |
Filed: |
June 4, 1998 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60081186 |
Apr 9, 1998 |
|
|
|
Current U.S.
Class: |
514/278 |
Current CPC
Class: |
A61K 31/4355 20130101;
A61K 31/00 20130101; A61K 31/58 20130101 |
Class at
Publication: |
514/278 |
International
Class: |
A61K 031/56; A61K
031/44 |
Claims
1. A method for inhibiting paracrine and/or autocrine signals
produced by a hedgehog proteins comprising contacting a cell
senstive to the hedgehog protein with a hedgehog antagonist in a
sufficient amount to reduce the sensitivity of the cell to the
hedgehog protein, wherein the hedgehog antagonist is a organic
molecule having a molecule weight less than 750 amu.
2. A method for inhibiting paracrine and/or autocrine signals
produced by a hedgehog proteins comprising contacting a cell
senstive to the hedgehog protein with a hedgehog antagonist, or
anaolog thereof, in a sufficient amount to reduce the sensitivity
of the cell to the hedgehog protein.
3. The method of claim 1, wherein the steroidal alkaloid is
represented in the general forumlas (I), or unsaturated forms
thereof and/or seco-, nor- or homo-derivatives thereof: 30wherein,
as valence and stability permit, R.sub.2, R.sub.3, R.sub.4, and
R.sub.5, represent one or more substitutions to the ring to which
each is attached, for each occurrence, independently represent
hydrogen, halogens, alkyls, alkenyls, alkynyls, aryls, hydroxyl,
.dbd.O, .dbd.S, alkoxyl, silyloxy, amino, nitro, thiol, amines,
imines, amides, phosphoryls, phosphonates, phosphines, carbonyls,
carboxyls, carboxamides, anhydrides, silyls, ethers, thioethers,
alkylsulfonyls, arylsulfonyls, selenoethers, ketones, aldehydes,
esters, or --(CH.sub.2).sub.m--R.sub.8; R.sub.6, R.sub.7, and
R'.sub.7, are absent or represent, independently, halogens, alkyls,
alkenyls, alkynyls, aryls, hydroxyl, .dbd.O, .dbd.S, alkoxyl,
silyloxy, amino, nitro, thiol, amines, imines, amides, phosphoryls,
phosphonates, phosphines, carbonyls, carboxyls, carboxamides,
anhydrides, silyls, ethers, thioethers, alkylsulfonyls,
arylsulfonyls, selenoethers, ketones, aldehydes, esters, or
--(CH.sub.2).sub.m--R.sub.8, or R.sub.6 and R.sub.7, or R.sub.7 and
R'.sub.7, taken together form a ring or polycyclic ring, e.g. which
is susbstituted or unsubstituted, with the proviso that at least
one of R.sub.6, R.sub.7, or R'.sub.7 is present and includes a
primary or secondary amine; R.sub.8 represents an aryl, a
cycloalkyl, a cycloalkenyl, a heterocycle, or a polycycle, and m is
an integer in the range 0 to 8 inclusive.
4. The method of claim 3, wherein: R.sub.2 and R.sub.3, for each
occurrence, is an --OH, alkyl, --O-alkyl, --C(O)-alkyl, or
--C(O)--R.sub.8; R.sub.4, for each occurrence, is an absent, or
represents --OH, .dbd.O, alkyl, --O-alkyl, --C(O)-alkyl, or
--C(O)--R.sub.8; R.sub.6, R.sub.7, and R'.sub.7 each independently
represent, hydrogen, alkyls, alkenyls, alkynyls, amines, imines,
amides, carbonyls, carboxyls, carboxamides, ethers, thioethers,
esters, or --(CH.sub.2).sub.m--R.sub.8, or R.sub.7, and R'.sub.7
taken together form a furanopiperidine, such as
perhydrofuro[3,2-b]pyridine, a pyranopiperidine, a quinoline, an
indole, a pyranopyrrole, a naphthyridine, a thiofuranopiperidine,
or a thiopyranopiperidine with the proviso that at least one of
R.sub.6, R.sub.7, or R'.sub.7 is present and includes a primary or
secondary amine; R.sub.8 represents an aryl, a cycloalkyl, a
cycloalkenyl, a heterocycle, or a polycycle, and preferably R.sub.8
is a piperidine, pyrimidine, morpholine, thiomorpholine,
pyridazine,
5. The method of claim 1, wherein the steroidal alkaloid is
represented in the general formula (II), or unsaturated forms
thereof and/or seco-, nor- or homo-derivatives thereof: 31wherein
R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, and R'.sub.7
are as defined above, and X represents O or S, though preferably
O.
6. The method of claim 1, wherein the steroidal alkaloid is
represented in the general formula (III), or unsaturated forms
thereof and/or seco-, nor- or homo-derivatives thereof: 32wherein
R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.8 are as defined
above; A and B represent monocyclic or polycyclic groups; T
represent an alkyl, an aminoalkyl, a carboxyl, an ester, an amide,
ether or amine linkage of 1-10 bond lengths; T' is absent, or
represents an alkyl, an aminoalkyl, a carboxyl, an ester, an amide,
ether or amine linkage of 1-3 bond lengths, wherein if T and T' are
present together, than T and T' taken together with the ring A or B
form a covelently closed ring of 5-8 ring atoms; R9 represent one
or more substitutions to the ring A or B, which for each
occurrence, independently represent halogens, alkyls, alkenyls,
alkynyls, aryls, hydroxyl, .dbd.O, .dbd.S, alkoxyl, silyloxy,
amino, nitro, thiol, amines, imines, amides, phosphoryls,
phosphonates, phosphines, carbonyls, carboxyls, carboxamides,
anhydrides, silyls, ethers, thioethers, alkylsulfonyls,
arylsulfonyls, selenoethers, ketones, aldehydes, esters, or
--(CH.sub.2).sub.m--R.sub.8; and n and m are, independently, zero,
1 or 2; with the proviso that A and R.sub.9, or T, T' B and
R.sub.9, taken together include at least one primary or secondary
amine.
7. The method of claim 1, wherein the steroidal alkaloid is
represented in the general formula (IV), or unsaturated forms
thereof and/or seco-, nor- or homo-derivatives thereof: 33wherein
R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6 and R.sub.9 are as
defined above; R.sub.22 is absent or represents an alkyl, an
alkoxyl or --OH.
8. The method of claim 1, wherein the steroidal alkaloid is
represented in the general formula (V) or unsaturated forms thereof
and/or seco-, nor- or homo-derivatives thereof: 34wherein R.sub.2,
R.sub.3, R.sub.4, R.sub.6 and R.sub.9 are as defined above;
9. The method of claim 1, wherein the steroidal alkaloid is
represented in the general formula (VI), or unsaturated forms
thereof and/or seco-, nor- or homo-derivatives thereof: 35wherein
R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.9 are as defined
above;
10. The method of claim 1 wherein the steroidal alkaloid is
represented in the general formula (VII) or unsaturated forms
thereof and/or seco-, nor- or homo-derivatives thereof: 36wherein
R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.9 are as defined
above.
11. The method of claim 1, wherein the steroidal alkaloid does not
substantially interfere with the biological activity of such
steroids as aldosterone, androstane, androstene, androstenedione,
androsterone, cholecalciferol, cholestane, cholic acid,
corticosterone, cortisol, cortisol acetate, cortisone, cortisone
acetate, deoxycorticosterone, digitoxigenin, ergocalciferol,
ergosterol, estradiol-17-.alpha., estradiol-17-.beta., estriol,
estrane, estrone, hydrocortisone, lanosterol, lithocholic acid,
mestranol, .beta.-methasone, prednisone, pregnane, pregnenolone,
progesterone, spironolactone, testosterone, triamcinolone and their
derivatives.
12. The method of claim 1, wherein the steroidal alkaloid does not
specifically bind a nuclear hormone receptor.
13. The method of claim 1, wherein the steroidal alkaloid does not
specfically bind estrogen or testerone receptors.
14. The method of claim 1, wherein the steroidal alkaloid has no
estrogenic activity at therapeutic concentrations.
15. The method of claim 1, wherein the steroidal alkaloid inhibit
hedgehog-mediated signal transduction with an ED.sub.50 of 1 mM or
less.
16. The method of claim 1, wherein the steroidal alkaloid inhibit
hedgehog-mediated signal transduction with an ED.sub.50 of 1 .mu.M
or less.
17. The method of claim 1, wherein the steroidal alkaloid inhibit
hedgehog-mediated signal transduction with an ED.sub.50 of 1 nM or
less.
18. The method of claim 1, wherein the cell is contacted with the
steroidal alkaloid in vitro.
19. The method of claim 1, wherein the cell is contacted with the
steroidal alkaloid in vivo.
20. The method of claim 1, wherein the steroidal alkaloid is
administered as part of a therapeutic or cosmetic application.
21. The method of claim 1, wherein the therapeutic or cosmetic
application is selected from the group consisting of 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 primative gut,
regulation of hematopoietic function, regulation of skin and hair
growth, etc.
22. A pharmaceutical preparation comprising steroidal alkaloid is
represented in the general forumlas (I), or unsaturated forms
thereof and/or seco-, nor- or homo-derivatives thereof: 37wherein,
as valence and stability permit, R.sub.2, R.sub.3, R.sub.4, and
R.sub.5, represent one or more substitutions to the ring to which
each is attached, for each occurrence, independently represent
hydrogen, halogens, alkyls, alkenyls, alkynyls, aryls, hydroxyl,
.dbd.O, .dbd.S, alkoxyl, silyloxy, amino, nitro, thiol, amines,
imines, amides, phosphoryls, phosphonates, phosphines, carbonyls,
carboxyls, carboxamides, anhydrides, silyls, ethers, thioethers,
alkylsulfonyls, arylsulfonyls, selenoethers, ketones, aldehydes,
esters, or --(CH.sub.2).sub.m--R.sub.8; R.sub.6, R.sub.7, and
R'.sub.7, are absent or represent, independently, halogens, alkyls,
alkenyls, alkynyls, aryls, hydroxyl, .dbd.O, .dbd.S, alkoxyl,
silyloxy, amino, nitro, thiol, amines, imines, amides, phosphoryls,
phosphonates, phosphines, carbonyls, carboxyls, carboxamides,
anhydrides, silyls, ethers, thioethers, alkylsulfonyls,
arylsulfonyls, selenoethers, ketones, aldehydes, esters, or
--(CH.sub.2).sub.m--R.sub.8, or R.sub.6 and R.sub.7, or R.sub.7 and
R'.sub.7, taken together form a ring or polycyclic ring, e.g.,
which is susbstituted or unsubstituted, with the proviso that at
least one of R.sub.6, R.sub.7, or R'.sub.7 is present and includes
a primary or secondary amine; R.sub.8 represents an aryl, a
cycloalkyl, a cycloalkenyl, a heterocycle, or a polycycle; and m is
an integer in the range 0 to 8 inclusive.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
application No. 60/081,186, filed Apr. 9, 1998.
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 various Hedgehog proteins consist of a signal peptide, a
highly conserved N-terminal region, and a more divergent C-terminal
domain. In addition to signal sequence cleavage in the secretory
pathway (Lee, J. J. et al. (1992) Cell 71:33-50; Tabata, T. et al.
(1992) Genes Dev. 2635-2645: Chang, D. E. et al. (1994) Development
120:3339-3353), Hedgehog precursor proteins undergo an internal
autoproteolytic cleavage which depends on conserved sequences in
the C-terminal portion (Lee et al. (1994) Science 266:1528-1537;
Porter et al. (1995) Nature 374:363-366). This autocleavage leads
to a 19 kD N-terminal peptide and a C-terminal peptide of 26-28 kD
(Lee et al. (1992) supra; Tabata et al. (1992) supra; Chang et al.
(1994) supra; Lee et al. (1994) supra; Bumcrot, D. A., et al (1995)
Mol. Cell. Biol. 15:2294-2303; Porter et al. (1995) supra; Ekker,
S. C. et al. (1995) Curr. Biol. 5:944-955; Lai, C. J. et al. (1995)
Development 121:2349-2360). The N-terminal peptide stays tightly
associated with the surface of cells in which it was synthesized,
while the C-terminal peptide is freely diffusible both in vitro and
in vivo (Porter et al. (1995) Nature 374:363; Lee et al. (1994)
supra; Bumcrot et al. (1995) supra; 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. (1996) 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).
[0006] HH has been implicated in short- and longe 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.
[0007] 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 neighbouring
tissues. Recent evidence indicates that Shh is involved in these
interactions.
[0008] 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. (993) 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).
[0009] In the CNS, Shh from the notochord and the floorplate
appears to induce ventral cell fates. When ectopically expressed,
Shh leads to a ventralization of large regions of the mid- and
hindbrain in mouse (Echelard et al. (1993) supra; Goodrich, L. V.
et al. (1996) Genes Dev. 10:301-312), Xenopus (Roelink, H. et al.
(1994) supra; Ruiz i Altaba, A. et al. (1995) Mol. Cell. Neurosci.
6:106-121), and zebrafish (Ekker et al. (1995) supra; Krauss et al.
(1993) supra; Hammerschmidt, M., et al. (1996) Genes Dev.
10:647-658). In explants of intermediate neuroectoderm at spinal
cord levels, Shh protein induces Doorplate 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
Doorplate observed in vitro (Placzek, M. et al. (1993) Development
117:205-218), and the midline positioning of the floorplate
immediately above the notochord in vivo. Lower concentrations of
Shh released from the notochord and the Doorplate presumably induce
motor neurons at more distant ventrolateral regions in a process
that has been shown to be contact-independent in vitro (Yamada, T.
et al. (1993) Cell 73:673-686). In explants taken at midbrain and
forebrain levels, Shh also induces the appropriate ventrolateral
neuronal cell types, dopaminergic (Heynes, M. et al. (1995) Neuron
15:35-44; Wang, M. Z. et al. (1995) Nature Med. 1:1184-1188) and
cholinergic (Ericson, J. et al. (1995) Cell 81:747-756) precursors,
respectively, indicating that Shh is a common inducer of ventral
specification over the entire length of the CNS. These observations
raise a question as to how the differential response to Shh is
regulated at particular anteroposterior positions.
[0010] 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).
[0011] 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 chick 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.
[0012] 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 affect 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).
[0013] 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 urogential 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).
[0014] 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.
SUMMARY OF THE INVENTION
[0015] The present invention makes availables assays and reagents
inhibiting paracrine and/or autocrine signals produced by a
hedgehog protein comprising contacting a cell sensitive to the
hedgehog protein with a steroidal alkaloid, or other small
molecule, in a sufficient amount to reduce the sensitivity of the
cell to the hedgehog protein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1. Structures of the synthetic compounds AY 9944 and
triparanol, of the plant steriodal alkaloids jervine, cyclopamine
and tomatidine, and of cholesterol.
[0017] FIG. 2. Holoprosencephaly induced in chick embryos exposed
to jervine (4). (A) SEM of external facial features of an untreated
embryo. (B, C, D and E) Embryos exposed to 10 .mu._M jervine with
variable loss of midline tissue and resulting fusion of the paired,
lateral olfactory processes (olf), optic vesicles (Opt), and
maxillary (Mx) and mandibular (Mn) processes. A complete fusion of
the optic vesicles (E) lead to true cyclopia.
[0018] FIG. 3. Synthetic and plant derived teratogens block
endogenous Shh signaling in explanted chick tissues (41). (A)
Midline tissue was removed from stage 9-10 chick embryos at a level
just rostral to Hensen's node (white dashed line), and further
dissected (black dashed lines) to yield an explant containing an
endogenous source of Shh signal (notochord) and a responsive tissue
(neural plate ectoderm). After two days of culture in a collagen
gel matrix, the neural ectoderm expresses markers of floor plate
cells (HNF3.beta., rhodamine) and motor neurons (Isl-1, FITC) in
untreated control explants (B) and explants cultured with the
non-teratogenic alkaloid tomatidine (50 .mu.M, C). Intermedia doses
of the teratogenic compounds AY 9944 (0.5 .mu.M, D), triparanol
(0.25 .mu.M, E), jervine (0.5 .mu.M, F) and cyclopamine (0.25
.mu.M, G) block induction of HNF3.beta., which requires a high
level of Shh pathway activation, while permitting induction of
Isl-1, which requires a lower level of Shh pathway activation (see
text). Higher doses of the teratogenic compounds AY 9944 (4.0
.mu.M, H), triparanol (1.0 .mu.M, I), jervine (4.0 .mu.M, J) and
cyclopamine (1.0 .mu.M, K) and fully inhibit HNF3.beta. and Isl-1
induction.
[0019] FIG. 4. Teratogenic compounds do not inhibit Shh
autoprocessing in vivo (47). Stably transfected HK293 cells
expressing Shh protein under ecdysone-inducible control (lanes 1,
2, 3) were treated with jervine (lanes 4, 5) cyclopamine (lanes 6,
7), tomatidine (lanes 10, 11), AY 9944 (lanes 12, 13) or triparanol
(lanes 14, 15) and cell lysates were immunoblotted to assess the
efficiency of autoprocessing. As seen in the untreated control
(lane 3). Shh in treated cells is efficiently processed with little
or no detectable accumulation of precursor protein (M.sub.r45 kD).
The processed amino-terminal product (Shh-N.sub.p) is cell
associated and migrates faster than Shh-N protein from the media of
cultured cells transfected with a construct carrying an open
reading frame truncated after Gly.sub.198 (lane 8; Shh-N.sub.p and
Shh-N both loaded in lanes 9 and 17). This faster migration and the
lack of detectable protein in the culture medium (not shown)
indicate that Shh-N.sub.p from treated cells likely carries a
sterol adduct. The slower migrating species resulting from
tomatidine treatment is .about.1.9 kD larger, suggestive of a minor
inhibition of signal sequence cleavage (see asterisk; lanes 10,
11). Immunoblotted actin for each lane is shown as a loading
control.
[0020] FIG. 5. Plant steriodal alkaloids do not inhibit or
participate in Hh autoprocessing in vitro (5). (A) Coomassie
blue-stained SDS-polyacrylamide gel showing in vitro autocleavage
reactions of the baterically expressed His.sub.6Hh-C protein
(.about.29 kD) incubated for 3 hours at 30.degree. C. with no
sterol additions (lane 1) or 12 .mu.M cholesterol to stimulate the
autoprocessing reaction and generate a .about.25 kD Hh-C product
(lanes 2-27 and a .about.5 kD NH.sub.2-terminal product (not
resolved on this gel). The addition of jervine (lanes 3-6),
cyclopamine (lanes 8-11) and tomatidine (lanes 13-16) does not
interfere with autoprocessing, even when added in 27-fold excess to
cholesterol (lanes 6, 11 and 16). (B) Coomassie blue-stained
SDS-polyacrylamide gel showing that the His.sub.6Hh-C autocleavage
reaction does not proceed when carried out in the absence of sterol
(lane 1), or in the presence of jervine (lanes 2-5), cyclopamine
(lanes 6-9) and tomatidine (lanes 10-13), even at 324 .mu.M
concentrations of these steriodal alkaloid (lanes 5, 9 and 13). (C)
Coomassie blue-stained SDS0 polyacrylamide gel of His.sub.6Hh-C
autocleavage reactions carried out in the absence of sterols (lane
1), with 50 mM dithiothreitol (lane 2), 12 .mu.M cholesterol (lane
3) 12 .mu.M7 dehydrocholesterol (lane 4) 12 .mu.M desmosterol (lane
5), 12 .mu.M muristerone (lanes 9, 10). The 27-carbon cholesterol
precursors (lanes 4-6) stimulate His.sub.6Hh-C autocleavage
reactions carried out in the absence of sterols (lane 1), with 50
mM dithiothreitol (lane 2), 12 .mu.M cholesterol (lane 3) 12 .mu.M
7 dehydrocholesterol (lane 4) 12 .mu.M lathosterol (lane 6), 12 and
350 .mu.M lanosterol (lanes 7, 8) and 12 and 350 .mu.M muristerone
(lanes 9, 10). The 27-carbon cholesterol precursors (lanes 4-6)
stimulate His.sub.6Hh-C autoprocessing as efficiently as
cholesterol (lane 3). The amino-terminal product migrates as a
.about.7 kD species (lane 2) when generated in the presence of 50
mM dithiothreitol and as a .about.5 kD species (lanes 3-6) with a
sterol adduct. Lanosterol (lanes 7 and 8) and muristerone (lanes 9
and 10) do not stimulate autoprocessing above background (lane
1).
[0021] FIG. 6. Teratogenic compounds inhibit neural ectoderm
response to exogenous Shh-N protein (41). (A) Intermediate neural
plate ectoderm, free of notochord and other tissues, was dissected
as shown (dashed lines) from stage 9-10 chick embryos at a level
just rostral to Hensen's node (see FIG. 3A). (B) Explanted
intermediate neural plate tissue cultured in a collagen gel matrix
for 20 hours expresses the dorsal marker Pax7 (FITC) and not the
floor plate marker HNF3.beta. (Rhodamine). (C) Addition of
recombinant, purified Shh-N at 2 nM suppresses Pax7 expression. (D)
Markers of motor neuron (Isl-1, FITC) and floor plate cell
(HNF3.beta., rhodamine) fates are induced upon explant culture for
40 hours in the presence of 6.25 nM Shh-N. (E) At 25 nM Shh-N,
HNF3.beta. expression expands at the expense of Isl-1 expression,
which is lost. The repression of Pax7 expression by 2 nM Shh-N is
inhibited by (F) 0.5 .mu.M AY 9944, (G) 0.25 .mu.M triparanol, (H)
0.125 .mu.M jervine and (1) 0.0625 .mu.M cyclopamine, but not by
(J) 50 .mu.M tomatidine. Induction of HNF3.beta. is blocked while
induction of Isl-1 at 25 NM Shh-N is maintained or expanded at
intermediate levels of AY 9949 (1.0 .mu.M, K), triparanol (0.25
.mu.M, L), jervine (0.25 .mu.M, M), and cyclopamine (0.125 .mu.M,
N). Tomatidine at 25 nM displays a slight inhibitory effect with
decrease in HNF3.beta. expression and an increase in the number
Isl-1 expressing cells. HNF3.beta. and Isl-1 induction are
completely blocked at 2-fold higher doses of inhibitors AY 9944
(2.0 .mu.M, P), triparanol (0.5 .mu.M, Q), jervine (0.5, .mu.M, R)
and cyclopamine (0.25 .mu.M, S). Tomatidine at 50 .mu.M (T)
markedly reduces HNF3.beta. induction and enhances Isl1-1
induction. Note that for each teratogenic compound the
concentrations required to block complete the response to 2 nM
Shh-N (F-I) are lower than those required to block completely the
response to 25 nM Shh-N (P-S). Also note that the response to 25 nM
Shh-N is only partially inhibited (K-N) at concentrations of
teratogen 2 fold lower than those required to block this response
completely. See text for further comment.
[0022] FIG. 7. Jervine does not inhibit neural ectoderm response to
BMP7 (41). (A) Ventral neural plate ectoderm was dissected as shown
(dashed lines) from stage 9-10 chick embryos at a level just
rostral to Hensen's node (see FIG. 3A). (B) Ventral neural plate
explants cultured for 24 hours in a collagen gel matrix do not give
rise to any migratory cells that can be visualized by
immunostaining for the HNK-1 antigen. (C) Addition of 100 ng/ml
BMP7 induces formation of numerous HNK-1 positive cells that
migrate out from the explant (borders outlined by white dashed
line). (D) Induction of migratory HNK-1 positive cells by 100 ng/ml
BMP7 is not inhibited by the presence of 10 .mu.M jervine, nor by
addition of the other plant-derived compounds (10 .mu.M
cyclopamine. 50 .mu.M tomatidine; data not shown).
DETAILED DESCRIPTION OF THE INVENTION
[0023] Hedgehog (hedgehog) proteins comprise a family of secreted
signaling molecules essential for patterning a variety of
structures in animal embryo genesis, and play a role in regulating
cell proliferation and specifying cell identity in diverse systems
in adults.
[0024] During biosynthesis, hedgehog undergoes an autocleavage
reaction, mediated by its carboxyl-terminal domain, that produces a
lipid-modified amino-terminal fragment responsible for all known
hedgehog signaling activity. In addition to peptide bond cleavage,
hedgehog autoprocessing causes the covalent attachment of a
lipophilic adduct to the COOH-terminus of N-terminal hedgehog
fragment. This modification is critical for the spatially
restricted tissue localization of the hedgehog signal; in its
absence, the signaling domain exerts an inappropriate influence
beyond its site of expression. It has recently been reported,
Porter et al. (1996) Science 274:255, that cholesterol is the
lipophilic moiety covalently attached to the amino-terminal
signaling domain during autoprocessing and that the
carboxyl-terminal domain acts as an intramolecular cholesterol
transferase. This use of cholesterol to modify the hedgehog
signaling proteins is consistent with some of the effects that
perturbed cholesterol biosynthesis can have on animal development.
See also Volhard et al. (1998) Nature 287 795; Mohleret al. (1988)
Genetics 120:1061; Lee et al. (1992) Cell 71:33; Tabata et al.
(1992) Genes Dev 6:2635; Tashiro et al. (1993) Genel 24:183; P. W.
Ingham, Nature 366:560 (1993); Mohler et al. Development 115:957
(1992); Ma et al. Cell 75:927 (1993); Heberlein et al. ibid, p.913;
Echelard et al., Ibid, p. 1417; Riddle et al. ibid., p. 1401;
Krauss et al., ibid., p. 1431; Roelink et al., ibid 76, 761 (1994);
Chang et al., Development 120:3339 (1994); Basler et al. Nature
368:208 (1994); Tabata et al. Cell 76:89 (1994); Heemskerk et al.,
ibid., p. 449; Fan et al., ibid. 79:1175 (1994); Johnson et al.,
ibid., p. 1165; Hynes et al., Neuron 15:35 (1995); Ekker et al.,
Development 121:2337 (1995); Macdonald et al., ibid. p. 3267; Ekker
et al., Curr. Biol 5:944 (1995); Lai et al. Development 121, 2349
(1995); Ericson et al., Cell 81:747 (1995). Chiang et al., Nature
83:407 (1996); Bitgood et al. Curr Biol. 6:298 (1996); Vortkamp et
al., Science 273:613 (1996); Lee et al., ibid. 266:1528 (1994);
Porter et al., Nature 374: 363 (1995); Porter et al. Cell 86:21
(1996).
[0025] I. Overview
[0026] The present invention relates to the discovery that signal
transduction pathways dependent on hedgehog proteins can be
inhibited, at least in part, by compounds which disrupt the
cholesterol modification of hedgehog proteins and/or which inhibit
the bioactivity of hedgehog proteins. In particular, Applicants
believe that they are the first to demonstrate that a small
molecule, e.g., having a molecular weight less than 2500 amu, is
capable of inhibiting at least some of the biological activities of
hedgehog proteins.
[0027] One aspect of the present invention relates to the use of
steroidal alkaloids, and analogs thereof, to interfere with
paracrine and/or autocrine signals produced by the hedgehog
proteins, particularly cholesterol-modified (CM) forms of the
proteins. As set out in more detail below, we have observed that
members of the steroidal alkaloid class of compounds, such as the
Veratrum-derived compound jervine, disrupt cholesterol-mediated
activities of the hedgehog proteins.
[0028] While not wishing to bound by any particular theory, the
ability of jervine and other steroidal alkaloids to inhibit
hedgehog signalling may be due to the ability of such molecules to
interact with the sterol sensing domain(s) of the hedgehog
receptor, patched, or at least to intefere with the ability of a
hedgehog protein, e.g., a cholesterol-modified protein, to interact
with its receptor, or other molecules associated with the receptor,
or proteins otherwise involved in hedgehog-mediated signal
transduction.
[0029] Alternatively, or in addition to such a mechanism of action,
the effects of jervine on hedgehog signaling could be the result of
perturbations of cholesterol homeostasis which affect
cholesterol-mediated autoprocessing of the hedgehog protein and or
the activity or stability of protein. In particular, as described
in the appended examples, Jervine and other of the steroidal
alkaloids are so-called "class 2" inhibitors of cholesterol
biosynthesis, that is they inhibit the inward flux of sterols. As
described by Lange and Steck (1994) J Biol Chem 269: 29371-4, these
inhibitors immediately inhibit plasma membrane cholesterol
esterification and progressively induce
3-hydroxy-3-methylglutaryl-coenzyme A reductase activity and sterol
biosynthesis. The change in the relative cholesterol levels can
effect, e.g., the activity and/or stability of ptc. According to
the present invention, the subject methods may be carried out
utilizing other agents which perturb cholesterol homeostasis in a
manner similar to jervine.
[0030] It is, therefore, specifically contemplated that other small
molecules, steroidal and non-steroidal in structure, which
similarly intefere with cholesterol dependent aspects of ptc
activity will likewise be capable of disrupting hedgehog-mediated
signals. In preferred embodiments, the subject inhibitors are
organic molecules having a molecular weight less than 2500 amu,
more preferably less than 1500 amu, and even more preferagly less
than 750 amu, and are capable of inhibiting at least some of the
biological activities of hedgehog proteins.
[0031] Thus, the methods of the present include the use of
steroidal alkaloids, and other small molecules, which antagonize
hedgehog signalling in the regulation of repair and/or functional
performance of a wide range of cells, tissues and organs, and have
therapeutic and cosmetic applications ranging from regulation of
neural tissues, bone and cartilage formation and repair, regulation
of spermatogenesis, regulation of smooth muscle, regulation of
lung, liver and other organs arising from the primative gut,
regulation of hematopoietic function, regulation of skin and hair
growth, etc. Accordingly, the methods and compositions of the
present invention include the use of the subject inhibitors for all
such uses as antagonists of hedgehog proteins may be implicated.
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).
[0032] In one aspect, the present invention provides pharmaceutical
preparations comprising, as an active ingredient, an inhibitor of
cholesterol-mediated hedgehog bioactivity, such as described
herein.
[0033] The subject treatments using hedgehog antagonists can be
effective for both human and animal subjects. Animal subjects to
which the invention is applicable extend to both domestic animals
and livestock, raised either as pets or for commercial purposes.
Examples are dogs, cats, cattle, horses, sheep, hogs and goats.
[0034] II. Definitions
[0035] For convience, certain terms employed in the specfication,
examples, and appended claims are collected here.
[0036] The term "hedgehog polypeptide" encompasses preparations of
hedgehog proteins and peptidyl fragments thereof, both agonist and
antagonist forms as the specific context will make clear.
[0037] An "effective amount" of, e.g., a hedgehog antagonist, with
respect to the subject method of treatment, refers to an amount of
the antagonist in a preparation which, when applied as part of a
desired dosage regimen brings about, e.g., a change in the rate of
cell proliferation and/or the state of differentiation of a cell
and/or rate of survival of a cell according to clinically
acceptable standards for the disorder to be treated or the cosmetic
purpose.
[0038] A "patient" or "subject" to be treated by the subject method
can mean either a human or non-human animal.
[0039] The "growth state" of a cell refers to the rate of
proliferation of the cell and/or the state of differentiation of
the cell.
[0040] The terms "steroid" and "'steroid-like" are used
interchangeable herein and refer to a general class of polycyclic
compounds possessing the skeleton of cyclopentanophenanthrene or a
skeleton derived therefrom by one or more bond scissions or ring
expansions or contractions. The rings may be substituted at one or
more positions, to create derivatives that adhere to the rules of
valence and stability, such as by methyl or other lower alkyl
groups, hydroxyl groups, alkoxyl groups and the like.
[0041] 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.
[0042] The term "epithelialization" refers to healing by the growth
of epithelial tissue over a denuded surface.
[0043] 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.
[0044] 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.
[0045] 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.
[0046] The term "nail" refers to the horny cutaneous plate on the
dorsal surface of the distal end of a finger or toe.
[0047] 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.
[0048] 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, 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; and "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.
[0049] The term "nasal epithelial tissue" refers to nasal and
olfactory epithelium.
[0050] "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.
[0051] "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.
[0052] "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.
[0053] "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.
[0054] "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.
[0055] A "wound to eye tissue" refers to severe dry eye syndrome,
corneal ulcers and abrasions and ophthalmic surgical wounds.
[0056] 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.
[0057] The term "carcinoma" refers to a malignant new growth made
up of epithelial cells tending to infiltrate surrounding tissues
and to give rise to metastases. Exemplary carcinomas include:
"basal cell carcinoma", which is an epithelial tumor of the skin
that, while seldom metastasizing, has potentialities for local
invasion and destruction; "squamous cell carcinoma", which refers
to carcinomas arising from squamous epithelium and having cuboid
cells; "carcinosarcoma", which include malignant tumors composed of
carcinomatous and sarcomatous tissues; "adenocystic carcinoma",
carcinoma marked by cylinders or bands of hyaline or mucinous
stroma separated or surrounded by nests or cords of small
epithelial cells, occurring in the mammary and salivary glands, and
mucous glands of the respiratory tract; "epidermoid carcinoma",
which refers to cancerous cells which tend to differentiate in the
same way as those of the epidermis; i.e., they tend to form prickle
cells and undergo cornification; "nasopharyngeal carcinoma", which
refers to a malignant tumor arising in the epithelial lining of the
space behind the nose; and "renal cell carcinoma", which pertains
to carcinoma of the renal parenchyma composed of tubular cells in
varying arrangements. Another carcinomatous epithelial growth is
"papillomas", which refers to benign tumors derived from epithelium
and having a papillomavirus as a causative agent; and
"epidermoidomas", which refers to a cerebral or meningeal tumor
formed by inclusion of ectodermal elements at the time of closure
of the neural groove.
[0058] As used herein, the term "psoriasis" refers to a
hyperproliferative skin disorder which alters the skin's regulatory
mechanisms. In particular, lesions are formed which involve primary
and secondary alterations in epidermal proliferation, inflammatory
responses of the skin, and an expression of regulatory molecules
such as lymphokines and inflammatory factors. Psoriatic skin is
morphologically characterized by an increased turnover of epidermal
cells, thickened epidermis, abnormal keratinization, inflammatory
cell infiltrates into the dermis layer and polymorphonuclear
leukocyte infiltration into the epidermis layer resulting in an
increase in the basal cell cycle. Additionally, hyperkeratotic and
parakeratotic cells are present.
[0059] 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.
[0060] As used herein, "proliferating" and "proliferation" refer to
cells undergoing mitosis.
[0061] As used herein, "transformed cells" refers to cells which
have spontaneously converted to a state of unrestrained growth,
i.e., they have acquired the ability to grow through an indefinite
number of divisions in culture. Transformed cells may be
characterized by such terms as neoplastic, anaplastic and/or
hyperplastic, with respect to their loss of growth control.
[0062] 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.
[0063] 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 select moieties which are
hydrolyzed under physiological conditions to provide the desired.
In other embodiments, the prodrug is converted by an enzymatic
activity of the host animal.
[0064] 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.
[0065] 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.
[0066] 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 chain, C.sub.3-C.sub.30 for branched
chain), 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.
[0067] 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
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.
[0068] The term "aralkyl", as used herein, refers to an alkyl group
substituted with an aryl group (e.g. an aromatic or heteroaromatic
group).
[0069] 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.
[0070] 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.
[0071] 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, 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.
[0072] 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.
[0073] 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, pyrazolc,
isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine,
indolizine, isoindole, indole, indazole, purine, quinolizine,
isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline,
quinazoline, cinnoline, pteridinc, 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, 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.
[0074] 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, 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.
[0075] The term "carbocycle", as used herein, refers to an aromatic
or non-aromatic ring in which each atom of the ring is carbon.
[0076] 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--.
[0077] 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: 1
[0078] 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.
[0079] The term "acylamino" is art-recognized and refers to a
moiety that can be represented by the general formula: 2
[0080] wherein R.sub.9 is as defined above, and R'.sub.11
represents a hydrogen, an alkyl, an alkenyl or
--(CH.sub.12).sub.m--R.sub.8, where m and R.sub.8 are as defined
above.
[0081] The term "amido" is art recognized as an amino-substituted
carbonyl and includes a moiety that can be represented by the
general formula: 3
[0082] wherein R.sub.9, R.sub.10 are as defined above. Preferred
embodiments of the amide will not include imides which may be
unstable.
[0083] 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, ethyl
thio, and the like.
[0084] The term "carbonyl" is art recognized and includes such
moieties as can be represented by the general formula: 4
[0085] 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 "thiolcarbonyl" group. Where X is
a sulfur and R.sub.11 or R'.sub.11 is not hydrogen, the formula
represents a "thiolester." Where X is a sulfur and R.sub.11 is
hydrogen, the formula represents a "thiolcarboxylic acid." Where X
is a sulfur and R.sub.11' is hydrogen, the formula represents a
"thiolformate." On the other hand, where X is a bond, and R.sub.11
is not hydrogen, the above formula represents a "ketone" group.
Where X is a bond, and R.sub.11 is hydrogen, the above formula
represents an "aldehyde" group.
[0086] 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.
[0087] The term "sulfonate" is art recognized and includes a moiety
that can be represented by the general formula: 5
[0088] in which R.sub.41 is an electron pair, hydrogen, alkyl,
cycloalkyl, or aryl.
[0089] The terms triflyl, tosyl, mesyl, and nonaflyl are
art-recognized and refer to trifluoromethanesulfonyl,
p-toluenesulfonyl, methanesulfonyl, and nonafluorobutanesulfonyl
groups, respectively. The terms triflate, tosylate, mesylate, and
nonaflate are art-recognized and refer to trifluoromethanesulfonate
ester, p-toluenesulfonate ester, methanesulfonate ester, and
nonafluorobutanesulfonate ester functional groups and molecules
that contain said groups, respectively.
[0090] The term "sulfate" is art recognized and includes a moiety
that can be represented by the general formula: 6
[0091] in which R.sub.41 is as defined above.
[0092] The term "sulfonamido" is art recognized and includes a
moiety that can be represented by the general formula: 7
[0093] in which R.sub.9 and R'.sub.11 are as defined above.
[0094] The term "sulfamoyl" is art-recognized and includes a moiety
that can be represented by the general formula: 8
[0095] in which R.sub.9 and R.sub.10 are as defined above.
[0096] The terms "sulfoxido" or "sulfinyl", as used herein, refers
to a moiety that can be represented by the general formula: 9
[0097] in which R.sub.44 is selected from the group consisting of
hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl,
aralkyl, or aryl.
[0098] A "phosphoryl" can in general be represented by the formula:
10
[0099] wherein Q1 represented S or O, and R.sub.46 represents
hydrogen, a lower alkyl or an aryl. When used to substitute, e.g.
an alkyl, the phosphoryl group of the phosphorylalkyl can be
represented by the general formula: 11
[0100] 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".
[0101] A "phosphoramidite" can be represented in the general
formula: 12
[0102] wherein R.sub.9 and R.sub.10 are as defined above, and
Q.sub.2 represents O, S or N.
[0103] A "phosphonamidite" can be represented in the general
formula: 13
[0104] 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.
[0105] 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.
[0106] 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.
[0107] 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.
[0108] 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.
[0109] 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 are 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.
[0110] Contemplated equivalents of the compounds described above
include compounds which otherwise correspond thereto, and which
have the same general properties thereof (e.g. the ability to
inhibit hedgehog signaling), wherein one or more simple variations
of substituents are made which do not adversely affect the efficacy
of the compound. In general, the compounds of the present invention
may be prepared by the methods illustrated in the general reaction
schemes as, for example, described below, or by modifications
thereof, using readily available starting materials, reagents and
conventional synthesis procedures. In these reactions, it is also
possible to make use of variants which are in themselves known, but
are not mentioned here.
[0111] 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.
[0112] 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.
[0113] 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.
[0114] 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).
[0115] A list of many 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.
[0116] The term "ED.sub.50" means the dose of a drug which produces
50% of its maximum response or effect. Alternatively, the dose
which produces a pre-determined response in 50% of test subjects or
preparations.
[0117] The term "LD.sub.50" means the dose of a drug which is
lethal in 50% of test subjects.
[0118] The term "therapeutic index" refers to the therapeutic index
of a drug defined as LD.sub.50/ED.sub.50.
[0119] The term "agonist", with respect to hedgehog, refers to a
compound that mimics the action of a native hedgehog protein.
[0120] The term "antagonist", with respect to hedgehog bioactivity,
refers to a compound that inhibits hedgehog-mediated signal
transduction. In the context of the present invention, such
antagonists can include compounds which mimic the activity of
jervine, having such characteristics as the ability to disrupt
cholesterol homoeostasis such as through inhibition of sterol
trafficking (e.g., a a class 2 inhibitor), the ability to bind to a
hedgehog receptor site and inhibit the simultaneous binding of
hedgehog to the receptor, or, by non-competitive and/or allosteric
effects of the like, inhibit the response of the cell to hedgehog
which does bind.
[0121] The term "competitive antagonist" refers to a compound that
binds to a receptor site; its effects can be overcome by increased
concentration of the agonist.
[0122] As used herein, "steroid hormone receptor superfamily"
refers to the class of related receptors comprised of
glucocorticoid, mineralocorticoid, progesterone, estrogen,
estrogen-related, vitamin D3, thyroid, v-erb-A, retinoic acid and
E75 (Drosophila) receptors. As used herein "steroid hormone
receptor" refers to members within the steroid hormone receptor
superfamily. In higher organisms, the nuclear hormone receptor
superfamily includes approximately a dozen distinct genes that
encode zinc finger transcription factors, each of which is
specifically activated by binding a ligand such as a steroid,
thyroid hormone (T3) or retinoic acid (RA).
[0123] III. Exemplary Compounds of the Invention.
[0124] As described in further detail below, it is contemplated
that the subject methods can be carried out using a variety of
different steroidal alkaloids, as well as non-steroidal small
molecules, which can be readily identified, e.g. by such drug
screening assays as described herein. The above notwithstanding, in
a preferred embodiment, the methods and compositions of the present
invention make use of compounds having a steroidal alkaloid ring
system. Steroidal alkaloids have a fairly complex nitrogen
containing nucleus. Two exemplary classes of steroidal alkaloids
for use in the subject methods are the Solanum type and the
Veratrum type.
[0125] There are more than 50 naturally occuring veratrum alkaloids
including veratramine, cyclopamine, cycloposine, jervine, and
muldamine occurring in plants of the Veratrum spp. The Zigadenus
spp., death camas, also produces several veratrum-type of steroidal
alkaloids including zygacine. In general, many of the veratrum
alkaloids (e.g., jervine, cyclopamine and cycloposine) consist of a
modified steroid skeleton attached spiro to a furanopiperidine. A
typical veratrum-type alkaloid may be represented by: 14
[0126] An example of the Solanum type is solanidine. This steroidal
alkaloid is the nucleus (i.e. aglycone) for two important
glycoalkaloids, solanine and chaconine, found in potatoes. Other
plants in the Solanum family including various nightshades,
Jerusalem cherries, and tomatoes also contain solanum-type
glycoalkaloids. Glycoalkaloids are glycosides of alkaloids. A
typical solanum-type alkaloid may be represented by: 15
[0127] Based on these structures, and the possibility that certain
unwanted side effects can be reduced by some manipulation of the
structure, a wide range of steroidal alkaloids are contemplated as
potential hedgehog antagonists for use in the subject method. For
example, compounds useful in the subject methods include steroidal
alkaloids represented in the general forumlas (I) or unsaturated
forms thereof and/or seco-, nor- or homo-derivatives thereof:
16
[0128] wherein, as valence and stability permit,
[0129] R.sub.2, R.sub.3, R.sub.4, and R.sub.5, represent one or
more substitutions to the ring to which each is attached, for each
occurrence, independently represent hydrogen, halogens, alkyls,
alkenyls, alkynyls, aryls, hydroxyl, .dbd.O, .dbd.S, alkoxyl,
silyloxy, amino, nitro, thiol, amines, imines, amides, phosphoryls,
phosphonates, phosphines, carbonyls, carboxyls, carboxamides,
anhydrides, silyls, ethers, thioethers, alkylsulfonyls,
arylsulfonyls, selenoethers, ketones, aldehydes, esters, or
--(CH.sub.2).sub.m--R.sub.8;
[0130] R.sub.6, R.sub.7, and R'.sub.7, are absent or represent,
independently, halogens, alkyls, alkenyls, alkynyls, aryls,
hydroxyl, .dbd.O, .dbd.S, alkoxyl, silyloxy, amino, nitro, thiol,
amines, imines, amides, phosphoryls, phosphonates, phosphines,
carbonyls, carboxyls, carboxamides, anhydrides, silyls, ethers,
thioethers, alkylsulfonyls, arylsulfonyls, selenoethers, ketones,
aldehydes, esters, or --(CH.sub.2).sub.m--R.sub.8, or
[0131] R.sub.6 and R.sub.7, or R.sub.7 and R'.sub.7, taken together
form a ring or polycyclic ring, e.g., which is susbstituted or
unsubstituted, with the proviso that at least one of R.sub.6,
R.sub.7, or R'.sub.7 is present and includes a primary or secondary
amine;
[0132] R.sub.8 represents an aryl, a cycloalkyl, a cycloalkenyl, a
heterocycle, or a polycycle; and
[0133] m is an integer in the range 0 to 8 inclusive.
[0134] In preferred embodiments,
[0135] R.sub.2 and R.sub.3, for each occurrence, is an --OH, alkyl,
--O-alkyl, --C(O)-alkyl, or --C(O)--R.sub.8;
[0136] R.sub.4, for each occurrence, is an absent, or represents
--OH, .dbd.O, alkyl, --O-alkyl, --C(O)-alkyl, or
--C(O)--R.sub.8;
[0137] R.sub.6, R.sub.7, and R'.sub.7 each independently represent,
hydrogen, alkyls, alkenyls, alkynyls, amines, imines, amides,
carbonyls, carboxyls, carboxamides, ethers, thioethers, esters, or
--(CH.sub.2).sub.m--R.sub.8, or
[0138] R.sub.7, and R'.sub.7 taken together form a
furanopiperidine, such as perhydrofuro[3,2-b]pyridine, a
pyranopiperidine, a quinoline, an indole, a pyranopyrrole, a
naphthyridine, a thiofuranopiperidine, or a
thiopyranopiperidine
[0139] with the proviso that at least one of R.sub.6, R.sub.7, or
R'.sub.7 is present and includes a primary or secondary amine;
[0140] R.sub.8 represents an aryl, a cycloalkyl, a cycloalkenyl, a
heterocycle, or a polycycle, and preferably R.sub.8 is a
piperidine, pyrimidine, morpholine, thiomorpholine, pyridazine.
[0141] In certain preferred embodiments, the definitions outlined
above apply, and the subject compounds are represented by general
formula Ia or unsaturated forms thereof and/or seco-, nor- or
homo-derivatives thereof: 17
[0142] In preferred embodiments, the subject hedgehog antagonists
can be represented in one of the following general formulas (II) or
unsaturated forms thereof and/or seco-, nor- or homo-derivatives
thereof: 18
[0143] wherein R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6,
R.sub.7, and R'.sub.7 are as defined above, and X represents O or
S, though preferably O.
[0144] In certain preferrred embodiments, the definitions outlined
above apply, and the subject compounds are represented by general
formula IIa or unsaturated forms thereof and/or seco-, nor- or
homo-derivatives thereof: 19
[0145] In certain embodiments, the subject hedgehog antagonists are
represented by the general formula (III) or unsaturated forms
thereof and/or seco-, nor- or homo-derivatives thereof: 20
[0146] wherein
[0147] R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.8 are as
defined above;
[0148] A and B represent monocyclic or polycyclic groups;
[0149] T represent an alkyl, an aminoalkyl, a carboxyl, an ester,
an amide, ether or amine linkage of 1-10 bond lengths;
[0150] T' is absent, or represents an alkyl, an aminoalkyl, a
carboxyl, an ester, an amide, ether or amine linkage of 1-3 bond
lengths, wherein if T and T' are present together, than T and T'
taken together with the ring A or B form a covelently closed ring
of 5-8 ring atoms;
[0151] R9 represent one or more substitutions to the ring A or B,
which for each occurrence, independently represent halogens,
alkyls, alkenyls, alkynyls, aryls, hydroxyl, .dbd.O, .dbd.S,
alkoxyl, silyloxy, amino, nitro, thiol, amines, imines, amides,
phosphoryls, phosphonates, phosphines, carbonyls, carboxyls,
carboxamides, anhydrides, silyls, ethers, thioethers,
alkylsulfonyls, arylsulfonyls, selenoethers, ketones, aldehydes,
esters, or --(CH.sub.2).sub.m--R.sub.9; and
[0152] n and m are, independently, zero, 1 or 2;
[0153] with the proviso that A and R.sub.9, or T, T' B and R.sub.9,
taken together include at least one primary or secondary amine.
[0154] In certain preferred embodiments, the definitions outlined
above apply, and the subject compounds are represented by general
formula IIIa or unsaturated forms thereof and/or seco-, nor- or
homo-derivatives thereof: 21
[0155] For example, the subject methods can utilize hedgehog
antagonists based on the veratrum-type steroidal alkaloids jervine,
cyclopamine, cycloposine, mukiamine or veratramine, e.g., which may
be represented in the general formula (IV) or unsaturated forms
thereof and/or seco-, nor- or homo-derivatives thereof: 22
[0156] wherein
[0157] R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6 and R.sub.9 are
as defined above;
[0158] R.sub.22 is absent or represents an alkyl, an alkoxyl or
--OH.
[0159] In certain preferred embodiments, the definitions outlined
above apply, and the subject compounds are represented by general
formula IVa or unsaturated forms thereof and/or seco-, nor- or
homo-derivatives thereof: 23
[0160] In even more preferred embodments, the subject antagonists
are represented in the formulas (V) or unsaturated forms thereof
and/or seco-, nor- or homo-derivatives thereof: 24
Formula V
[0161] wherein R.sub.2, R.sub.3, R.sub.4, R.sub.6 and R.sub.9 are
as defined above;
[0162] In certain preferred embodiments, the definitions outlined
above apply, and the subject compounds are represented by general
formula Va or unsaturated forms thereof and/or seco-, nor- or
homo-derivatives thereof: 25
[0163] Another class of hedgehog antagonists can be based on the
veratrum-type steroidal alkaloids resmebling verticine and
zygacine, e.g., represented in the general formulas (VI) or
unsaturated forms thereof and/or seco-, nor- or homo-derivatives
thereof: 26
[0164] wherein R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.9 are
as defined above.
[0165] In certain preferred embodiments, the definitions outlined
above apply, and the subject compounds are represented by general
formula VIa or unsaturated forms thereof and/or seco-, nor- or
homo-derivatives thereof: 27
[0166] Still another class of potential hedgehog antagonists are
based on the solanum-type steroidal alkaloids, e.g., solanidine,
which may be represented in the general formula (VII) or
unsaturated forms thereof and/or seco-, nor- or homo-derivatives
thereof: 28
[0167] wherein R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.9 are
as defined above.
[0168] In certain preferred embodiments, the definitions outlined
above apply, and the subject compounds are represented by general
formula VIIa or unsaturated forms thereof and/or seco-, nor- or
homo-derivatives thereof: 29
[0169] In certain embodiments, the subject antagonists can be
chosen on the basis of their selectively for the hedgehog pathway.
This selectivity can for the hedgehog pathway versus other
steroid-mediated pathways (such as testosterone or estrogen
mediated activities), as well as selectivity for particular
hedgehog pathways, e.g., which isotype specific for hedgehog (e.g.,
Shh. Ihh, Dhh) or the patched receptor (e.g., ptc-1, ptc-2). For
instance, the subject method may employ steroidal alkaloids which
do not substantially interfere with the biological activity of such
steroids as aldosterone, androstane, androstene, androstenedione,
androsterone, cholecalciferol, cholestane, cholic acid
corticosterone, cortisol, cortisol acetate, cortisone, cortisone
acetate, deoxycorticosterone, digitoxigenin, ergocalciferol,
ergosterol, estradiol-17-.alpha., estradiol-17-.beta., estriol,
estrane, estrone, hydrocortisone, lanosterol, lithocholic acid,
mestranol, .beta.-methasone, prednisone, pregnane, pregnenolone,
progesterone, spironolactone, testosterone, triamcinolone and their
derivatives, at least so far as those activities are unrelated to
ptc related signaling.
[0170] In one embodiment, the subject steroidal alkaloid for use in
the present method has a k.sub.d for members of the nuclear hormone
receptor superfamily of greater than 1 .mu.M, and more preferably
greater than 1 mM, e.g., it does not bind estrogen., testosterone
receptors or the like. Preferably, the subject hedgehog antagonist
has no estrogenic activity at physiological concentrations (e.g.,
in the range of 1 ng-1 mg/kg).
[0171] In this manner, untoward side effects which may be
associated certain members of the steroidal alkaloid class can be
reduced. For example, using the drug screening assays described
herein, the application of combinatorial and medicinal chemistry
techniques to the steroidal alkaloids provides a means for reducing
such unwanted negative side effects including personality changes,
shortened life spans, cardiovascular diseases and vascular
occlusion., organ toxicity, hyperglycemia and diabetes, Cushnoid
features, "wasting" syndrome, steroidal glaucoma, hypertension,
peptic ulcers, and increased susceptibility to infections. For
certain embodiments, it will be benefical to reduce the teratogenic
activity relative to jervine, as for example, in the use of the
subject method to selectively inhibit spermatogenesis.
[0172] In preferred embodiment, the subject antagonists are
steroidal alkaloids other than spirosolane, tomatidine, jervine,
etc.
[0173] In certain preferred embodiments, the subject inhibitors
inhibit hedgehog-mediated signal transduction with an ED.sub.50 of
1 mM or less, more preferably of 1 .mu.M or less, and even more
preferably of 1 nM or less.
[0174] In certain embodiments, the subject inhibitors inhibit
hedgehog-mediated signal transduction with an ED.sub.50 of 1 mM or
less, more preferably 1 .mu.M or less, and even more preferably 1
nM or less.
[0175] In particular embodiments, the steroidal alkaloid is chosen
for use because it is more selective for one hedgehog isoform over
the next, e.g., 10 fold, and more preferably at least 100 or even
1000 fold more selective for one hedgehog pathway (Shh, Ihh, Dhh)
over another. Likewise, a steroidal alkaloid can be 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.
[0176] IV. Exemplary Applications of Method and Compositions
[0177] Another aspect of the present invention relates to a method
of modulating a differentiated state, survival, and/or
proliferation of a cell responsive to a hedgehog protein, by
contacting the cells with a hedgehog antagonist according to the
subject method and as the circumstances may warrant. For instance,
it is contemplated by the invention that, in light of the present
finding of an apparently broad involvement of hedgehog proteins in
the formation of ordered spatial arrangements of differentiated
tissues in vertebrates, the subject method could be used as part of
a process for generating and/or maintaining an array of different
vertebrate tissue both in vitro and in vivo. The hedgehog
antagonist, whether inductive or anti-inductive with respect
proliferation or differentiation of a given tissue, can be, as
appropriate, any of the preparations described above, including
veratrum-type alkaloids and solanum-type alkaloids.
[0178] For example, 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 neuronal cells at
various stages of differentiation, and can be employed, for
instance, in cell cultures designed to test the specific activities
of other trophic factors. In such embodiments of the subject
method, the cultured cells can be contacted with a hedgehog
antagonist of the present invention in order to alter the rate of
proliferation of neuronal stem cells in the culture and/or alter
the rate of differentiation, or to maintain the integrity of a
culture of certain terminally-differentiated neuronal cells by
preventing loss of differentiation. 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. For example, hedgehog polypeptides may
be useful in establishing and maintaining the olfactory neuron
cultures described in U.S. Pat. No. 5,318,907 and the like.
[0179] 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
effected by contact with hedgehog antagonists 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 antagonist.
[0180] 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 be proliferated, 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.
[0181] 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.
[0182] 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.
[0183] 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.
[0184] 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.
[0185] 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. 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.
[0186] 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.
[0187] 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.
[0188] 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.
[0189] 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.
[0190] 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.
[0191] 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 antagonist.
[0192] To further illustrate other uses of the subject hedgehog
antagonists, it is noted that intracerebral grafting has emerged as
an additional approach to central nervous system therapies. For
example, one approach to repairing damaged brain tissues involves
the transplantation of cells from fetal or neonatal animals into
the adult brain (Dunnett et al. (1987) J Exp Biol 123:265-289; and
Freund et al. (1985) J Neurosci 5:603-616). Fetal neurons from a
variety of brain regions can be successfully incorporated into the
adult brain, and such grafts can alleviate behavioral defects. For
example, movement disorder induced by lesions of dopaminergic
projections to the basal ganglia can be prevented by grafts of
embryonic dopaminergic neurons. Complex cognitive functions that
are impaired after lesions of the neocortex can also be partially
restored by grafts of embryonic cortical cells. The subject method
can be used to regulate the growth state in the culture, or where
fetal tissue is used, especially neuronal stem cells, can be used
to regulate the rate of differentiation of the stem cells.
[0193] 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
antagonsits employed in the present method to culture such stem
cells can be to regulate differentiation of the uncommitted
progenitor, or to regulate further restriction of the developmental
fate of a committed progenitor cell towards becoming a
terminally-differentiated neuronal cell. For example, the present
method can be used in vitro to regulate the differentiation of
neural crest cells into glial cells, schwann cells, chromaffin
cells, cholinergic sympathetic or parasympathetic neurons, as well
as peptidergic and serotonergic neurons. The hedgehog antagonists
can be used alone, or can be used in combination with other
neurotrophic factors which act to more particularly enhance a
particular differentiation fate of the neuronal progenitor
cell.
[0194] In addition to the implantation of cells cultured in the
presence of the subject hedgehog antagonists, yet another aspect of
the present invention concerns the therapeutic application of a
hedgehog antagonist to regulate the growth state of neurons and
other neuronal cells in both the central nervous system and the
peripheral nervous system. The ability of hedgehog protein to
regulate neuronal differentiation during development of the nervous
system and also presumably in the adult state indicates that, in
certain instances, the subject hedgehog antagonists can be expected
to facilitate control of adult neurons with regard to maintenance,
functional performance, and aging of normal cells; repair and
regeneration processes in chemically or mechanically lesioned
cells; and treatment of degeneration in certain pathological
conditions. In light of this understanding, the present invention
specifically contemplates applications of the subject method to the
treatment protocol of (prevention and/or reduction of the severity
of) neurological conditions deriving from: (i) acute, subacute, or
chronic injury to the nervous system, including traumatic injury,
chemical injury, vascular injury and deficits (such as the ischemia
resulting from stroke), together with infectious/inflammatory and
tumor-induced injury; (ii) aging of the nervous system including
Alzheimer's disease; (iii) chronic neurodegenerative diseases of
the nervous system, including Parkinson's disease, Huntington's
chorea, 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. The subject antagonists can be used
in conjunction with a therapy involving hedgehog agonists to
control the timing and rates of proliferation and/or
differentiation of the affected neuronal cells.
[0195] 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 and antagonists 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.
[0196] In another embodiment, the subject method can be used in the
treatment of neoplastic or hyperplastic transformations such as may
occur in the central nervous system. For instance, the hedgehog
antagonists can be utilized to cause such transformed cells to
become either post-mitotic or apoptotic. The present method may,
therefore, be used as part of a treatment for, e.g., malignant
gliomas, medulloblastomas, neuroectodermal tumors, and
ependymomas.
[0197] Yet another aspect of the present invention concerns the
observation in the art that hedgehog proteins are morphogenic
signals involved in other vertebrate organogenic pathways in
addition to neuronal differentiation as described above, having
apparent roles in other endodermal patterning, as well as both
mesodermal and endodermal differentiation processes. Thus, it is
contemplated by the invention that compositions comprising hedgehog
antagonists can also be utilized for both cell culture and
therapeutic methods involving generation and maintenance of
non-neuronal tissue.
[0198] In one embodiment, the present invention makes use of the
discovery that hedgehog proteins, such as Sonic hedgehog, are
apparently involved in controlling the development of stem cells
responsible for formation of the digestive tract, liver, lungs, and
other organs which derive from the primitive gut. Shh serves as an
inductive signal from the endoderm to the mesoderm, which is
critical to gut morphogenesis. Therefore, for example, hedgehog
antagonists of the instant method can be employed for regulating
the development and maintenance of an artificial liver 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.
[0199] 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.
[0200] In yet another embodiment, the subject method can be
employed therapeutically to regulate such organs after physical,
chemical or pathological insult. For instance, therapeutic
compositions comprising hedgehog antagonists can be utilized in
liver repair subsequent to a partial hepatectomy.
[0201] 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, Apclqvist 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 homcobox 1), an essential regulator of early pancreatic
development. Apclqvist 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.
[0202] In the context of the present invention, it is contemplated
therefore that the subject hedgehog inhibitors can be used to
control the regulate the proliferation and/or differentiation of
pancreatic tissue both in vivo and in vitro.
[0203] There are a wide variety of pathological cell proliferative
and differentiative conditions for which the inhibitors of the
present invention may provide therapeutic benefits, with the
general strategy being, for example, the correction of abberrant
insulin expression, or modulation of differentiation. More
generally, however, the present invention relates to a method of
inducing and/or maintaining a differentiated state, enhancing
survival and/or affecting proliferation of pancreatic cells, by
contacting the cells with the subject inhibitors. For instance, it
is contemplated by the invention that, in light of the apparent
involvement of hedgehog protein(s) 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 ptc 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.
[0204] 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 hedgehog signaling may be indicated in disease
progression, the subject inhibitors, can be used to enhance
regeneration of the tissue after anti-tumor therapy.
[0205] Moreover, manipulation of ptc 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 the hedgehog 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
ptc 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 coactive 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
embyonal traits in mature .beta.-cells can be observed. By
utilizing agents which alter ptc signal transduction, the the
action of endogenous hedgehog protein on the differentiation path
or proliferative index of the cells can be regulated.
[0206] 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
ptc function to affect tissue differentiation can be utilized as a
means of maintaining graft viability.
[0207] 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.
[0208] Fujita et al. (1997) Biochem Biophys Res Commun 238:658
reported that Sonic hedgehog is expressed in human lung squamous
carcinoma and adenocarcinoma cells. The expression of Sonic
hedgehog was also detected in the human lung squamous carcinoma
tissues, but not in the normal lung tissue of the same patient.
They also observed that Sonic hedgehog stimulates the incorporation
of BrdU into the carcinoma cells and stimulates their cell growth,
while anti-Shh-N inhibited their cell growth. These results suggest
that a Sonic hedgehog signal is involved in the cell growth of such
transformed lung tissue and therefore indicates that the subject
method can be used as part of a treatment of lung carcinoma and
adenocarcinomas, and other proliferative disorders involving the
lung epithelia.
[0209] In still another embodiment of the present invention,
compositions comprising hedgehog antagonists can be used in the in
vitro generation of skeletal tissue, such as from skeletogenic stem
cells, as well as the in vivo treatment of skeletal tissue
deficiencies. The present invention particularly contemplates the
use of hedgehog antagonists to regulate the rate of chondrogenesis
and/or osteogenesis. By "skeletal tissue deficiency", it is meant a
deficiency in bone or other skeletal connective tissue at any site
where it is desired to restore the bone or connective tissue, no
matter how the deficiency originated, e.g. whether as a result of
surgical intervention, removal of tumor, ulceration, implant,
fracture, or other traumatic or degenerative conditions.
[0210] 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.
[0211] In one embodiment of the present invention, the subject
method comprises treating the afflicted connective tissue with a
therapeutically sufficient amount of a hedgehog antagonist,
particularly an antagonist selective for Indian hedgehog, 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.
[0212] In an illustrative embodiment, the subject method can be
used as part of a therapeutic intervention in the treatment of
cartilage of a diarthroidal joint, such as a knee, an ankle, an
elbow, a hip, a wrist, a knuckle of either a finger or toe, or a
tempomandibular joint. The treatment can be directed to the
meniscus of the joint, to the articular cartilage of the joint, or
both. To further illustrate, the subject method can be used to
treat a degenerative disorder of a knee, such as which might be the
result of traumatic injury (e.g. a sports injury or excessive wear)
or osteoarthritis. The subject antagonists may be administered as
an injection into the joint with, for instance, an arthroscopic
needle. In some instances, the injected agent can be in the form of
a hydrogel or other slow release vehicle described above in order
to permit a more extended and regular contact of the agent with the
treated tissue.
[0213] 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.
[0214] 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 car 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.
[0215] In one embodiment of the subject method, the implants are
contacted with a hedgehog antagonist during certain stages of the
culturing process in order to manage the rate of differentiation of
chondrocytes and the formation of hypertrophic chrondrocytes in the
culture.
[0216] 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 out above with respect to tissue transplants, the artificial
transplants suffer from the same deficiency of not being derived in
a setting which is comparable to the actual mechanical environment
in which the matrix is implanted. The ability to regulate the
chondrocytes in the matrix by the subject method can allow the
implant to acquire characteristics similar to the tissue for which
it is intended to replace.
[0217] 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.
[0218] 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 antagonists
of the present invention can be employed as part of a method for
regulating the rate of bone loss in a subject. For example,
preparations comprising hedgehog antagonists can be employed, for
example, to control endochondral ossification in the formation of a
"model" for ossification.
[0219] In yet another embodiment of the present invention, a
hedgehog antagonist can be used to regulate spermatogenesis. The
hedgehog proteins, particularly Dhh, have been shown to be involved
in the differentiation and/or proliferation and maintenance of
testicular germ cells. Dhh expression is initiated in Sertoli cell
precursors shortly after the activation of Sry 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. The subject method can be utilized to block
the action of a naturally-occurring hedgehog protein. In a
preferred embodiment, the hedgehog antagonist inhibits the
biological activity of Desert hedgehog with respect to
spermatogenesis, and can be used as a contraceptive. In similar
fashion, hedgehog antagonists of the subject method are potentially
useful for modulating normal ovarian function.
[0220] The subject method also has wide applicability to the
treatment or prophylaxis of disorders afflicting epithelial tissue,
as well as in cosmetic uses. In general, the method can be
characterized as including a step of administering to an animal an
amount of a hedgehog antagonist effective to alter the growth state
of a treated epithelial tissue. The mode of administration and
dosage regimens will vary depending on the epithelial tissue(s)
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.
[0221] 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.
[0222] 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.
[0223] The present treatment can also be effective as part of a
therapeutic regimen for treating oral and paraoral ulcers, e.g.
resulting from radiation and/or chemotherapy. Such ulcers commonly
develop within days after chemotherapy or radiation therapy. These
ulcers usually begin as small, painful irregularly shaped lesions
usually covered by a delicate gray necrotic membrane and surrounded
by inflammatory tissue. In many instances, lack of treatment
results in proliferation of tissue around the periphery of the
lesion on an inflammatory basis. For instance, the epithelium
bordering the ulcer usually demonstrates proliferative activity,
resulting in loss of continuity of surface epithelium. These
lesions, because of their size and loss of epithelial integrity,
dispose the body to potential secondary infection. Routine
ingestion of food and water becomes a very painful event and, if
the ulcers proliferate throughout the alimentary canal, diarrhea
usually is evident with all its complicating factors. According to
the present invention, a treatment for such ulcers which includes
application of an hedgehog antagonist can reduce the abnormal
proliferation and differentiation of the affected epithelium,
helping to reduce the severity of subsequent inflammatory
events.
[0224] The subject method and compositions can also be used to
treat wounds resulting from dermatological diseases, such as
lesions resulting from autoimmune disorders such as psoriasis.
Atopic dermititis refers to skin trauma resulting from allergies
associated with an immune response caused by allergens such as
pollens, foods, dander, insect venoms and plant toxins.
[0225] In other embodiments, antiproliferative preparations of
hedgehog antagonists can be used to inhibit lens epithelial cell
proliferation to prevent post-operative complications of
extracapsular cataract extraction. Cataract is an intractable eye
disease and various studies on a treatment of cataract have been
made. But at present, the treatment of cataract is attained by
surgical operations. Cataract surgery has been applied for a long
time and various operative methods have been examined.
Extracapsular lens extraction has become the method of choice for
removing cataracts. The major medical advantages of this technique
over intracapsular extraction are lower incidence of aphakic
cystoid macular edema and retinal detachment. Extracapsular
extraction is also required for implantation of posterior chamber
type intraocular lenses which are now considered to be the lenses
of choice in most cases.
[0226] However, a disadvantage of extracapsular cataract extraction
is the high incidence of posterior lens capsule opacification,
often called after-cataract, which can occur in up to 50% of cases
within three years after surgery. After-cataract is caused by
proliferation of equatorial and anterior capsule lens epithelial
cells which remain after extracapsular lens extraction. These cells
proliferate to cause Sommerling rings, and along with fibroblasts
which also deposit and occur on the posterior capsule, cause
opacification of the posterior capsule, which interferes with
vision. Prevention of after-cataract would be preferable to
treatment. To inhibit secondary cataract formation, the subject
method provides a means for inhibiting proliferation of the
remaining lens epithelial cells. For example, such cells can be
induced to remain quiescent by instilling a solution containing an
hedgehog antagonist preparation into the anterior chamber of the
eye after lens removal. Furthermore, the solution can be
osmotically balanced to provide minimal effective dosage when
instilled into the anterior chamber of the eye, thereby inhibiting
subcapsular epithelial growth with some specificity.
[0227] The subject method can also be used in the treatment of
comeopathies marked by corneal epithelial cell proliferation, as
for example in ocular epithelial disorders such as epithelial
downgrowth or squamous cell carcinomas of the ocular surface.
[0228] 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 proliferative diseases of retinal cells and regulate
photoreceptor differentiation.
[0229] Yet another aspect of the present invention relates to the
use of the subject method to control hair growth. Hair is basically
composed of keratin, a tough and insoluble protein; its chief
strength lies in its 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.
[0230] As is well knows 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.
[0231] In certain embodimemts, the subject method can be employed
as a way of reducing the growth of human hair as opposed to its
conventional removal by cutting, shaving, or depilation. For
instance, the present method can be used in the treatment of
trichosis characterized by abnormally rapid or dense growth of
hair, e.g. hypertrichosis. In an exemplary embodiment, hedgehog
antagonists can be used to manage hirsutism, a disorder marked by
abnormal hairiness. The subject method can also provide a process
for extending the duration of depilation.
[0232] Moreover, because a hedgehog antagonist will often be
cytostatic to epithelial cells, rather than cytotoxic, such agents
can be used to protect hair follicle cells from cytotoxic agents
which require progression into S-phase of the cell-cycle for
efficacy, e.g. radiation-induced death. Treatment by the subject
method can provide protection by causing the hair follicle cells to
become quiescent, e.g., by inhibiting the cells from entering S
phase, and thereby preventing the follicle cells from undergoing
mitotic catastrophe or programmed cell death. For instance,
hedgehog antagonists can be used for patients undergoing chemo- or
radiation-therapies which ordinarily result in hair loss. By
inhibiting cell-cycle progression during such therapies, the
subject treatment can protect hair follicle cells from death which
might otherwise result from activation of cell death programs.
After the therapy has concluded, the instant method can also be
removed with concommitant relief of the inhibition of follicle cell
proliferation.
[0233] The subject method can also be used in the treatment of
folliculitis, such as folliculitis decalvans, folliculitis
ulerythematosa reticulata or keloid folliculitis. For example, a
cosmetic prepration of an hedgehog antagonist can be applied
topically in the treatment of pseudofolliculitis, a chronic
disorder occurring most often in the submandibular region of the
neck and associated with shaving, the characteristic lesions of
which are erythematous papules and pustules containing buried
hairs.
[0234] In another aspect of the invention, the subject method can
be used to induce differentiation 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.
[0235] For instance, the pharmaceutical preparations of the
invention are intended for the treatment of hyperplastic epidermal
conditions, such as keratosis, as well as for the treatment of
neoplastic epidermal conditions such as those characterized by a
high proliferation rate for various skin cancers, as for example
basal cell carcinoma or squamous cell carcinoma. The subject method
can also be used in the treatment of autoimmune diseases affecting
the skin, in particular, of dermatological diseases involving
morbid proliferation and/or keratinization of the epidermis, as for
example, caused by psoriasis or atopic dermatosis.
[0236] Many common diseases of the skin, such as psoriasis squamous
cell carcinoma, keratoacanthoma and actinic keratosis are
characterized by localized abnormal proliferation and growth. For
example, in psoriasis, which is characterized by scaly, red,
elevated plaques on the skin, the keratinocytes are known to
proliferate much more rapidly than normal and to differentiate less
completely.
[0237] In one embodiment, the preparations of the present invention
are suitable for the treatment of dermatological ailments linked to
keratinization disorders causing abnormal proliferation of skin
cells, which disorders may be marked by either inflammatory or
non-inflammatory components. To illustrate, therapeutic
preparations of a hedgehog antagonist, e.g., which promotes
quiescense or differentiation can be used to treat varying forms of
psoriasis, be they cutaneous, mucosal or ungual. Psoriasis, as
described above, is typically characterized by epidermal
keratinocytes which display marked proliferative activation and
differentiation along a "regenerative" pathway. Treatment with an
antiproliferative embodiment of the subject method can be used to
reverse the pathological epidermal activiation and can provide a
basis for sustained remission of the disease.
[0238] A variety of other keratotic lesions are also candidates for
treatment with the subject method. Actinic keratoses, for example,
are superficial inflammatory premalignant tumors arising on
sun-exposed and irradiated skin. The lesions are erythematous to
brown with variable scaling. Current therapies include excisional
and cryosurgery. These treatments are painful, however, and often
produce cosmetically unacceptable scarring. Accordingly, treatment
of keratosis, such as actinic keratosis, can include application,
preferably topical, of a hedgehog antagonist composition in amounts
sufficient to inhibit hyperproliferation of epidermal/epidermoid
cells of the lesion.
[0239] Acne represents yet another dermatologic ailment which may
be treated by the subject method. Acne vulgaris, for instance, is a
multifactorial disease most commonly occurring in teenagers and
young adults, and is characterized by the appearance of
inflammatory and noninflammatory lesions on the face and upper
trunk. The basic defect which gives rise to acne vulgaris is
hypercornification of the duct of a hyperactive sebaceous gland.
Hypercornification blocks the normal mobility of skin and follicle
microorganisms, and in so doing, stimulates the release of lipases
by Propinobacterium acnes and Staphylococcits epidermidis bacteria
and Pitrosporum ovale, a yeast. Treatment with an antiproliferative
hedgehog antagonist, particularly topical preparations, may be
useful for preventing the transitional features of the ducts, e.g.
hypercomification, which lead to lesion formation. The subject
treatment may further include, for example, antibiotics, retinoids
and antiandrogens.
[0240] The present invention also provides a method for treating
various forms of dermatitis. Dermatitis is a descriptive term
referring to poorly demarcated lesions which are either pruritic,
erythematous, scaley, blistered, weeping, fissured or crusted.
These lesions arise from any of a wide variety of causes. The most
common types of dermatitis are atopic, contact and diaper
dermatitis. For instance, seborrheic dermatitis is a chronic,
usually pruritic, dermatitis with erythema, dry, moist, or greasy
scaling, and yellow crusted patches on various areas, especially
the scalp, with exfoliation of an excessive amount of dry scales.
The subject method can also be used in the treatment of stasis
dermatitis, an often chronic, usually eczematous dermatitis.
Actinic dermatitis is dermatitis that due to exposure to actinic
radiation such as that from the sun, ultraviolet waves or x- or
gamma-radiation. According to the present invention, the subject
method can be used in the treatment and/or prevention of certain
symptoms of dermatitis caused by unwanted proliferation of
epithelial cells. Such therapies for these various forms of
dermatitis can also include topical and systemic corticosteroids,
antipurities, and antibiotics.
[0241] Ailments which may be treated by the subject method are
disorders specific to non-humans, such as mange.
[0242] In still another embodiment, the subject method can be used
in the treatment of human cancers, particularly basal cell
carcinomas and other tumors of epithelial tissues such as the skin.
For example, hedgehog antagonists can be employed, in the subject
method, as part of a treatment for basal cell nevus syndrome
(BCNS), and other other human carcinomas, adenocarcinomas, sarcomas
and the like.
[0243] In one aspect, the present invention provides pharmaceutical
preparations and methods for controlling the formation of
megakaryocyte-derived cells and/or controlling the functional
performance of megakaryocyte-derived cells. For instance, certain
of the compositions disclosed herein may be applied to the
treatment or prevention of a variety hyperplastic or neoplastic
conditions affecting platelets.
[0244] The hedgehog antagonists for use in the subject method may
be conveniently formulated for administration with a biologically
acceptable medium, such as water, buffered saline, polyol (for
example, glycerol, propylene glycol, liquid polyethylene glycol and
the like) or suitable mixtures thereof. The optimum concentration
of the active ingredient(s) in the chosen medium can be determined
empirically, according to procedures well known to medicinal
chemists. As used herein, "biologically acceptable medium" includes
any and all solvents, dispersion media, and the like which may be
appropriate for the desired route of administration of the
pharmaceutical preparation. The use of such media for
pharmaceutically active substances is known in the art. Except
insofar as any conventional media or agent is incompatible with the
activity of the hedgehog antagonist, its use in the pharmaceutical
preparation of the invention is contemplated. Suitable vehicles and
their formulation inclusive of other proteins are described, for
example, in the book Remington's Pharmaceutical Sciences
(Remington's Pharmaceutical Sciences. Mack Publishing Company,
Easton, Pa., USA 1985). These vehicles include injectable "deposit
formulations".
[0245] Pharmaceutical formulations of the present invention can
also include veterinary compositions, e.g. pharmaceutical
preparations of the hedgehog antagonists suitable for veterinary
uses, e.g., for the treatment of live stock or domestic animals,
e.g., dogs.
[0246] 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 antagonist at a particular target site.
[0247] 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, etc. administration
by injection, infusion or inhalation; topical by lotion or
ointment; and rectal by suppositories. Oral and topical
administrations are preferred.
[0248] 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.
[0249] 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.
[0250] These compounds may be administered to humans and other
animals for therapy by any suitable route of administration,
including orally, nasally, as by, for example, a spray, rectally,
intravaginally, parenterally, intracisternally and topically, as by
powders, ointments or drops, including buccally and
sublingually.
[0251] 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.
[0252] 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.
[0253] 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 hedegehog antagonist
employed, the age, sex, weight, condition, general health and prior
medical history of the patient being treated, and like factors well
known in the medical arts.
[0254] 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.
[0255] In general, a suitable daily dose of a compound of the
invention will bc 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.
[0256] 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.
[0257] The term "treatment" is intended to encompass also
prophylaxis, therapy and cure.
[0258] 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.
[0259] The compound of the invention can be administered as such or
in admixtures with pharmaceutically acceptable 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.
[0260] V. Pharmaceutical Compositions
[0261] While it is possible for a compound of the present invention
to be administered alone, it is preferable to administer the
compound as a pharmaceutical formulation (composition). The
hedgehog antagonists according to the invention may be formulated
for administration in any convenient way for use in human or
veterinary medicine.
[0262] 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.
[0263] 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 inhibiting a
hedgehog signaling pathway 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.
[0264] 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.
[0265] The phrase "pharmaceutically-acceptable carrier" as used
herein means a pharmaceutically-acceptable material, composition or
vehicle, such as a liquid or solid filler, diluent, excipient,
solvent or encapsulating material, involved in carrying or
transporting the subject antagonists from one organ, or portion of
the body, to another organ, or portion of the body. Each carrier
must be "acceptable" in the sense of being compatible with the
other ingredients of the formulation and not injurious to the
patient. Some examples of materials which can serve as
pharmaceutically-acceptable carriers include: (1) sugars, such as
lactose, glucose and sucrose; (2) starches, such as corn starch and
potato starch; (3) cellulose, and its derivatives, such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)
powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8)
excipients, such as cocoa butter and suppository waxes; (9) oils,
such as peanut oil, cottonseed oil, safflower oil, sesame oil,
olive oil, corn oil and soybean oil; (10) glycols, such as
propylene glycol; (11) polyols, such as glycerin, sorbitol,
mannitol and polyethylene glycol; (12) esters, such as ethyl oleate
and ethyl laurate; (13) agar; (14) buffering agents, such as
magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)
pyrogen-free water; (17) isotonic saline; (18) Ringer's solution;
(19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other
non-toxic compatible substances employed in pharmaceutical
formulations.
[0266] As set out above, certain embodiments of the present
hedgehog antagonists may contain a basic functional group, such as
amino or alkylamino, and are, thus, capable of forming
pharmaceutically-acceptable salts with pharmaceutically-acceptable
acids. The term "pharmaceutically-acceptable salts" in this
respect, refers to the relatively non-toxic, inorganic and organic
acid addition salts of compounds of the present invention. These
salts can be prepared in situ during the final isolation and
purification of the compounds of the invention, or by separately
reacting a purified compound of the invention in its free base form
with a suitable organic or inorganic acid, and isolating the salt
thus formed. Representative salts include the hydrobromide,
hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate,
valerate, oleate, palmitate, stearate, laurate, benzoate, lactate,
phosphate, tosylate, citrate, maleate, fumarate, succinate,
tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and
laurylsulphonate salts and the like. (See, for example, Berge et
al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:1-19)
[0267] 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.
[0268] 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).
[0269] 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.
[0270] 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.
[0271] 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.
[0272] 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.
[0273] 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.
[0274] 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.
[0275] 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.
[0276] 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.
[0277] 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.
[0278] 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.
[0279] 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.
[0280] It is known that sterols, such as cholesterol, will form
complexes with cyclodextrins. Thus, in preferred embodiments, where
the inhibitor is a steroidal alkaloid, it may be formulated with
cyclodextrins, such as .alpha.-, .beta.- and .gamma.-cyclodextrin,
dimethyl-.beta. cyclodextrin and
2-hydroxypropyl-.beta.-cyclodextrin.
[0281] Formulations of the pharmaceutical compositions of the
invention for rectal or vaginal administration may be presented as
a suppository, which may be prepared by mixing one or more
compounds of the invention with one or more suitable nonirritating
excipients or carriers comprising, for example, cocoa butter,
polyethylene glycol, a suppository wax or a salicylate, and which
is solid at room temperature, but liquid at body temperature and,
therefore, will melt in the rectum or vaginal cavity and release
the active hedgehog antagonist.
[0282] 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.
[0283] 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.
[0284] 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.
[0285] 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.
[0286] Transdermal patches have the added advantage of providing
controlled delivery of a compound of the present invention to the
body. Such dosage forms can be made by dissolving or dispersing the
hedgehog antagonists in the proper medium. Absorption enhancers can
also be used to increase the flux of the hedgehog antagonists
across the skin. The rate of such flux can be controlled by either
providing a rate controlling membrane or dispersing the compound in
a polymer matrix or gel.
[0287] Ophthalmic formulations, eye ointments, powders, solutions
and the like, are also contemplated as being within the scope of
this invention.
[0288] 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.
[0289] 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.
[0290] 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.
[0291] 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.
[0292] 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.
[0293] 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.
[0294] 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.
[0295] Alternatively, an intermediate concentrate or feed
supplement containing the active ingredient can be blended into the
feed. The way in which such feed premixes and complete rations can
be prepared and administered are described in reference books (such
as "Applied Animal Nutrition". W.H. Freedman and CO., San
Francisco, U.S.A., 1969 or "Livestock Feeds and Feeding" O and B
books. Corvallis, Oreg., U.S.A., 1977).
[0296] VI. Synthetic Schemes and Identification of Active
Antagonists
[0297] The subjects steroidal alkaloids, 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.
[0298] a. Combinatorial Libraries
[0299] 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 antagonists lead compounds, as well as to refine the
specificity, toxicity, and/or cytotoxic-kinetic profile of a lead
compound. For instance, hedgehog bioactivity assays as described
above can be used to screen a library of the subject compounds for
those having antagonist activity toward all or a particular
hedgehog isoform or activity.
[0300] 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.
[0301] 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.
[0302] A variety of techniques are available in the art for
generating combinatorial libraries of small organic molecules such
as the subject hedgehog antagonists. 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; 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 antagonists can be synthesized and screened for particular
activity or property.
[0303] In an exemplary embodiment, a library of candidate hedgehog
antagonists 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 e.g., located at one of the
positions of the candidate antagonists 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" on a lawn of hedgehog-sensitive
cells for which an inhibitor is sought. The diversomers can be
released from the bead, e.g. by hydrolysis. Beads surrounded by
areas of no, or diminished, hedgehog sensitivity (e.g., to
exogeneously added hedgehog protein), e.g. a "halo", can be
selected, and their tags can be "read" to establish the identity of
the particular diversomer.
[0304] b. Screening Assays
[0305] There are a variety of assays availble for determining the
ability of a compound to inhibit hedgehog-mediated signaling, 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 steroidal and non-steroidal compounds which have similar
activity to jervine with respect inhibition of hedgehog
signals.
[0306] The availability of purified and recombinant hedgehog
polypeptides facilitates the generation of assay systems which can
be used to screen for drugs, such as small organic molecules, which
are antagonists of the normal cellular function of a hedgehog,
particularly its role in the pathogenesis of cell proliferation
and/or differentiation. In one embodiment, the assay evaluates the
ability of a compound to modulate binding between a hedgehog
polypeptide and a hedgehog receptor such as patched. In other
embodiments, the assay merely scores for the ability of a test
compound to alter hedgehog-mediated signal transduction. In this
manner, a variety of antagonists can be identified. A variety of
assay formats will suffice and, in light of the present disclosure,
will be comprehended by skilled artisan.
[0307] Assays which are performed in cell-free systems, such as may
be derived with purified or semi-purified proteins, are often
preferred as "primary" screens in that they can be generated to
permit rapid development and relatively easy detection of an
alteration in a molecular target which is mediated by a test
compound. Moreover, the effects of cellular toxicity and/or
bioavailability of the test compound can be generally ignored in
the in vitro system, the assay instead being focused primarily on
the effect of the drug on the molecular target as may be manifest
in an alteration of binding affinity with receptor proteins.
[0308] While not wishing to be bound by any particular theory,
should jervine and other steroidal alkaloids exert their activity
in the hedgehog signal pathway by interfering with
cholesterol-derived hedgehog, e.g., through interaction with the
sterol sensing domain of patched (see, e.g. Loftus et al. (1997)
Science 277:232), an exemplary screening assay for a hedgehog
antagonist comprises contacting a compound of interest with a
mixture including a hedgehog receptor protein (e.g., a cell
expressing the patched receptor) and a hedgehog protein under
conditions in which the receptor is ordinarily capable of binding
the hedgehog protein, or at least jervine. To the mixture is then
added a composition containing a test compound. Detection and
quantification of receptor/hedgehog and/or receptor/jervine
complexes provides a means for determining the test compound's
efficacy at inhibiting (or potentiating) complex formation between
the receptor protein and the hedgehog polypeptide or the jervine
antagonist. The efficacy of the compound can be assessed by
generating dose response curves from data obtained using various
concentrations of the test compound, and by comparing the results
to that obtained with jervine. Moreover, a control assay can also
be performed to provide a baseline for comparison. In the control
assay, isolated and purified hedgehog polypeptide is added to the
receptor protein, and the formation of receptor/hedgehog complex is
quantitated in the absence of the test compound.
[0309] In an illustrative embodiment, the polypeptide utilized as a
hedgehog receptor can be generated from the patched protein, and in
particular, includes the steroid sensing domain, e.g. and a soluble
portion of the protein including a functional steroid sensing
domain. Accordingly, an exemplary screening assay includes all or a
suitable portion of the patched protein which can be obtained from,
for example, the human patched gene (GenBank U43148) or other
vertebrate sources (see GenBank Accession numbers U40074 for
chicken patched and U46155 for mouse patched), as well as from
drosophila (GenBank Accession number M28999) or other invertebrate
sources. The patched protein can be provided in the screening assay
as a whole protein (preferably expressed on the surface of a cell),
or alternatively as a fragment of the full length protein, e.g.
which includes the steroid sensing domain and/or at least a portion
which binds to hedgehog polypeptides, e.g. as one or both of the
substantial extracellular domains (e.g. corresponding to residues
Asn120-Ser438 and/or Arg770-Trp1027 of the human patched protein.
For instance, the patched protein can be provided in soluble form,
as for example a preparation of one of the extracellular domains,
or a preparation of both of the extracellular domains which are
covalently connected by an unstructured linker (see, for example,
Huston et al. (1988) PNAS 85:4879; and U.S. Pat. No. 5,091,513). In
other embodiments, the protein can be provided as part of a
liposomal preparation or expressed on the surface of a cell. The
patched protein can derived from a recombinant gene, e.g., being
ectopically expressed in a heterologous cell. For instance, the
protein can be expressed on oocytes, mammalian cells (e.g., COS,
CHO, 3T3 or the like), or yeast cell by standard recombinant DNA
techniques. These recombinant cells can be used for receptor
binding, signal transduction or gene expression assays. Stone et
al. (1996) Nature 384:129-34; and Marigo et al. (1996) Nature
384:176-9 illustrate binding assays of human hedgehog to patched,
such as a chicken patched protein ectopically expressed in Xenopus
laevis oocytes. The assay system of Marigo et al. for example, can
be adapted to the present drug screening assays. As illustrated in
that reference, Shh binds to the patched protein in a selective,
saturable, dose-dependent manner, thus demonstrating that patched
is a receptor for Shh.
[0310] Complex formation between the hedgehog polypeptide or
jervine and a hedgehog receptor may be detected by a variety of
techniques. For instance, modulation of the formation of complexes
can be quantitated using, for example, detectably labelled proteins
such as radiolabelled, fluorescently labelled, or enzymatically
labelled hedgehog polypeptides, by immunoassay, or by
chromatographic detection.
[0311] Typically, for cell-free assays, it will be desirable to
immobilize either the hedgehog receptor or the hedgehog polypeptide
or jervine molecule to facilitate separation of receptor complexes
from uncomplexed forms of one of the protein, as well as to
accommodate automation of the assay. In one embodiment, a fusion
protein can be provided which adds a domain that allows the protein
to be bound to a matrix. For example,
glutathione-S-transferase/receptor (GST/receptor) fusion proteins
can be adsorbed onto glutathione sepharose beads (Sigma Chemical,
St. Louis, Mo.) or glutathione derivatized microtitre plates, which
are then combined with jervine or the hedgehog polypeptide, e.g. an
.sup.35S-labeled hedgehog polypeptide, and the test compound and
incubated under conditions conducive to complex formation, e.g. at
physiological conditions for salt and pH, though slightly more
stringent conditions may be desired. Following incubation, the
beads are washed to remove any unbound ligand, and the matrix
bead-bound radiolabel determined directly (e.g. beads placed in
scintillant), or in the supernatant after the complexes are
dissociated. Alternatively, the complexes can be dissociated from
the bead, separated by SDS-PAGE gel, and the level of hedgehog
polypeptide or jervine found in the bead fraction quantitated from
the gel using standard techniques (HPLC, gel electrophoresis,
etc).
[0312] Where the desired portion of the hedgehog receptor (or other
hedgehog binding molecule) cannot be provided in soluble form,
liposomal vesicles can be used to provide manipulatable and
isolatable sources of the receptor. For example, both authentic and
recombinant forms of the patched protein can be reconstituted in
artificial lipid vesicles (e.g. phosphatidylcholine liposomes) or
in cell membrane-derived vesicles (see, for example. Bear et al.
(1992) Cell 68:809-818; Newton et al. (1983) Biochemistry
22:6110-6117; and Reber et al. (1987) J Biol Chem
262:11369-11374).
[0313] In addition to cell-free assays, such as described above,
the compounds of the subject invention can also be tested in
cell-based assays. In one embodiment, cell which are sensitive to
hedgehog induction, e.g. patched-expressing cells or other cells
sensitive to hedgehog induction, can be contacted with a hedgehog
protein and a test agent of interest, with the assay scoring for
anything from simple binding to the cell to inhibition in hedgehog
inductive responses by the target cell in the presence and absence
of the test agent.
[0314] In addition to characterizing cells that naturally express
the patched protein, cells which have been genetically engineered
to ectopically express patched can be utilized for drug screening
assays. As an example, cells which either express low levels or
lack expression of the patched protein, e.g. Xenopus laevis
oocytes, COS cells or yeast cells, can be genetically modified
using standard techniques to ectopically express the patched
protein. (see Marigo et al., supra).
[0315] The resulting recombinant cells, e.g., which express a
functional patched receptor, can be utilized in receptor binding
assays to identify agonist or anatagonsts of hedgehog binding.
Binding assays can be performed using whole cells. Furthermore, the
recombinant cells of the present invention can be engineered to
include other heterolgous genes encoding proteins involved in
hedgehog-dependent siganl pathways. For example, the gene products
of one or more of smoothened, costal-2, fused, and/or suppressor of
fused can be co-expressed with patched in the reagent cell, with
assays being sensitive to the functional reconstituion of the
hedgehog signal transduction cascade.
[0316] Alternatively, liposomal preparations using reconstituted
patched protein can be utilized. Patched protein purified from
detergent extracts from both authentic and recombinant origins can
be reconstituted in in artificial lipid vesicles (e.g.
phosphatidylcholine liposomes) or in cell membrane-derived vesicles
(see, for example, Bear et al. (1992) Cell 68:809-818; Newton et
al. (1983) Biochemistry 22:6110-6117; and Reber et al. (1987) J
Biol Chem 262:11369-11374). The lamellar structure and size of the
resulting liposomes can be characterized using electron microscopy.
External orientation of the patched protein in the reconstituted
membranes can be demonstrated, for example, by immunoelectron
microscopy. The hedgehog protein binding activity of liposomes
containing patched and liposomes without the protein in the
presence of candidate agents can be compared in order to identify
potential modulators of the hedgehog-patched interaction.
[0317] The hedgehog protein used in these cell-based assays can be
provided as a purified source (natural or recombinant in origin),
or in the form of cells/tissue which express the protein and which
are co-cultured with the target cells, and is preferably a
cholesterol-derived form. In addition to binding studies, by
detecting changes in intracellular signals, such as alterations in
second messengers or gene expression, in patched-expressing cells
contacted with a test agent, candidate hedgehog antagonists can be
identified.
[0318] A number of gene products have been implicated in
patched-mediated signal transduction, including patched, the
transcription factor cubitus interruptus (ci), the serine/threonine
kinase fused (fu) and the gene products of costal-2, smoothened and
suppressor of fused.
[0319] 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 antagonists of hedgehog.
[0320] 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 hedgehog signaling. To identify potential regulatory elements
responsive to hedgehog signaling present in the transcriptional
regulatory sequence of a target gene, nested deletions of genomic
clones of the target gene can be constructed using standard
techniques. See, for example, Current Protocols in Molecular
Biology, Ausubel, F. M. et al. (eds.) Greene Publishing Associates,
(1989); U.S. Pat. No. 5,266,488; Sato et al. (1995) J Biol Chem
270:10314-10322; and Kube et al. (1995) Cytokine 7:1-7. A nested
set of DNA fragments from the gene's 5'-flanking region are placed
upstream of a reporter gene, such as the luciferase gene, and
assayed for their ability to direct reporter gene expression in
patched expressing cells. Host cells transiently transfected with
reporter gene constructs can be scored for the regulation of
expression of the reporter gene in the presence and absence of
hedgehog to determine regulatory sequences which are responsive to
patched-dependent signalling.
[0321] 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 second messengers generated by
induction with hedgehog protein. Typically, the reporter gene
construct will include a reporter gene in operative linkage with
one or more transcriptional regulatory elements responsive to the
hedgehog activity, with the level of expression of the reporter
gene providing the hedgehog-dependent detection signal. 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 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 hedgehog) 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 difference in the amount of
transcription indicates that the test compound has in some manner
altered the signal transduction activity of the hedgehog protein,
e.g., the test compound is a potential hedgehog antagonist.
[0322] As described in further detail below, in preferred
embodiments the gene product of the reporter is detected by an
intrinsic activity associated with that product. For instance, the
reporter gene may encode a gene product that, by enzymatic
activity, gives rise to a detection signal based on color,
fluorescence, or luminescence. In other preferred embodiments, the
reporter or marker gene provides a selective growth advantage,
e.g., the reporter gene may enhance cell viability, relieve a cell
nutritional requirement, and/or provide resistance to a drug.
[0323] Preferred reporter genes are those that are readily
detectable. The reporter gene may also be included in the construct
in the form of a fusion gene with a gene that includes desired
transcriptional regulatory sequences or exhibits other desirable
properties. Examples of reporter genes include, but are not limited
to CAT (chloramphenicol acetyl transferase) (Alton and Vapnek
(1979), Nature 282: 864-869) luciferase, and other enzyme detection
systems, such as beta-galactosidase, firefly luciferase (deWet et
al. (1987), Mol. Cell. Biol. 7:725-737); bacterial luciferase
(Engebrecht and Silverman (1984), PNAS 1: 4154-4158; Baldwin et al.
(1984), Biochemistry 23: 3663-3667); alkaline phosphatase (Toh et
al. (1989) Eur. J. Biochem. 182: 231-238, Hall et al. (1983) J.
Mol. Appl. Gen. 2: 101), human placental secreted alkaline
phosphatase (Cullen and Malim (1992) Methods in Enzymol.
216:362-368).
[0324] Transcriptional control elements which may be included in a
reporter gene construct include, but are not limited to, promoters,
enhancers, and repressor and activator binding sites. Suitable
transcriptional regulatory elements may be derived from the
transcriptional regulatory regions of genes whose expression is
induced after modulation of a hedgehog signal transduction pathway.
The characteristics of preferred genes from which the
transcriptional control elements are derived include, but are not
limited to, low or undetectable expression in quiescent cells,
rapid induction at the transcriptional level within minutes of
extracellular simulation, induction that is transient and
independent of new protein synthesis, subsequent shut-off of
transcription requires new protein synthesis, and mRNAs transcribed
from these genes have a short half-life. It is not necessary for
all of these properties to be present.
[0325] Moreover, a number of assays are known in the art for
detecting inhibitors of cholesterol biosynthesis and can be readily
adapted for determining if the subject hedgehog antagonists disrupt
cholesterol homeosynthesis, e.g., by inhibiting biosynthesis and/or
transport of sterols.
[0326] To illustrate, pores formed in the membranes of animal cells
by complexes of sterols and the polyene antibiotic amphotericin B
can efficiently kill the cells. Thus, in the absence of exogenous
sources of cholesterol, inhibitors of enzymes in the cholesterol
biosynthetic pathway render cells resistant to amphotericin B.
However, in the case of class 2 inhibitors, the increase in
cholesterol in the plasma membrane should in fact sensitive the
cells to amphotericin B killing. Preincubation of Chinese hamster
ovary cells with a test compound which disrupts cholesterol
homoeostasis is a manner similar to jervine, such as a test
steroidal alkaloid, will sensitize cells to amphotericin B killing.
This can be used, therefore, to assay test compounds and is
amenable to high through-put screening. A simple two-step protocol
in which cells are preincubated (15-24 h) with potential inhibitors
and then treated (3-6 h) with amphotericin B is described by
Krieger (1983) Anal Biochem 135:383-391, and is a sensitive method
for detecting direct (e.g., competitive) and regulatory inhibitors
of cholesterol biosynthesis. This protocol may prove useful in
detecting potential hedgehog antagonists.
[0327] SREBP cleavage-activating protein (SCAP) stimulates the
proteolytic cleavage of membrane-bound SREBPs, thereby initiating
the release of NH2-terminal fragments from cell membranes. The
liberated fragments enter the nucleus and stimulate transcription
of genes involved in synthesis and uptake of cholesterol and fatty
acids. Sterols repress cleavage of SREBPs, apparently by
interacting with the membrane attachment domain of SCAP. In one
embodiment, the ability of a test agent to interfere with sterol
transport in a manner similar to jervine can be assayed by
generation of activated SCAP or SREBP proteins. For instance, a
particularly desirable embodiment, due to its ability to be used in
high throughput screening, is a reporter gene based assay which
detects SREBP-dependent gene transcription. A variey of genes have
been described in the art as including SREBP responsive elements,
and which are candidate for generation of reporter gene constructs.
For example, Bist et al. (1997) PNAS 94 (20); 10693-8 describes the
presence of SREBP responsive elements in the Caveolin gene; Wang et
al. (1997) J Biol Chem 272:26367-74 describes such elements in the
FAS gene; Magana et al. (1996) J Biol Chem 271:32689-94 describe
the presence of SREBP-RE in the fatty-acid synthase gene; and
Ericsson et al. (1996) PNAS 93:945-50 describe SREBP binding sites
in the farnesyl diphosphate synthase gene. Thus, such reporter gene
constructs as the FAS promoter-luciferase reporter described by
Wang et al. supra, or the squalene synthase promoter-luciferase
reporter described by Guan et al. (1997) J Biol Chem 272:10295-302
can be utilized in an assay for detecting potential equivalents to
jervine. Briefly, in the absence of a class 2 inhibitor, sterol
transport occurs at some level and SREBP-dependent transcription
occurs at a certain rate. Inhibition of sterol trafficking by
jervine or the like results in an increase in sterol precursors in
the plasma membrane and a decrease of such precrusors in the
endoplasmic reticulum. The latter causes activation of
SCAP-mediated cleavage of SREBP. and a concomitant increase in
expression of SREBP-RE reporter gene. Detection of reporter gene
expression can be accomplished by any of a wide range of techniques
known in the art. For example, the reporter gene may one which
confers drug resistance, such as to zeocin or hygromycin. Jervine,
or another compound capable of inhibiting cholesterol homeostasis
in a similar manner, will cause increased resistance to the drug as
expression of the reporter gene is increased.
[0328] In still other embodiments, the ability of a test agent to
effect the activity of 3-hydroxy-3-methyl-glutaryl coenzyme A
(HMG-CoA) reductase can be used to detect compounds which, like
jervine, affect cholesterol homeostasis. Conditions of low
cholesterol or other sterols in the endoplasmic reticulum, such as
caused by class 2 inhibitors like jervine, result in activation of
HMG-CoA reductase. This activation can be detected, for instance,
by detecting increased expression of HMG-CoA (Chambers et al.
(1997) Am J Med Genet 68:322-7) or by detecting increase enzymatic
activity, such as in the HMG-CoA reductase reduction of the
substrate, [.sup.14C]HMG-CoA (see U.S. Pat. No. 5,753,675).
[0329] Exemplification
[0330] 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.
[0331] In order to demonstrate an effect on Shh signaling, we chose
the chick (38) as a more tractable experimental system than the
rodents, sheep and other mammals in which teratogen-induced HPE
predominantly has been studied (14, 15, 16, 29, 39). Chick embryos
are easily cultured and manipulated and, as seen in FIG. 2,
exposure of these embryos to jervine at the intermediate to
definitive streak state (40) induced external malformations
characteristic of HPE (similar results were obtained with
cyclopamine; data not shown). The severity of these defects varied
among treated embryos, as seen in panels B-E by the degree of loss
of midline structures and approximation of paired lateral
structures. These midline deficits thus result in the fusion of the
mandibular and maxillary processes as well as the optic vesicles
and olfactory processes, with consequent cyclopia and formation of
a proboscis-like structure consisting of fused nasal chambers in
the most severely affected embryos (FIG. 2E).
[0332] As seen in FIG. 2 in ovo treatment produced variable defects
and some embryos displayed normal morphology, even at the highest
concentrations tested (50 .mu.M, jervine 5/10 and cyclopamine 2/10,
data now shown). The variability of these effects may be due to
imprecise embryonic staging and difficulties in applying these
hydrophobic compounds uniformly. To reduce this variability and
better evaluate the potential effects of teratogenic compounds on
Shh signaling we established an explant assay that allowed for
precise tissue staging and more uniform application of the
teratogen (41). As shown in FIG. 3A, medial neural plate with
notochord was explanted from a region just rostral to Hensen's
node. At this level, the medial neural plte does not yet express
floor plate cell (HNF3.beta.) or motor neuron (Isl-1) markers (42,
43, data not shown), although the notochord does express Shh (44,
45, data not shown). As seen in FIG. 3B, after a 40 hour incubation
the neutral plate expresses HNF3.beta. and Isl-1. Expression of
these markers has been shown to depend upon Shh signaling, both in
vivo and in vitro (2, 45), and these midline explants thus
constitute an integrated assay of Shh signaling, comprising both
inducing and target tissues.
[0333] To determine whether synthetic and plant-derived teratogens
block Shh signaling we exposed midline explants to varying
concentrations of the drugs AY 9944 and triparanol and to the
steroidal alkaloids cyclopamine and jervine. As can be seen in
FIGS. 3D-K, all of these compounds affect Shh signaling, with a
complete loss of HNF3 and Isl-1 expression consistently caused by
sufficiently high concentrations (FIGS. 3E,G,J,K). At
concentrations several-fold below those required for complete
inhibition, all of the teratogenic compounds are able to block
HNTF3.beta. expression while retaining and often enhancing Isl-1
expression (FIGS. 3D,F,H,H). These effects are fully consistent
with inhibition of Shh signaling (see below). In contract, the
structurally related but not teratogenic steroidal alkaloid
tomatidine (see FIG. 1, ref. 46, data not shown ) is unable to
block expression of HNF3.beta. and Isl-1, even at concentrations
two orders of magnitude higher than the inhibitory concentrations
of jervine and cyclopamine (FIG. 3C).
[0334] Inhibitory Compounds do not Block Shh Processing
[0335] Because the midline explants contain both inducing and
responding tissues, we set out to distinguish possible effects of
these inhibitory compounds on signal production versus possible
effects on signal response. The Shh protein undergoes an
intramolecular processing reaction that involves internal cleavage
and gives rise to an amino-terminal product (Shh-Np responsible for
all known signaling activities. The first step of the
autoprocessing reaction, mediated by the carboxy-terminal sequences
within the precursor, entails an internal rearrangement at the site
of cleavage to replace the scissile peptide bond by the thioester
involving a Cys side chain. In the second step cholesterol supplies
the nucleophile (the 3.beta.-OH) that attacks the thioester
intermediate, and remains covalently attached as an adduct to
Shh-Np (11, 13). Autoprocessing thus is required to release active
signal and the cholesterol adduct restricts the tissue distribution
of the signal by causing it to associate with the cell surface
(12,13).
[0336] Given this role of cholesterol in the giogenesis of Hedgehog
proteins, an effect of these compounds on Shh-Np production is a
particularly appealing possibility since jervine and cyclopamine
structurally resemble cholesterol (FIG. 1) and AY 9944 and
triparanol inhibit specific late-acting cholesterol biosynthetic
enzymes (17, 18, 19, 22). To examine potential effects of these
compounds on Shh processing we utilized HK293 cells cultured in
lipid-depleted serum and carrying a stable integrated construct for
expression of Shh under ecdysone-inducible control (47). Shh
protein expression in these cells can be induced by addition of
muristerone A, an ecdysone analog. As observed in embryos this
protein is efficiently processed (FIG. 4A, lanes 1 and 2), with
little or no detectable accumulation of precursor (M.sub.r 45 kD).
Addition of jervine, cyclopamine, tomatidine, AY 9944, or
triparanol during the 24-hour induction period did not diminish
Shh-N.sub.p production nor induce accumulation of unprocessed
precursor, even at doses 5-fold higher than those required to
completely inhibit Shh signaling (FIG. 4A, lanes 4-13). All of the
amino-terminal cleavage product generated in the presence of these
compounds is detected in cell lysates, not the culture medium (data
not shown), and has the same electrophoretic mobility as
cholesterol-modified Shh-N.sub.p. These observations are consistent
with the presence of a sterol adduct in the amino-terminal cleavage
product, since lack of such an adduct is associated with release
into the medium and with decreased electrophoretic mobility (the
unprocessed amino-terminal fragment is designated Shh-N to
distinguish it from processed Shh-Np; see lanes 8, 9, 17). We also
failed to observe any change in efficiency of Shh processing or
behavior of Shh-NP in transiently-transfected COS-7 or QT6 CELLS
treated with these compounds (48). We also have observed that chick
embryos treated with jervine after floor plate induction displayed
the normal apical localization of Shh protein within floor plate
cells (49).
[0337] Because of their structural similarity to cholesterol, we
also investigated the potential effects of the plant compounds on
an in vitro autoprocessing reaction utilizing purified components.
The protein utilized in this reaction is derived by replacement of
all but six codons of the Drosophila Shh amino-terminal coding
region with sequences encoding a hexahistidine purification tag
(10). The resulting 29 kDa protein, His6Hh-C, in purified form
undergoes autoprocessing in a cholesterol-dependent manner to yield
a 25 kD product (50). As seen in FIG. 5A neither jervine,
cyclopamine, nor tomatidine inhibit this cholesterol-stimulated
autoprocessing reaction, even at concentrations 27-fold higher than
that of cholesterol. Given the presence of 3.beta.-OH in each of
the plant compounds (FIG. 1), we also tested their ability to
replace cholesterol in providing the nucleophilic group during
processing. As seen in FIG. 5B. no appreciable cleavage is
stimulated by addition of these compounds in the absence of
cholesterol.
[0338] The observation that cholesterol synthesis inhibitors such
as AY 9944 and triparanol do not inhibit processing raises the
possibility that cholesterol biosynthetic precursors, which
accumulate in treated cells (see below), may participate in the
reaction. FIG. 5C shows that the in vitro reaction can be driven by
desmosterol, 7-dehydrocholesterol (7DHC), and lathosterol with
efficiencies similar to that of cholesterol. Desmosterol and 7DHC
are the major precursors reported to accumulate in cells treated
with triparanol and AY 9944, respectively. Lanosterl, a 30 carbon
cholesterol precursor, on the other hand is unable to participate
in the reaction, perhaps due to steric interference by the two
methyl groups attached to the C4 carbon near the 3-hydroxyl. In
other studies of this in vitro reaction we have observed a
requirement for an unhindered hydroxyl at the 3.beta. position on a
sterol nucleus, although neither the 8-carbon side chian nor the
number or position(s) of the double bond(s) in the sterol nucleus
appear to critically affect efficiency (51). These observations
suggest that all 27 carbon sterol intermediates in the biosynthetic
pathway are potential adducts in the autoprocessing reaction, and
may account for the unimpaired efficiency of processing in the
presence of distal synthesis inhibitors. Thus, although the extent
of Shh processing in cultured cells and its localization in vivo
appears to be unaffected by these inhibitory compounds (FIG. 4), we
can not rule out the possibility that the sterol adduct may differ
and that such an abnormally modified signal may have distinct
biological properties.
[0339] Inhibitory Compounds Specifically Affect the Response to Shh
Signaling
[0340] Since our studies of processing provided n evidence for an
inhibitory effect of these compounds on Shh signal production, we
examined the alternative possibility that these compound affect
response of target tissues. For these studies we utilized an
intermediate neural plate explant lacking any endogenous source of
inducing signal (41, see FIG. 6A). Recombinant Shh-N protein (45,
52, 53, 54), lacking a sterol adduct, suppresses molecular markers
such as Pax7 (55, see FIGS. 6B, C), normally expressed in dorsal
cell types, and induces ventral markers such as Isl-1 and
HNF3.beta. (FIGS. 6D,E), normally expressed in motor neurons and
floor plate cells. These cellular responses are elicited in a
concentration-dependent manner, with repression of Pax7 observed at
concentrations of Shh-N that are insufficient for induction of
HNF38 (ref. 55.2 nM. FIGS. 6B,C). Isl-1 and HNF3.beta. occurring at
the expense of Isl-1 (note that the induction of Isl-1 at 6.25 nM
Shh-N in FIG. 6D is abolished at 25 nM in 6E).
[0341] The teratogenic compounds are able to block completely the
repression of Pax7 (at 2 nM Shh-N, FIGS. 6F-I) and the induction of
Isl-1 and HNF3.beta. (at 25 nM Shh-N; FIG. 6)-S). In addition
tomatidine produces partial inhibition, but only at concentrations
100200 fold higher than those required for complete inhibition by
jervine and cyclopamine (FIG. 6T). A complete inhibition of the 24
nM response to Shh-N requires does of teratogenic compounds 2-4
higher than those required to completely block the 2 nM response;
inhibition of responses to higher concentrations of Shh-N requires
higher drug concentrations. Another dose dependent effect can be
noted in FIGS. 6K-N, where drug concentrations two fold below the
thresholds required for complete inhibition of the 25 nM response
(induction of HNF3.beta.) result in retention or expansion of Isl-1
expression. A similar expansion of Isl-1 at intermediate drug
concentrations was seen for midline explants (FIGS. 3D-G),
indicating that at a fixed level of stimulation by Shh-N, distinct
degrees of pathway activation can be produced by distinct inhibitor
concentrations.
[0342] To further examine the specificity of these compounds we
tested their effects on induction of a neural crest-like phenotype
by BMP7. The BMP7 signaling protein is expressed in ectodermal
cells adjacent to the neural plate, and appears to function in
induction of neural crest and dorsal neural tube cell fates 956).
To avoid contamination with endogenous lateral signals, the
explants used for these studies were taken from the ventral neural
plate, but excluded the notochord and the midline (FIG. 7A).
Addition of BMP7 protein induced formation of migratory cells that
express the HNK-1 surface antigen (compare FIGS. 7B,C), features
characteristic of neutral crest cells (56). Neither cell migration
nor expression HNK-1 were blocked by addition of jervine at 10
.mu.M (FIG. 7D), a concentration exceeding that required for a
complete block of Shh-N signaling. Similar results were obtained
with tomatidine and with cyclopamine. These compounds also failed
to inhibit formation of migratory HNK-1 positive cells from
explants containing dorsal neural plate and contiguous epidermal
ectoderm (49), which serves as an endogenous source of BMP activity
(56).
[0343] Drug Effects upon Cholesterol Homeostatis
[0344] Pervious reports indicate that triparanol and AY 944 cause
the accumulation of cholesterol precursors (predominantly
desmosterol and 7-dehycholesterol (7DI-IC) by specifically
inhibiting late-acting enzymes of cholesterol biosynthesis
(desmosterol .DELTA.24-reductase and 7DHC .DELTA.7-reductase,
respectively, 17, 18, 19, 22), and a preliminary analysis of
jervine also revealed an effect upon cholesterol biosynthesis (30).
A direct comparison of the effects of these compounds on human
primary lymphoblast cultures (57) revealed that all of them,
including tomatidine, cause a relative decrease in cholesterol
levels and an increase in the levels of other sterols (Table I, ref
58).
[0345] Table 1. Teratogenic compounds disrupt cholesterol
homeostasis in cultured cells. Cholesterol biosynthesis is
inhibited in primary human lymphoblasts cultured in the presence of
the teratogenic compounds and tomatidine (58). The sterol profiles
(57) from these cultures reveal the accumulation of multiple 27-,
28- and 29-carbon sterol precursors of cholesterol (59, 60).
Esterification of PM-labeled [3H]-cholesterol in rat hepatoma cells
is also inhibited by all of the compounds (63).
1TABLE 1 Effects of synthetic and plant-derived compounds on
cholesterol homeostasis. AY9944 Triparanol Jervine Cyclopamine
Tomatidine (.mu.M) (.mu.M) (.mu.M) (.mu.M) (.mu.M) Control 0.25 0.5
1.0 0.25 0.5 1.0 1.25 2.5 5.0 1.25 2.5 5.0 1.25 2.5 5.0 A.
Cholesterol Biosynthesis Assay Total Sterols (.mu.g/mg protein) 9.6
7.6 8.3 8.3 4.1 5.1 3.1 8.8 8.4 10 8.4 8.5 8.3 7.8 7.8 5.6 Percent
Sterols Cholesterol 95 30 33 34 56 45 51 90 90 88 87 76 68 54 42 32
Non-Cholesterol Sterols 1. C27 Sterols a. Desmosterol 1.9 9.1 8.7
6.7 2.5 2.4 2.7 4.2 7.1 11 b. 7 Dehydrodesmosterol 3.5 3.0 1.9 6.0
4.1 2.9 0.8 0.8 0.8 0.5 c. Cholesta-7,24-dien- 1.8 1.9 1.6 3.1 2.4
2.6 0.5 0.5 0.6 0.9 1.6 0.9 0.9 0.7 3.beta.-ol d. Zymosterol 9.3 27
23 1.7 2.0 2.3 2.3 4.5 4.7 e. Cholesta-8(14)-en-3.beta.-ol 9.7 14
20 9.1 8.7 7.3 1.0 1.7 2.3 0.9 2.5 2.7 6.7 8.9 8.7 f. 7
Dehydrocholesterol 50 36 16 1.5 1.4 1.3 2.4 4.2 6.3 19 14 9.8 g.
Lathosterol 1.3 6.2 7.3 7.9 4.9 4.7 3.6 h. C27 Sterol 1 (mw 384)
5.3 7.0 13 20 i. C27 Sterol 2 (mw 382) 6.0 4.1 4.7 j. C27 Oxysterol
1 (mw 400) 1.0 2.4 4.5 k. C27 Oxysterol 2 (mw 400) 2.0 5.0 11 2.
C28 Sterols 0.7 1.1 2.6 4.4 2.6 1.5 1.2 0.7 0.7 1.2 1.3 1.8 1.6 3.
C29 Sterols 1 3.3 4.6 7.8 8.1 5.5 0.8 0.8 1.6 0.7 1.2 3.1 3.1
AY9944 Triparanol Jervine Cyclopamine Tomatidine Percent Inhibition
(incorporation (.mu.M) (.mu.M) (.mu.M) (.mu.M) (.mu.M) of label
into cholesteryl ester*) 2.5 5.0 10 2.5 5.0 10 2.5 5.0 10 2.5 5.0
10 2.5 5.0 10 B. Cholesterol Esterification Assay
.sup.3H-Cholesterol 39 56 68 49 57 79 24 44 50 48 67 79 31 33 39
.sup.14C-Oleic Acid 20 35 51 54 65 81 28 36 49 45 62 74 30 64 52
*The percent of label taken up that was converted to cholesteryl
ester was 8%/hour for .sup.3H-cholesterol and 3.6%/hour for
.sup.14C-oleic acid
[0346] The accumulating sterols largely comprise established
intermediates in the cholesterol biosynthetic pathway or closely
related species that might be generated by action of the
giosynthetic enzymes upon these intermediates (59). Tomatidine
would appear to be the exception to this general rule, with
accumulation to relatively high levels of several unusual sterols
(60).
[0347] Reduction of cholesterol levels coupled with an accumulation
of cholesterol biosynthetic precursors are effects observed for a
group of compounds that have been termed class 2 inhibitors of
cholesterol biosynthesis (61, 62). These compounds appear to act by
inhibiting sterol flux between the plasma membrane (PM) and the
endoplasmic reticulum (ER). Since cholesterol biosynthetic enzymes
are located in the ER. and sterol precursors of cholesterol are
highly concentrated in the PM, such a block in transport results in
an overall reduction of cholesterol levels. We measured the effects
of the synthetic and plant compounds upon esterification of
exogenously added 3H-labelled cholesterol (63) a process which
requires transport of PM cholesterol to the ER. We observed
inhibition of esterification oat levels ranging for 25-75% for
these compounds. An effect of AY 9944 on sterol transport
previously has been reported (23), but this is the least active of
the compounds we tested in inhibition of esterification. Our data
therefore suggest that transport inhibition may be a factor in the
effects of all of these compounds on sterol profiles, consistent
with the general accumulation of multiple cholesterol biosynthetic
precursors. In addition however, AY 9944 and triparanol cause
accumulation to high levels of 7DHC and desmosterol, respectively
consistent with the well-known effects of these compounds on the
7DHC A7-reductase and desmosteroal .DELTA.24-reductase enzymes.
[0348] Discussion
[0349] The teratogenic effects of distal inhibitors of cholesterol
biosynthesis have been known and studied for more than thirty years
(14, 15). Similarly, cyclopamine and jervine were identified about
thirty years ago as the plant compounds responsible for the
teratogenic effects of the range plant Veratrum californicum (28,
29). The most dramatic teratogenic effect of these compounds is the
induction of cyclopia and other features of severe
holoprosencephaly (HPE); the recent discovery that HPE is also
caused by mutations at the murine and human loci suggested the
possibility that these compounds may act to block the Shh signaling
pathway. Our studies have verified the HPE-inducing properties of
these compounds in chick embryos. We have further examined the
early molecular correlates of these teratogenic effects and have
demonstrated that these compounds block the induction by Shh
protein of ventral cell types in chick neural plate explants.
[0350] Despite the inhibitory effects of these teratogens on
cholesterol biosynthesis (17, 18, 19, 22, 30, see above), we found
that none of the compounds appears to interfere with Shh processing
in cultured cells, and that the plant alkaloids neither participate
in nor inhibit an in vitro Hh protein autoprocessing reaction
utilizing purified components. Instead, it is the response to Shh
signaling that is affected, as indicated by failure of exogenously
added Shh-N to induce ventral cell types in the presence of
teratogenic compounds. Furthermore, although exogenously added
Shh-N protein can induce endogenous Shh gene expression in neural
plate explants (64, 65), we have demonstrated a complete inhibition
of response by these teratogens at 2 nM Shh-N, a concentration at
which there is not induction of floor plate cells and therefore no
endogenous Shh expression. The inhibitory effects of these
compounds are dose-dependent, as demonstrated: (1) by maintenance
or even expansion of the Isl-1 intermediate fate at intermediate
inhibitor concentrations below those required for complete
inhibition; and (2), by the requirement for correspondingly higher
concentrations of teratogenic compounds to inhibit the response to
increasing levels of Shh-N protein. A further indication of the
specificity of these effects is the inability of these compounds to
block cell behaviors such as migration expression of Pax7, or
HNK-1, or the response to other inductive signals such as BMP7 at
concentrations that completely block the response to Shh
signaling.
[0351] Our studies of sterol synthesis and transport suggest that
these compounds are acting as class 2 inhibitors of cholesterol
biosynthesis (61). For several reasons, however, simple reduction
of cholesterol levels seem unlikely to account for the effects of
these compounds on Shh signaling. First, the non-teratogenic
compound tomatidine also displays potent inhibitory effects on
cholesterol synthesis. Second Shh signaling in explants is not
inhibited by 25-hydroxycholesterol, a hydroxysterol that blocks de
novo cholesterol biosynthesis (66). We can also rule out an
inhibitory role for specific sterol precursors that may accumulate
in drug-treated cells, since addition of 25-hydroycholsterol
together with inhibitory compounds should eliminate synthesis of
sterol precursors yet does not restore the ability to respond to
Shh signaling (67). An alternative mechanism to simple reduction of
cholesterol would be a disruption of intracellular transport.
[0352] We have also shown that triparanol, jervine, and cyclopamine
are potent inhibitors of PM cholesterol esterification, consistent
with their classification as class 2 inhibitors. Consistent with
transport disruption as the mechanism of drug action in inhibiting
Shh signaling, we have found that several other previously
characterized class 2 compounds also are able to inhibit the
response to Shh signaling in explants (68). Tomatidine, however,
also blocks esterification, indicating that general inhibition of
this transport pathway is not sufficient for an inhibitory effect
on the Shh response. We are currently investigating the possibility
that this pathway comprises multiple steps that are differentially
affected by tomatidine and the teratogenic compounds, and that only
those steps not essential for the Shh response are affected by
tomatidine. The unusual sterols that accumulate in
tomatidine-treated cells are associated with peroxisomal sterol
metabolism (60), consistent with such a differential effect of
tomatidine on intracellular sterol transport.
[0353] In light of these drug effects on cholesterol homeostatis,
it is interesting to note the presence of a sterol sensing domain
(SSD) within Ptc, a key regulator of the Shh signaling pathway
(33). The Ptc SSD initially was detected as a region of similarity
to the Niemann-Pick C. Disease (NP-C) gene (31, 32). The similarity
between Ptc and the NP-C protein extends beyond the five
transmembrane spans of the SSD to include all twelve of the
proposed transmembrane spans of Ptc. The significance of this
sequence homology is not known, and the role of the SSD in NP-C is
not clear, although this protein is proposed to regulate
intracellular trafficking and loss of its function leads to
lysosomal cholesterol accumulation (69). The SSDs of other proteins
confer differential responses to high and low levels of
intracellular sterols. The HMGCoA reductase enzyme thus displays a
3-5 fold decrease in stability as sterol concentration rise, and
this behavior is dependent on the presence of the SSD. The SCAP
regulator protein at low (but not at high) sterol concentrations
stimulates the activity of the S2P metalloprotease, resulting in
cleaveage and activation of the SREBP transcription factor.
[0354] Those of the class 2 cholesterol synthesis inhibitors which
have been examined appear to increase HMGCoA reductase activity and
to stimulate the cleaveage of SREBP. Given the localization of
these two proteins to the ER, a likely mechanism for this effect is
that disruption of sterol transport from PM to ER by class 2
compounds induces a low sterol state in these ER proteins, despite
higher levels of cellular sterols overall. The teratogenic
compounds studied here all affect cholesterol synthesis and
transport, and it is conceivable that they alter the normal
distributions of sterols within intracellular compartments If the
function of Ptc is critically dependent upon the sterol
concentrations in particular compartment, skewed sterol
distributions in this compartment could act to perturb Ptc function
via its SSD. One other possibility is that the function of Ptc in
Shh signaling involves regulation of intracellular transport, as
has been suggested for the related NP-C protein. If this were true,
the perturbations of transport generated by these teratogenic
compounds might affect the transport functions of Ptc in such a
manner as to inhibit Shh signaling.
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[0394] 40. Fertile chick eggs (white leghorn) were placed in a
humidified incubator at 37.5.degree. C. in a rotating tray for 14
hours. The eggs were windowed at the air space and 250 .mu.l of a
sonicated 1 mg/ml jervine solution (Leibovitz's L15 medium, Gibco
BRL) was applied under the shell membrane. The window was taped and
the eggs incubated for an additional 4 days. The embryos were
dissected in phosphate buffered saline (PBS, pH 7.2). The heads
were removed form the trunk at the superior boarder of the heart
and fixed in 3% Glutaraldehyde (EM grade, Polysciences, Inc.) in
0.1M sodium cacodylate (Polysciences, Inc.), 3 mM CaC.sub.ls (pH
7.4) overnight at 4.degree. C. They were washed in 0.1M sodium
cacodylate (pH 7.4 placed in 2% osmium tetroxide (Polysciences,
Inc.), 0.1M sodium cacodylate (pH 7.4) for 2 hours and washed in
water. The samples were then dehydrated in a 50%, 70%, 90% and 100%
ethanol series. Samples were critical point dried in liquid
CO.sub.2 (CPD Model 10, Polaron), sputtter coated with
gold-palladium (Denton Desk II unit) and viewed on an Amray 1810
SEM operated at 20 kV.
[0395] 41. Hamburger and Hamilton stage 9-10 (8-10 somites) embryos
were used for all explant assays. Dissections were carried out in
Leibovitz's L15 medium (Gibco BRL). Midline tissue just rostral to
Hensen's node and well caudal to the last somite was removed with
fine scissors. The neural ectoderm was separated from the lateral
plate mesoderm and endoderm with dispase (Boehringer Mannheim,
grade II 2.4 U/ml) treatment and then washed in L15. Midline,
intermediate and ventral neural plate explants were further
dissected with tungsten needles as diagrammed in FIGS. 3A, 6A and
7A. Dissected tissues were transferred to a chambered coverglass
(Nunc) in a drop of collagen (vitrogen 100, Collagen Biomaterials.
Palo Alto, CA) containing 1.times. modified Eagle's medium (Gibco
BRL) and 24 mM NaH.sub.2CO.sub.3 (final pH 7.4-7.6), and warmed to
37.5.degree. C. for 30 minutes (in the absence of CO.sub.2) for
gelation. Explants were cultured in 400 .mu.l of F12 Nutrient
Mixture (Ham) with glutamine (Gibco BRL), containing N-2 supplement
(1.times., Gibco BRL) and 100 U/ml penicillin and 100 ug/ml
streptomycin in a 5% CO.sub.2, humidified incubator at 37.degree.
C. AY 9944, triparanol, jervine, cyclopamine and tomatidine (all
from 10 mM stocks in 95% ethanol, except AY 9944 which is water
soluble), purified Shh-N and BMP 7 were added at the initiation of
the cultures. All of the explants were cultured for 40-48 hours
except for the intermediate neural plate explants assayed for pax7
repression, which were cultured for 20-22 hours. At the end of the
incubation period, explants were fixed in 4.0% formaldehyde (EM
grade, Polysciences, Inc.) in PBS for 1 hour at 4.degree. C.,
washed with PBS and then stained with a secondary antibody for 2
hours at room temperature. Rabbit anti-rat HNF3.beta. (K2) 1:2000,
mouse anti-ISL1 (40.2D6) 1:1000, mouse anti-pax7 1:10, mouse
anti-rat HNK-1/N-CAM (sigma Biosciences) 1:1000, FITC-conjugated
donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc.)
1:100 and LRSC-conjugated donkey anti-rabbit IgG (Jackson
ImmunoResearch Laboratories, Inc.) 1:300 were all diluted in PBTS.
The explants were examined with an Olympus IX60 inverted microscope
using a planapo objective with a 1.4 numerical aperture. Images
were generated by confocal laser scanning microscopy with a
cripton-argon laser exciting at 488 and 568 nm with emissions at
450-550 and 550-650 nm and utilizing Oz with Intervission software
(Noran) on a Silicon Graphics Inc. platform.
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[0401] 47. HK293 cells, stably transfected with Shh using the
Ecdysonc-Inducible Mammalian Expression System (invitrogen). were
plated in 6-well culture plates (Flacon, well area 9.6 cm.sup.2) in
Dulbecco's modified Eagle's medium (DMEM, Gibco). 10% fetal bovine
serum (FBS), 400 .mu.g/ml Zeocin Invitrogen), 2 mM L-glutamine, 100
U/ml Penecillin, 100 .mu.g/ml Stregtomycin, 350 .mu.g/ml G418
(Invitrogen) at 30-40% confluency and grown at 37.degree. C. The
following day, the media was changed to one that contained 10%
dilapidated serum (K. M. Gibson et al, J. Lipid Res. 31, 515
(1990)) and 1% ITS (Sigma) and otherwise was the same as above.
After 24 hours, the cells were induced to express Shh with the
addition of 1 .mu.M muristerone A (Invitrogen). AY 9944,
triparanol, jervine, cyclopamine and tomatidine (all from 10 mM
stocks in 95% ethanol, except AY 9944 which is water soluble) were
added to the cultures at the time of induction. The control cells
received 0.475% ethanol to equal the maximum ethanol concentration
in the 50 .mu.M steriodal alkaloid treatments. After an additional
24 hours, the culture supernatants were removed and the cells were
lysed in the plate with 3.times. SDS-PAGE cell lysis buffer (3%
SDS), diluted two-fold with water and boiled. Lysate samples (and
in a separate experiment supernatant samples, for which the data is
not shown) were loaded onto SDS-12% polyacrylamide gels for
analysis, immunoblotted with primary antibodies for Shh-N and actin
(Amersham) and horseradish peroxidase-conjugated secondary
antibodies (Jackson ImmunoResearch Laboratories, Inc.), and
visualized with a with luminescent substrate (Pierce).
[0402] 48. Shh processing in transiently transfected cells is
ineffecient, with accumulation of 50-80% of Shh protein as
unprocessed precursor. Even in these circumstances, we did not
observe any effect of jervine, cyclopamine, or tomatidine upon Shh
processing efficiency.
[0403] 49. Unpublished data.
[0404] 50. The in vitro studies of Hh autoprocessing used a
baterially expressed derivative of the Drosophila Hh protein
(Porter 96A). The reactions were carried out as described (Porter
96B), except that the sterols and steroidal alkaloids were dried
down from an ethanol or chloroform stock and resuspended in a 0.2%
Triton-X 100 solution in a bath sonicator prior to addition to the
reaction mixture.
[0405] 51. Other sterols that participate in the reaction with
similar efficiency to cholesterol are .beta.-sitosterol,
5-androsten-3.beta.-ol, ergosterol. 4.beta.-hydroxycholesterol.
19-hydroxycholesterol. 20.alpha.-hydroxycholesterol,
22(S)-hydroxycholesterol, 22(R)-hydroxycholesterol and
25-hydroxycholesterol. Epicholesterol, cholesterol acetate,
.alpha.-ecdysone, 20-OH ecdysone and thiocholesterol are unable to
participatein the reaction.
[0406] 52. C. -M. Fan, M. Tessier-Lavigne, Cell 79, 1175-1186
(1994).
[0407] 53. M. Hynes, et al., Neuron 15, 35-44 (1995).
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(1995).
[0409] 55. J. Ericson, S. Morton, A. Kawakami, H. Roelink, T. M.
Jessell, Cell 87, 661-673 (1996).
[0410] 56. K. F. Liem, G. Tremml, H. Roelink, T. M. Jessell, Cell
82, 969-979 (1995).
[0411] 57. Pooled human lymphoblasts were washed with serum free
RPMI-1640, then plated in 35 mm microwells in RPMI-1640 with 15%
delipidated FBS (Gibson 90) and cultured at 37.degree. C. in a 5%
CO, humidified atmosphere for 12 hours. AY 9944, triparanol,
jervine, cyclopamine or tomatidine was then added and the cells
were incubated for five days, after which the neutral sterols were
extracted and analyzed as described by R. I. Kelley (Clin. Chim.
Acta 236, 45 (1995)). Briefly, pelleted cells were saponified at
60.degree. C. in 4% (w/v) KOH in 90% ethanol with epicoprostanol as
carrier, mixed with an equal volume of water and extracted three
times in hexane. The hexane extracts were dried under nitrogen,
derivatized with bistrimethylsilyltrifluoroacetamide (BSTFA,
Pierce) in pyridine and analyzed by selected ion monitoring gas
chromatography/mass-spectrometry (SIM-GC/MS), utilizing a Hewlett
Packard (HP) 5890A splitless injection port, a 0.2 mm.times.25 m
HP-1 methylsilicone (0.33 .mu.m liquid phase) capillary column and
a HP 5970A mass selective dector operated in electron impact mode
at 70 eV with an ion source temperature of 200.degree. C.
[0412] 58. For determining their effects on sterol composition, AY
9944 and triparanol were used at 0.5.mu.M and jervine, cyclopamine,
and tomatidine were used at 10 .mu.M. Doses lower than these
produced more normal sterol profiles; higher doses increased the
relative levels of cholesterol precursors but also reduced cell
growth during the five day incubation period of this assay.
[0413] 59. Sterols 1a, 1c-g, 2a,b and 3a,b are all intermediates in
normal cholesterol biosynthesis, and 1b is thought to derive from
1a (G. Salen et al., J. Lipid Res. 37, 1169 (1996)).
[0414] 60. Sterol 1h is associated with peroxisomal sterol
synthesis and is particularly prominent in tomatidine treated
cells. Sterol 4 is seen only in normal cells treated with
tomatidine, but not in tomatidine-treated cells from Zellweger's
Syndrome patients, which lack peroxisomes. Sterol 4 is an apparent
dihydroxy-ketosterol whose structure is not yet fully resolved.
[0415] 61. Y. Lange, T. L. Steck, J. Biol. Chem. 269, 29371-29374
(1994).
[0416] 62. Y. Lange, T. L. Steck, Trends in Cell Biol. 6, 205-208
(1996).
[0417] 63. Esterification of plasma membrane [.sup.3H] cholesterol
in hepatoma cells was assaved according to Lange and Steck.
Briefly, AH22 Hepatoma cells were cultured in 25 cm.sup.2 flasks to
.about.89-90% confluency in DMEM 10% FBS at 37.degree. C. The cells
were washed in PBS and then labeled with 1.38 .mu.Ci [.sup.3H]
cholesterol (3.17.times.10.sup.-5 mmol cholesterol) in PBS for 10
minutes at 37.degree. C. The [.sup.3H]cholesterol was in a vortexed
solution of 2.5% Triton WR-1339, 2.5 mM NaPi (pH 7.5) and 0.125 M
sucrose. The cells were then washed in PBS with 0.5 mg/ml bovne
serumalbumin (BSA) and incubated for 1.5 hours at 37.degree. C. in
DMEM 10%FBS without or with AY 9944, triparanol, jervine,
cyclopamine or tomatidine. The cells were detached with trypsin,
washed and suspended in 1 ml PBS. The sterols were then extracted
with 2.5 ml of chloroform:methanol (2:1), dried on a speed vacuum
concentrator, resuspendedin 50 .mu.l of chloroform and spotte don
solica gel G coated TLC plates (Merck). Cholesteryl esters and
cholesterol were fractionated with a heptane:ether:acetic acid
solvent (20:5:1), dried, visualized with I.sub.2 vapor, scraped and
counted directly in an aqueous scintilation counting cocktail
(Econo-Safe, Research Products International Corp.)
[0418] 64. E. Marti, D. A. Bumcrot, R. Takada, A. P. McMahon,
Nature 375, 32)2325 (1995).
[0419] 65. Thomas M. Jessell, personal communication.
[0420] 66. None of the explant responses to treatment with 2 nM or
25 nM Shh-N were affected by additional of 25-OH cholesterol at 25
.mu.M. 25-OH cholesterol is a potent inhibitor of HMG CoA reductase
and at the concentrations used blocks de novo cholesterol synthesis
in chick embryos and in cultured cell systems (data not shown; S.
C. Miller and G. Melnykovych, J. Lipid Res. 25, 991 (1984); J. J.
Bell, T. E. Sargeant and J. A. Watson, J. Bio. Chem. 251, 1745
(1976)).
[0421] 67. Addition of 25 .mu.M 25-hydroxycholesterol to explant
cultures did not reverse the inhibitory effects of any of the
teratogenic compounds.
[0422] 68. Class 2 cholesterol synthesis inhibitors at the given
concentrations block the response of intermediate neural plate
explants to 25 nM Shh-N, without affecting signaling by BMP7: U
18666A 0.25 .mu.M, chloroquine 50 .mu.M, imipramine 75 .mu.M,
progesterone 20 .mu.M.
[0423] 69. P. G. Pentchev, et al. Biochimica et Biophysica Acta
1225, 235-243 (1994).
[0424] All of the references cited above are hereby incorporated by
reference herein.
Equivalents
[0425] 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.
* * * * *